Important words and concepts from Stryer Biochemistry, Fourth Edition (5/31/01):

by Stephen T. Abedon (abedon.1@osu.edu) for Biochemistry 511 at the Ohio State University

 

 

Course-external links are in brackets

Click here to access text's website

Table of Contents are found below

 

Text Table of Contents with Links to Text Website and Other Notes

(Key: Covered, Partially Covered, Not Covered, Unsure)

(note difference in number of chapters covered between profs-when in doubt, follow text using the detailed syllabus = "[topic #]" for guidance)

 

(I)                Molecular Design of Life

(1)                           [topic 1?] [alt 01] Prelude [text site]

(2)                           [topic 02] [alt 02] Protein Structure and Function [text site] (see "amino acids") [amino acid structure quiz]

(3)                           [topic 02] [alt 02] Exploring Proteins [text site]

(4)                           [topic 19] [alt 08] DNA and RNA: Molecules of Heredity [text site] [nucleic acid structure quiz]

(5)                           [topic 20] [alt 10] Flow of Genetic Information [text site]

(6)                           Exploring Genes [text site]

(II)             Proteins: Conformation, Dynamics, and Function

(7)                           [topic 03] [alt 03] Portrait of an Allosteric Protein [text site]

(8)                           [topic 05] [alt 04] Enzymes: Basic Concepts and Kinetics [text site]

(9)                           [topic 06] [alt 05] Catalytic Strategies [text site]

(10)                       [alt 06] Regulatory Strategies [text site]

(11)                       [topic 08] [alt 10] Membrane Structure and Dynamics [text site]

(12)                       Membrane Channels and Pumps [text site]

(13)                       Signal Transduction Cascades [text site]

(a)    (we will put this off until second half of course and then on an as-needed basis)

(14)                       Antibodies and T-Cell Receptors [text site]

(15)                       Molecular Motors [text site]

(16)                       Protein Folding and Design [text site]

(III)          Metabolic Energy: Generation and Storage

(17)                       [topic 07] [topic 09] [alt 11] Metabolism: Basic Concepts and Design [text site]

(18)                       [topic 10] [alt 07] Carbohydrates [text site] [carbohydrate structure quiz]

 

Should we have the midterm here?

 

(19)                       [topic 11] [alt 12] Glycolysis [text site] [week 6] [glycolysis structure & enzyme quiz]

(20)                       [topic 12] [alt 13] Citric Acid Cycle [text site] [week 6] [TCA cycle structure & enzyme quiz—week 7]

(21)                       [topic 13] [alt 14] Oxidative Phosphorylation [text site] [week 7]

(22)                       [topic 14] [alt 15] Pentose Phosphate Pathway and Gluconeogenesis (p. 569) [text site] [week 7]

(23)                       [topic 14] [topic 15]  [alt 16] Glycogen Metabolism [text site] [week 7]

(24)                       [topic 16] [alt 17] Fatty Acid Metabolism [text site] [week 8]

(25)                       [topic 17] [alt 18] Amino Acid Degradation and the Urea Cycle [text site] [week 8]

(26)                       [topic 18] Photosynthesis [text site] [week 8]

(IV)           Biosynthesis of Building Blocks

(27)                       Biosynthesis of Membrane Lipids and Steroids [text site]

(28)                       [alt 18] Biosynthesis of Amino Acids and Heme [text site]

(29)                       Biosynthesis of Nucleotides [text site]

(30)                       Integration of Metabolism [text site]

(V)              Genes: Replication and Expression

(31)                       [topic 19] DNA Structure, Replication, and Repair [text site] [week 9]

(32)                       Gene Rearrangements [text site] [week 9]

(33)                       RNA Synthesis and Splicing [text site] [week 9]

(34)                       [topic 21] Protein Synthesis [text site] [week 9]

(35)                       Protein Targeting [text site]

(36)                       [topic 22] Control of Gene Expression in Prokaryotes [text site] [week 10]

(37)                       Eukaryotic Chromosomes and Gene Express [text site]

 

The following constitute random jottings.

They do not necessarily constrain the amount material you are responsible for:

The Amino Acids

(from pages 20-23 of Stryer)

 

Name

[1],[2]

[3]

Structure[4]

Basic Properties

pK[5]

Glycine

G

Gly

-H

Hydrophobic[6]

 

Alanine

A

Ala

-CH3

Hydrophobic

 

Valine

V

Val

(see text)

Hydrophobic

 

Leucine

L

Leu

(see text)

Hydrophobic

 

Isoleucine

I

Ile

(see text)

Hydrophobic, two chiral carbons

 

Proline

P

Pro

(see text)

Cyclic, not terribly hydrophobic

 

Phenylalanine

F

Phe

(see text)

Hydrophobic, bulky

 

Tyrosine

Y

Tyr

(see text)

Less hydrophobic (than Phe), bulky

10.0, rarely ionized

Tryptophan

W

Trp

(see text)

Hydrophobic, bulky (indole ring)

 

Cysteine

C

Cys

-C-SH

Hydrophobic, highly reactive (disulfide linkages)

8.5, rarely ionized

Methionine

M

Met

-C-C-S-CH3

Hydrophobic (start a.a.)

 

Serine

S

Ser

-CH2OH

Hydrophilic, reactive

 

Threonine

T

Thr

(see text)

Hydrophilic, reactive, two chiral carbons

 

Lysine

K

Lys

-C-C-C-C-NH3+

Highly hydrophilic, positively charged

10.0, ionized

Arginine

R

Arg

(see text)

Highly hydrophilic, positively charged

12.0, ionized

Histidine

H

His

(see text)

Highly hydrophilic, positive or neutral

6.5, varies

Aspartate

D

Asp

-C-COO-

Highly hydrophilic, negatively charged

4.4, ionized

Glutamate

E

Glu

-C-C-COO-

Highly hydrophilic, negatively charged

4.4, ionized

Asparagine

N

Asn

(see text)

Uncharged

 

Glutamine

Q

Gln

(see text)

Uncharged

 

 

 

 

 

 

 

Aspartic acid

or Aspartate

B

Asx

(see text)

Uncharged or charged

 

Glutamic acid

or Glutamate

B

Glx

(see text)

Uncharged or charged

 

·        Called a-amino acids (p. 18-19)

·        20 naturally occurring for which there are codons in DNA & RNA, there are actually many hundreds of amino acids due to post-translational modification

·        Zwitter ions (p. 19)

·        Cyclic form of proline causes rigidity that impacts on protein (secondary) structure

·        pK carboxyl terminus = 3.3; pK amino terminus = 8.0

·        Amino acid mnemonics:

1.      most of the following is from http://www.mednote.co.kr/Yellownote/BIOCHMNEMON.htm:

Structure of Amino Acids

To memorize the molecular structure of some of the 20 amino acids

Glycine

chiral no (this amino acid lacks a chiral carbon) [7]

Alanine

has a methyl group all alone.

Valine

is shape like a V

Isoleucine

Two (rhymes with leu) chiral carbons

Phenylalanine

is simply a phenyl group added to alanine.

Tyrosine

Is shaped like a tie with a tire (phenyl) in it.

Tryptophan

Is shaped like a toe attached to a fan.

Tryptophan

Has an (one) N in it's proximal ring.

Tryptophan

Two rings look like a W (which is its one-letter abbreviation)

Methionine

Has a methyl group attached to a thiol (sulfur) molecule

Serine

has a c (carbon) and a ring (oxygen).

Threonine

Has three branches, including one OH branch.

Lysine

Is long and sharp. Also, lyse means cut (like a knife).

Histadine

forms a backwards d.

Histadine

Has two Ns (in its ring).

Asparagine

looks like and sound like aspartic acid but

Glutamine

has an amino group glued to its side chain. Also, it looks and sounds like glutamic acid but has an amine group attached instead of -OH.

2.      the following is from http://iubio.bio.indiana.edu/R530517-533430-/news/bionet/general/9501.newsm:

 
 ____________________________ mnemonic - "Has a Saab"
|
|                ____________ mnemonic - "Salesmen Take Good And Valuable
|               |                         Lessons In Marketing Caravans     
|               |                         For Your Weekends.  Price
|               |                         Diminishes Enjoyment (but) No
|               |                         Question Helps Kick Ratrace."
|               |
 
H Hydroxyl______S serine               Weakly    Hydrophobic 
               T threonine            Polar          . 
                                         .           /|\ 
A Aliphatic_____G glycine               /|\           | 
               A alanine                |            |
               V valine                 |            |
               L luecine                |            |
               I isoleucine             |            |
                                         |            |
S Sulphur_______M methionine             |            |
   containing  C cysteine               |            |
                                         |            |
A Aromatic______F phenylalanine          |            |
               Y tyrosine               |            |
               W tryptophan             |            |
                                         |            |
S Secondary_____P proline                |            |
                                         |            |
A Acidic________D aspartic acid          |            |
               E glutamic acid          |            |
                                         |            |
A Amide_________N asparagine             |            |
               Q glutamine              |            |
                                        \|/           |
B Basic_________H histidine              .           \|/
               K lysine              Strongly        .
               R arginine             Polar     Hydrophilic

 

3.      the ten essential amino acids mnemonic: These Ten Valuable Amino acids Have Long Preserved Life In Man = Threonine, Tryptophan, Valine, Arginine, Histidine, Lysine, Phenylalanine, Leucine, Isoleucine, Methionine-see http://www.technion.ac.il/medicine/Students/Mnemonics.htm) (note: I’ve included this here just for the heck of it—there is no need to learn which amino acids are essential in man unless you are interested in doing so, in which case we can include this a question on the exam… let me know)

 

Detailed Syllabus Supplied by Biochemistry Department in Columbus

(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

(Key: Detailed syllabus, Stryer, My notes, Stryer online (or links), Alternative syllabus notes)

 

(1)               Topic 1 (Life as a Biochemical Process)

(A)           Origin of Life

(B)            Oparin Theory

(C)           Primitive Environment

(D)           Hill Experiment

(E)            Genesis of a Cell (Origin of Cells)

(2)               Topic 2 (Introduction to Proteins) [Bio 113 chapter 5]

(A)           Amino Acids

(i)                  See below.

(B)            Protein Structures (Review?)

(i)                  Proteins are a unique class of macromolecules in being able to specifically recognize and interact with highly diverse molecules. The repertoire of 20 kinds of side chains enables proteins to fold into distinctive structures and form complementary surfaces and clefts. The catalytic power of enzymes comes from their capacity to bind substrates in precise orientations and to stabilize transition states in the making and breaking of chemical bonds. Conformational changes transmitted between distant sites in protein molecules are at the heart of the capacity of proteins to transduce energy and information. (summary)

(ii)                Proteins are able to interact specifically with such a wide range of molecules because they are highly proficient at forming complementary surfaces and clefts. The rich repertoire of side chains on these surfaces and in these clefts enables proteins to form hydrogen bonds, electrostatic bonds, and van der Waals bonds with other molecules... The catalytic power of proteins comes from their capacity to bind substrate molecules in precises orientations and to stabilize transition states in the making and breaking of chemical bonds... Another recurring catalytic devise is the use of charged groups to polarize substrates and stabilize transition states. (p. 39)

(C)           Amino Acids

(i)                  Amino Acids As Building Blocks

(ii)                Amino Acid Structure and Chemical and Physical Characteristics

(iii)               Amino Acids Ionization

(a)    See "amino acids" above

(iv)             

(D)           Henderson-Hasselbalch Equation (p. 42)

(a)    This is found in a review of pH and buffering found on page 42

(b)   The pK of an acid is the pH at which it is half dissociated

(c)    pH = pK + log{[A-]/[HA]} = Henderson-Hasselbalch equation

(d)   One important take-home message is that pK approximately defines the point of ionization in terms of pH

·        Think of this a the pH as which an ionizable group deprotonates

·        Above this point the proton is lost

·        Below this point the proton is gained

·        If the group is ionized when a proton is present, then it will be ionized when pH is above the pK

·        If the group is ionized when a proton is absent, then it will be ionized when pH is below the pK

(e)    The second important take-home message is that the H-H equation may be employed to determine the pH of buffers

(E)            H3PO4 and H2CO3 as Buffers (Why Locate Here?)

(i)                  ???

(F)            Peptide Bonds (p. 24, 27-28)

(a)    Peptide bond formed by condensation reaction (see figure 2-20)

(b)   Recall what a condensation reaction is (=loss of water molecule between carboxyl group and amino group of adjacent amino acids)

(c)    Recall that condensation reactions are exergonic (require input of energy to go forward)

(d)   Peptide bond is rigid due to partial double-bond character (resonance with carbonyl attached to involved carbon): no free rotation

(e)    Naturally occurring peptide bonds in peptides and proteins form as trans isomers-carbonyl oxygen to a carbon across peptide bond (proline is exception)

(f)     ...the peptide unit is rigid and planar (Figure 2-28). The hydrogen of the substituted amino group is nearly always trans (opposite) to the oxygen of the carbonyl group. The only exceptions are X-Pro peptide bonds (X denotes any residue), which can be cis (on the same side) or trans. The bond between the carbonyl carbon atom and the nitrogen atom of the peptide unit is not free to rotate because this link has partial double-bond character (Figure 2-29)... In contrast, the link between the a carbon atom and the carbonyl carbon atom is a pure single bond. The bond between the a carbon atom and the peptide nitrogen also is a pure single bond. Consequently, there is a large degree of rotational freedom about these bonds on either side of the rigid peptide unit (Figure 2-30). The rigidity of the peptide bond enables proteins to have well-defined three-dimensional forms. The freedom of rotation on either side of the peptide unit is equally important because it allows proteins to fold in many different ways. (p. 27)

(G)           Primary Structures

(a)    Peptide bonds and amino acid sequence. In all proteins, amino acids are joined through peptide (amide) bonds between the a-carboxyl group of one amino acid and the a-amino group of the next. In writing the amino acid sequence of a protein, the residue with the free a-amino group (not in a peptide bond) is listed first and that with the free a-carboxyl group last. Thus Ala-Gly-Glu and Glu-Gly-Ala are two distinct peptides.

(b)   Main chains and side chains of polypeptides. The linear, unbranched main chain of a polypeptide includes the a-amino group, the a carbon, and the a-carboxyl group of each amino acid residue. The sequence of amino acids, each with its particular side chain, determines the unique characteristics of each polypeptide. Linkages between side chains in polypeptides are rare. The most common type is a disulfide link between two cysteine residues.

(c)    Relation between amino acid sequence and polypeptide function. Because each protein is encoded by a distinct DNA sequence, its amino acid sequence is unique. The sequence of amino acids ultimately determines all the complex structural and functional properties of the protein. Each protein folds into a unique three-dimensional structure, which is essential for biological function. Disruption of this three-dimensional structure for any reason almost always impairs the protein's function.

(d)   Modification of amino acids and cleavage of polypeptides. Proteins are initially assembled only from the 20 L-amino acids introduced earlier in the chapter, but sometimes amino acid side chains are modified after assembly is complete. For example, some proline residues in collagen are hydroxylated, a modification that permits additional hydrogen bonds among adjacent collagen chains in connective tissues. Some newly synthesized polypeptides are cleaved or shortened (groups of amino acids removed) before they take on their active functions. Examples here are insulin and some of the digestive enzymes.

(e)    Primary structure by convention written with terminal amino group on left (=beginning) and carboxyl on right (p. 24)

(f)     Within a peptide and amino acid remnant (i.e., what is left after peptide bond formation) is called a residue

(g)    A polypeptide chain consists of a regularly repeating part, called the main chain, and a variable part [the amino acid R groups), comprising the distinctive side chains (Figure 2-22). The main chain is sometimes called the backbone. (p. 24)

(h)    Number of amino acids distinguishes peptides (<50) from proteins (>50)

(ii)                Sanger's Determination of Insulin (p. 25)

(a)    In 1953, Frederick Sanger determined the amino acid sequence of insulin, a protein hormone (Figure 2-25). This work is a landmark in biochemistry because it showed for the first time that a protein has a precisely defined amino acid sequence. Moreover, it demonstrated that insulin consists only of L amino acids in peptide linkage between a-carboxyl groups. This accomplishment stimulated other scientists to carry out sequence studies of a wide variety of proteins. Indeed, the complete amino acid sequence of more than 10,000 proteins are now known. The striking fact is that each protein has a unique, precisely defined amino acid sequence." (p. 25)

(iii)               Use of Sanger's Procedures in Problem, i.e., "N" and "C" Terminal Determination

(a)    The amino-terminal residue of a protein or peptide can be identified by labeling it with a compound that forms a stable covalent link (p. 54)

(iv)              Overlap (p. 56-57)

(a)    "Peptides much longer than about 50 residues cannot be reliably sequenced by Edmund method because not quite all peptides in the reaction mixture release the amino acid derivative in each step" (meaning that the amino acid removed per step becomes increasingly impure with increasing rounds of Edmund degradation)

(b)   This problem is gotten around by breaking up proteins into into <50 amino acid fragments, purifying those fragments, and then sequencing them

(c)    Trypsin and other proteases as well as chemical means of protein fragmenting (cutting at specific sites) are employed

(d)   The overlap method (which may also be employed in DNA sequencing) means that the order of fragments ultimately is determined by sequencing overlapping peptide fragments (produced by employing different fragmenting methods) and then inferring the overall sequence from the position of overlaps

(v)                Edmund's Reagent (p. 55) and Sequentator (p. 55)

(a)    The Edmund degradation sequentially removes one residue at a time from the amino end of a peptide. (p. 55)

(b)   The removed amino acid may then be identified

(c)    A Sequenator is an automated method of protein sequencing employing Edmund degradations

(d)   The amino acid composition of a protein can be ascertained by hydrolyzing it into its constituent amino acids in 6 N HCL at 110°C. They can be separated by ion-exchange chromatography and quantified by reacting them with ninhydrin or fluorescamine. Amino acid sequences can be determined by Edman degradation, which removes one amino acid at a time from the amino end of a peptide... (summary)

(e)    Determining amino acid composition. The initial step is hydrolysis of the peptide bonds with concentrated hydrochloric acid, separation of the free amino acids by ion-exchange chromatography, and quantification of each residue type by reaction with colored or fluorescent dyes (ninhydrin or fluorescamine). The composition of the peptide and the molar ratio of amino acids are now established.

(f)     Sequencing. The Edman degradation (figures 3-18 and 3-19) establishes the amino-terminal residue and the order of the succeeding residues. In this sequence of reactions, the amino-terminal residue is chemically labeled, then the labeled residue is cleaved from the peptide and identified by chromatography. The shortened peptide is passed again through the cycle. In this way up to 50 residues can be identified within a few hours.

(g)    Sequencing of larger proteins. Because the yield at each step of the Edman technique is less than 100%, succeeding cycles yield increasing amounts of "contaminating" derivatives. Thus very large proteins are cleaved into peptides of 50 amino acids or less, each peptide is sequenced, and the order of the peptides in the protein is established.

(H)           Techniques for Protein Purification (Chapter 3)

(a)    The basis for protein purification. Every cell has a variety of proteins, and the first step is to separate the protein to be studied. All proteins have a distinct number and sequence of amino acids, and thus differ in size, shape, and net charge. Biochemists exploit these differences to separate and purify the protein that interests them. The purer the protein, the easier and more reliable will be the determination of its primary structure.

(b)   During purification, the investigator needs a method for periodically checking whether the protein of interest is still present and, in most cases, still functional. For example, the presence and activity of an enzyme can be monitored by a specific assay that measures its catalysis of a particular reaction.

(c)    The three basic techniques for purifying proteins are electrophoresis, chromatography, and ultracentrifugation. You should become familiar with the general principles of these techniques and what each technique can achieve. For example, why are two proteins of similar size but different primary structure more likely to be successfully separated by ion exchange chromatography than by ultracentrifugation?

(d)   Why no mention of electrophoresis in detailed syllabus?

(ii)                Ion Exchange Chromatography (p. 50)

(a)    This is a method of separation is based on protein net charge

(b)   In a column of negatively charged polymer beads, a positively charged protein binds to the negatively charged beads, while a negatively charged protein passes through the column. Proteins are released from the beads when a solution containing a competing positively charged ion is passed through the column.

(c)    Proteins can be separated on the basis of their net charge by ion-exchange chromatography. If a protein has a net positive charge at pH 7, it will usually bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not (Figure 3-9 [p. 50]). A positively charged protein bound to such a column can then be eluted (released) by increasing the concentration of sodium chloride or another salt in the eluting buffer. Sodium ions compete with positively charged groups on the protein for binding to column. Proteins that have a low density of net positive charge will tend to emerge first, followed by those having a higher charge density. Factors other than net charge, such as affinity for the supporting matrix, can also influence the behavior of proteins on ion-exchange columns. (p. 50)

(d)   Note the dependence of mobility over column on charge density

(e)    Note that elution from an ion-exchange column is not equivalent to the "salting out" of a protein

(f)     Negatively charged proteins (anionic proteins) can be separated by chromatography on positively charged diethyl-aminoethyl-cellulose (DEAE-cellulose) columns.

(g)    Conversely, positively charged proteins (cationic proteins) can be separated on negatively charged carboxymethyl-cellulose (CM-cellulose) columns.

(iii)               Gel-Filtration Chromatography (p. 49)

(a)    This is a method of separation based on protein size

(b)   The sample is applied to the top of a column consisting of porous beads made of an insoluble but highly hydrated polymer such as dextran or agarose (which are carbohydrates) or polyacrylamide... Small molecules can enter these beads, but larger ones cannot. The result is that small molecules are distributed both in the aqueous solution inside the beads and between them, whereas larger molecules are located only in the solution between the beads. Large molecules flow more rapidly through this column and emerge first because a smaller volume is accessible to them (see Figure 3-8 [p. 49]). It should be noted that the order of emergence of molecules from a column of porous beads is the reverse of the order in gel electrophoresis, in which a continuous polymer impedes the movement of large molecules (see Figure 3-4 [p. 47, but really see Figure 3.1, p. 46]). Much larger quantities of protein can be separted by gel filtration than by gel electrophoresis, but at the price of lower resolution. (p. 49)

(c)    In other words, if you want to purify a protein particularly by size, then use gel-filtration chromotagraphy

·        Large proteins will come out of the column first

(d)   If you want to separate and then analyze small amounts of protein, then use gel electrophoresis

·        Small proteins will travel faster than larger proteins

(iv)              Affinity Chromatography (p. 50)

(a)    This is a method of separation based on protein binding affinities (qualities)

(b)   Some proteins have high affinity for particular chemical groups and bind to them via one or more types of noncovalent interactions. For example, an enzyme binds tightly to its substrate. A column containing beads with an attached (immobilized) substrate can bind the enzyme as it passes through, while other proteins pass freely. The enzyme is then removed by a solution with a high concentration of unbound substrate. This technique provides a high level of purification from contaminating proteins.

(c)    The high affinity of many proteins for specific chemical groups is exploited in affinity chromatography, in which proteins bind to columns containing beads bearing covalently linked substances, inhibitors, or other specifically recognized groups. (summary)

(d)   Affinity chromatography is another powerful and generally applicable means of purifying proteins. This technique takes advantage of the high affinity of many proteins for specific chemical groups... In general, affinity chromatography can be effectively used to isolate a protein that recognizes group X by (1) covalently attaching X or a derivative of it to a column, (2) adding a mixture of proteins to this column, which is then washed with a buffer to remove unbound proteins, and (3) eluting the desired protein by adding a high concentration of a soluble form of X. (p. 50)

(e)    See figure 3-10 (p. 50)

(I)              Primary, Secondary, Tertiary, and Quaternary Structures and their Characteristics

(a)    Primary structure is discussed above

·        Factors determining three-dimensional structure. We can't yet exactly predict the three-dimensional structure of a protein from its amino acid sequence, but we do have precise information about some three-dimensional structural features common to many proteins. These structures are determined to a large extent by the planar nature of the peptide bond, the freedom of rotation about the other two bonds in the peptide unit, and the nature of the side chains on the bonding and the neighboring amino acids.

·        Reduced, unfolded ribonuclease spontaneously forms the correct disulfide pairings and regains full enzymatic activity when oxidized by air after removal of mercaptoethanol and urea. (summary)

(b)   The two most important secondary structures are a-helices and b-sheets

·        These are both held together by H-bonds between the N-H and the C=O projecting from the main chain

·        a-helix

(i)                  The a helix is a rodlike structure. The tightly coiled polypeptide main chain forms the inner part of the rod, and the side chains extend outward in a helical array (Figures 2-31 and 2-32). The a helix is stabilized by hydrogen bonds between the NH and CO groups of the main chain. The CO group of each amino acid is hydrogen bonded to the NH group of the amino acid that is situated four residues ahead in the linear sequence (Figure 2-33). Thus, all the main-chain CO and NH groups are hydrogen bonded... The a-helical content of proteins ranges widely, from nearly none to almost 100%... The elucidation of the structure of the a helix is a landmark in molecular biology because it demonstrated that the conformation of a polypeptide chain can be predicted if the properties of its components are rigorously and precisely known. (p. 28-30)

(ii)                H-bonding is between chains coiled with a frequency of 3.6 amino acids per turn

(iii)               They may be left or right handed helices but in proteins are found only as right-handed helices

(iv)              R groups project out from axis

(v)                a Helix. This rod-like structure is stabilized by hydrogen bonds between each peptide NH and the peptide CO four residues ahead in the main chain. The helix winds in the right-hand direction. All NH and CO groups in the main chain (except those near each end of the helix) are hydrogen bonded. The R groups extend outward from the axis of the helix. Proline is the only common L-amino acid that cannot occur in the middle of an a helix (its amino nitrogen is not available for hydrogen bonding and its rigid five-membered ring causes steric hindrance in the helix backbone).

·        b-sheets

(i)                  ...the b pleated sheet is stabilized by hydrogen bonds between NH and CO groups in different polypeptide strands, whereas in the a helix the hydrogen bonds are between the NH and CO groups in the same strand. (p. 30)

(ii)                H-bonding is between parallel or antiparallel chains of amino acids

(iii)               Most proteins have compact, globular shapes owing to reversals in the direction of their polypeptide chains. Many of these reversals are accomplished by a common structural element called the b turn. The essence of this hairpin turn is that the CO group of residue n of a polypeptide is hydrogen-bonded to the NH group of the residue n + 3 (Figure 2-37). Thus, a polypeptide chain can abruptly reverse its direction. b turns often connect anitparallel b strands, hence their name. They are also known as reverse turns or hairpin bends. (p. 31)

(iv)              b Strand. The polypeptide chain is more fully extended in this structure, and hydrogen bonds most often form between the CO and NH groups of adjacent chains—either parallel (running in the same direction) or antiparallel (different directions)—from different regions of the polypeptide. Sometimes two or more strands form b sheets.

(v)                Globular proteins are folded structures. Bends or reverse turns serve to change the direction of the main chain, connecting regions of more regular structure such as b strands and a helices. For example, the b turn links antiparallel b strands. Glycine, with its small hydrogen side chain that allows great flexibility, and proline, whose ring causes a natural turn, are common in or near turns or loops.

·        Proline is the one amino acid that consistently does not participate in secondary structures (too bulky plus N not available for H bonding?)

(c)    Supersecondary... (use definition from Stryer site)

(d)   Tertiary

·        Tertiary structure. Interactions between residues that are farther apart in the primary structure determine tertiary structure. An example is the disulfide bond between two cysteine residues. As you'll see later in the text, enzymes often have active (catalytic) sites consisting of the side chains of residues widely separated in the primary sequence but brought together by protein folding. Large proteins can contain locally folded regions, or domains, of tertiary structure that play different roles in overall structure and function. The same domains, having similar roles, may be found in several different proteins.

·        Sulfide linkages: See Figures 2-23, 2-24, and 2-25

·        The polypeptide chain... folds spontaneously so that its hydrophobic side chains are buried and its polar, charged chains are on the surface. (p. 34)... The secret of burying a segment of main chain in a hydrophobic environment is to pair all the NH and CO groups by hydrogen bonding. This pairing is neatly accomplished in an a helix or b sheet. Van der Waals bonds between tightly packed hydrocarbon side chains also contribute to the stability of proteins. We can now understand why the set of 20 amino acids contain several that differ subtly in size and shape (see Figure 2-9). Nature can choose among them to fill the interior of a protein neatly and thereby maximize var der Waals interactions, which require intimate contact. (p. 34-35)

(e)    Quarternary (use definition from Stryer site)

·        Quaternary structure. Proteins with highly complex structural or functional roles may have two or more polypeptide chains, or subunits. In these oligomeric proteins, interactions between subunits—which are necessary to protein function—include hydrogen bonds, salt bridges, and hydrophobic interactions. Quaternary structure describes these noncovalent interactions among the subunits.

(ii)                Importance of H Bonds

(a)    The strong tendency of hydrophobic residues to be herded together by water drives the folding of soluble proteins. Proteins are stabilized by many reinforcing hydrogen bonds and ver der Waals interactions as well as hydrophobic interactions. (summary)

(b)   What are the forces that determine the three-dimensional architecture of proteins? ...all reversible molecular interactions in biological systems are mediated by three kinds of forces: electrostatic bonds, hydrogen bonds, and van der Waals bonds. We have already seen hydrogen bonds between main-chain NH and CO groups at work in forming [secondary structure]. In fact, side chains of 11 of the 20 fundamental amino acids also can participate in hydrogen bonding. It is convenient to group these amino acids according to their hydrogen-bonding potentials... (p. 33)

(iii)               Importance of Semi-Rigid Peptide Bonds

(a)    The flexibility of the main chain combined with the chemical diversity of R groups makes proteins masters of forming complementary interactions particularly between proteins and ligands, proteins and substrates, polypeptides and polypeptides (along their surfaces), and within the interior of proteins

(b)   On the other than hand, the rigidity of one-third of the main-chain bonds (i.e., the peptide bond) allows proteins to form and maintain relatively complex three-dimensional structures

(c)    In a sense, the idea that life reflects an approximation of the complexity of the solid phase within an approximation of the dynamism of the liquid phase is reflected in the flexibility of the main chain with the single bonds around which free rotation is possible accounting for flexibility and at least some of the dynamic nature of proteins and the rigidity of the peptide bond accounting for much of the structural inertness in the main chain necessary for the establishment and maintenance of complexity within the liquid phase

(d)   Proteins containing pairs of sites that are coupled to each other by conformational changes have the capacity to convert energy from one form to another. Suppose that a protein has a catalytic site that hydrolyzes adenosine triphosphate (ATP) to adenosine diphosphate (ADP), and energetically favored reaction (Figure 2-58). The change from a bound triphosphate to a diphosphate group induces a change at the catalytic site that is transmitted to a different binding site some distance away on the same protein. The role of this second site is to bind another protein when ADP is bound to the first site and to release it when ATP is again bound to the first site. Indeed, enzymes with these properties function as molecular motors that convert chemical bond energy into directed movemenet, as in muscle contraction. (p. 40)

(iv)              Pauling Contribution

(a)    Linus Pauling was involved in the prediction of protein secondary structure (a helix and b pleated sheet)

(b)   He was also involved in the discovery of the protein-level difference between normal and sickle-cell hemoglobin (p. 170)

(3)               Topics 3 & 4 (Hemoglobin as a Model Protein) [Bio 113 chapter 5]

(A)           Oxygen Transport

(a)    Oxygen is essential to metabolic processes that extract energy from fuel molecules. In vertebrates, O2 is delivered to cells by hemoglobin, a protein of red blood cells. Hemoglobin binds O2 in the lungs and unloads it in other tissues. Myoglobin serves as an emergency reservoir of O2 in muscle tissue.

(b)   Myoglobin and hemoglobin are the oxygen-carrying proteins in vertebrates. Myoglobin facilitates the transport of oxygen in muscle and serves as a reserve store of oxygen, whereas hemoglobin is the oxygen carrier in blood. (summary)

(B)            Hemoglobin and Myoglobin

(a)    Both myoglobin and hemoglobin have heme as a prosthetic group (a nonpolypeptide unit) that reversibly binds O2. Stryer describes the structure of heme and the role of ferrous iron (Fe2+) in O2 binding (page 148). Note that iron carries out its O2-binding function in heme only in the ferrous form.

(b)   See Figure 7.2 (p. 148). Note the structure of Heme (which is Protoporphyrin complexed with Fe) such that you can at least recognize it.

(c)    The myoglobin polypeptide. Kendrew's structural studies on myoglobin gave the first detailed picture of a globular protein. Both the rigidity and planarity of the main-chain peptide groups and the presence of a number of a helices confirmed the earlier ideas of Pauling and Corey (see chapter 2, pages 27-28). The compact molecule also illustrates many of the features of folded polypeptides that were later described in crystallographic studies of other proteins. These features include

·        the virtual absence of water in the interior of the folded protein, and

·        the role of proline in interrupting a-helical structure.

(C)           Real Molecules-Structure Function

(a)    The idea here is to try to understand how Hemoglobin structure translates into Hemoglobin function, particularly as a model for understanding of how protein structure in general translates into protein function.

(b)   Note as you study that much of the Hemoglobin protein consists of domains that either bind Heme or which are employed to transport information from one portion of the protein to another.

(c)    When understanding how Hemoglobin's structure translates into function, typically these latter amino acid residues are somewhat ignored while instead one focuses on the chemistry and spatial orientation of those amino acid side chains that specifically interact with substrates, as well as the various allosteric effects that result from their interaction with substrates (and other molecules).

(D)           X-Ray Crystallography

(a)    Hemoglobin chains. Hemoglobin exists in several different forms during the course of vertebrate development, including embryonic, fetal, and adult hemoglobins. The main adult hemoglobin (hemoglobin A, or Hb A) has two a and two b chains. Fetal hemoglobin (hemoglobin F, or Hb F), the most prominent form during the last six months of fetal life, has two a and two z (zeta) g (gamma) chains.

(b)   Perutz's x-ray studies of hemoglobin revealed a tetrameric molecule, with each chain, or subunit, containing a heme group. The four subunits are tightly packed together, held by noncovalent forces, with extensive contacts between each a chain and the two b chains.

(c)    Hemoglobin consists of four polypeptide chains, each with a heme group. Hemoglobin A, the predominant hemoglobin in adults, has the subunit structure a2b2. The three-dimensional structure of the a and b chains of hemoglobin is strikingly similar to that of myoglobin. (summary)

(E)            Spatial Appearance of Myoglobin

(a)    Myoglobin, a single polypeptide chain of 153 residues (18 kd), has a compact shape. The inside of myoglobin consists almost exclusively of nonpolar residues. About 75% of the polypeptide chain is a-helical. The single ferrous group is located in a nonpolar niche, which protects it from oxidation to the ferric form. (summary)

(b)   Similarities between hemoglobin and myoglobin. The three-dimensional structures of myoglobin and each type of hemoglobin subunit are quite similar (figure 7-17). Given that only about one-sixth of the amino acid sequence is identical in the myoglobin and hemoglobin chains, quite different primary sequences clearly can specify very similar three-dimensional structures.

(c)    Those residues that are highly conserved in the myoglobin and hemoglobin chains appear to be essential to the O2-carrying function or to crucial structural features. The conservation of Gly at the junction of the B and E helices illustrates the crucial role even the smallest amino acid can play in determining protein structure.

(d)   When comparing the three types of chains, another interesting feature is the nature of the residues inside the globular folded molecules. These residues can differ among the three types of chains, but all are hydrophobic.

(F)            Positions of Porphyrine Ring

(a)    Oxygenation of heme. Experiments on myoglobin, free heme groups, and picket-fence hemes have shown that the protein portion of myoglobin plays an essential role in the reversible O2-binding function of heme. Study the experiments described on pages 151-152 to understand why this is so.

(b)   Two hemes must sandwhich an O2 molecule for oxygen to irreversibly bind heme-this cannot occur when hemes are protected within clefts in proteins such as myoglobin and hemoglobin; thus, oxygen is allowed to only interact with the ferrous heme, without oxidizing it to ferric heme, thereby allowing only reversible interaction = O2 can leave, heme then available to bind subsequent O2.

(G)           Iron Coordination Number

(a)    These proteins contain tightly bound heme, a substituted porphyrin with a central iron atom. The ferrous (+2) state of heme binds O2, whereas the ferric (+3) state does not. (summary)

(b)   But only the ferrous state is present. One function of the protein surrounding the heme is to prevent the formation of the ferric state.

(H)           Role of Proximal and Distal Histidines

(a)    See Figures 7-2, 7-6, and 7-7 to get a feel for what is meant by proximal and distal histidines. Note that the proximal histidine binds to Fe (thereby filling up the fifth available Fe binding site="fifth coordination position"). Note that oxygen fills the sixth position. Note that the distal histidine does not bind Fe but instead interferes with the binding of other things to Fe, i.e., allows O2 to bind in a preferred angled configuration (not perpendicular to heme plane) while CO is forced also to bind in an angled configuration while it prefers perpendicular. (pp. 148-150).

(b)   Kendrew's studies also showed how myoglobin provides a suitable environment for the binding of O2 to the heme. Note that the F8 (proximal) and E7 (distal) His residues are the only polar side chains located inside the globular structure (figures 7-4 and 7-6).

(c)    The iron atom of the heme is directly bonded to a nitrogen atom of a histidine side chain. This proximal histidine occupies the fifth coordinate position. The sixth coordinate position on the other side of the heme plane is the binding site for O2. (summary)

(d)   The nearby distal histidine diminishes the binding of CO at the oxygen-binding site and inhibits the oxidation of heme to the ferric state. (summary)

(e)    ...the protein forces CO to bind at an angle rather than in line. This bent geometry in the globins weakens the interaction of CO with the heme. (p. 152)

(f)     Carbon monoxide binding. Note in figure 7-13 how the distal His (His E7) decreases the natural affinity of heme for the toxic CO molecule. Interference with the linear geometry required for optimal coordination and binding of CO means that under normal CO concentrations (CO produced in cells), less than 1% of myoglobin and hemoglobin are occupied by CO.

(I)              Hemoglobin Differences (???)

(J)              Subtleties Generated (???)

(K)           Curves of O2 Saturation

(a)    Kinetics of O2 binding. In oxidative metabolism, cells use O2 as an electron acceptor and produce protons and carbon dioxide. The role of hemoglobin is

·        to transport ample O2 from the lungs to the cells, and

·        to pick up H+ and CO2 from tissues for transport to the lungs.

(b)   The kinetics of O2 binding differs in myoglobin and hemoglobin. Hemoglobin binds O2 cooperatively: a hemoglobin molecule binds O2 more efficiently when one or more oxygens are already bound. Figure 7-20 shows the sigmoid kinetics of O2 binding to hemoglobin. Compare this with the hyperbolic biding curve for myoglobin.

(c)    As Stryer shows on pages 157-159, the expression for the O2 dissociation curve of myoglobin cannot be used to describe the kinetics of O2 binding by hemoglobin. Make sure you understand why this is so.

(d)   The Hill plot is used as a measure of cooperativity in hemoglobin. The Hill coefficient of 2.8 for hemoglobin shows that binding of O2 to one subunit facilitates binding of additional O2 to subunits of the same molecule. Again, make sure you understand how the Hill plot demonstrates cooperativity and why the Hill coefficient for myoglobin is 1.0

(e)    Myoglobin gives a Hill plot with n = 1.0 (Figure 7-21), which means that O2 molecules bind independently of each other, as indicated in equation 1 [and that makes perfect sense since each myoglobin protein can bind only a single O2]. In contrast, the Hill coefficient of 2.8 for hemoglobin [each of which can bind four O2, total] indicates that the binding of oxygen in hemoglobin is cooperative. Binding at one heme facilitates the binding of oxygen at the other hemes on the same tetramer. Conversely, the unloading of oxygen at one heme facilitates the unloading of oxygen at the others. In other words, the heme groups of a hemoglobin molecule communicate with each other. (p. 159)

(L)            Physiological and Structural Explanations

(a)    Changes in hemoglobin's quaternary structure. Stryer describes the structural changes among the four subunits when O2 binds to hemoglobin. Study the various depictions of the change in the tetramer when O2 binds to a heme group (figures 7-27, 7-29, 7-30). The important point is that the initial binding of O2 to one subunit increases the O2 affinity of hemes in the other subunits, so that subsequent binding occurs more rapidly.

(b)   When O2 binds to a heme, the iron moves into the plane of the porphyrin and makes it more planar. This movement tugs on the proximal His residue coordinated with the heme, and movement of this His shifts adjoining helices in the subunit. Overall, these movements affect more remote interactions with the other subunits to break salt bridges and to increase affinity for additional O2 molecules.

(c)    This idea of movement into the plane is readily apparent upon viewing Figure 7-32 (p. 163). Note that this movement results in a subtle shift of the main chain attached to the proximal histidine.

(d)   Oxyhemoglobin and deoxyhemoglobin structures differ in several ways:

·        The oxy form is more compact than the deoxy form.

·        The carboxyl-terminal residues of the oxy form have more freedom of rotation than the anchored carboxyl-terminal residues of the deoxy form.

·        The oxy form has eight fewer salt links than the deoxy form.

(e)    The difference in the number of salt links makes the deoxy form more taut or tense (rigid), as denoted by T, than the more relaxed oxy or R form.

(f)     With salt bridges (between subunits), taut (T), larger (less compact), not oxygenated.

(g)    Without salt bridges (between subunits), relaxed (R), smaller (more compact), oxygenated.

(M)          Bohr Effects

(a)    Hemoglobin as O2 transporter. As figure 7-22 shows, hemoglobin is an ideal O2-transport molecule.

·        Hemoglobin can release almost twice as much O2 in active muscle as can myoglobin.

·        Unlike myoglobin, hemoglobin binds H+ and CO2, and its ability to bind O2 is influenced by the binding of either of these small molecules.

·        Deep in tissues, where the CO2 and H+ concentrations are high, hemoglobin unloads O2 rapidly (the Bohr effect). In the lungs, where O2 concentration is high, hemoglobin binds O2 readily, as it unloads CO2 and H+.

(b)   CO2 and H+ binding. Binding of CO2 (in the form of carbonate) to hemoglobin stabilizes the T (deoxy) form through formation of salt bridges. This reduces the affinity of the protein for O2.

(c)    Deoxyhemoglobin binds H+ more effectively than does oxyhemoglobin. Changes in the local ionic environment of three residues in each subunit following the oxy to deoxy transition give these residues a greater affinity for protons.

(N)           BPG Action

(a)    2,3-Bisphosphoglycerate (BPG). Another small molecule that influences the ability of hemoglobin to bind O2 is BPG. By binding firmly to deoxyhemoglobin, BPG lowers the O2 affinity of hemoglobin by a factor of 25, thus promoting the unloading of O2 in tissues.

(b)   The finding that one BPG molecule binds to each hemoglobin tetramer suggested that BPG must bind at a place where it can interact with all four subunits. X-ray analysis eventually pinpointed the site of interaction of BPG with both b chains. BPG decreases O2 affinity by cross-linking the b chains, stabilizing the deoxy form.

(c)    Note how the lower affinity of hemoglobin F (F is for fetal) for BPG results from the difference in chemistry between the Ser side chain and the His side chain. (that is, fetal hemoglobin binds BPG less well, due to differences in its structure relative to hemoglobin A, so consequently fetal hemoglobin has a higher affinity for O2 than adult hemoglobin so can strip adult hemoglobin of its O2 cargo)

(O)           Allosteric Effects

(a)    ...new properties appear in tetrameric hemoglobin that are not present in monomeric myoglobin. Hemoglobin transports H+ and CO2 in addition to O2. Furthermore, their binding is regulated by allosteric interations, which are interactions between separate sites on the same protein. Indeed, hemoglobin is the best-understood allosteric protein. (summary)

(b)   Hemoglobin exhibits three kinds of allosteric effects.

·        First, the oxygen-binding curve of hemoglobin is sigmoidal, which means that the binding of oxygen is cooperative. The binding of oxygen to one heme facilitates the binding of oxygen to the other hemes in the same molecule.

·        Second, H+ and CO2 promote the release of O2 from hemoglobin, an effect that is physiologically important in enhancing the release of O2 in metabolically active tissues such as muscle. Conversely, O2 promotes the release of H+ and CO2 in the alveolar capillaries of the lungs. These allosteric linkages between the binding of H+, CO2, and O2 are known as the Bohr effect.

·        Third, the affinity of hemoglobin for O2 is further regulated by 2,3-bisphosphoglycerate (BPG), a small molecule with a very high density of negative charge. BPG binds tightly to deoxyhemoglobin but not to oxyhemoglobin. Hence, BPG lowers the oxygen affinity of hemoglobin. Fetal hemoglobin (a2g2) has a higher oxygen affinity than adult hemoglobin because it binds BPG less tightly. (summary)

(c)    The allosteric properties of hemoglobin arise from interactions between its a and b subunits. The T (tense) quaternary structure is constrained by salt links between different subunits, giving it a low affinity for O2. These intersubunit salt links are absent from the R (relaxed) form, which has a high affinity for O2. On oxygenation, the iron atom moves into the plane of the heme, pulling the proximal histidine with it. This motion cleaves some of the salt links, and equilibrium is shifted from T to R. BPG stabilizes the deoxy (T) state by binding to positively charged groups around the central cavity of hemoglobin. (summary)

(d)   Carbon dioxide, another allosteric agent, binds to the terminal amino groups of all four chains by forming readily reversible carbamate linkages (see chemical equation top of p. 165 to understand what carbamate is). The hydrogen ions participating in the Bohr effect are bound to several pairs of sites that have a more negatively charged environment in the deoxy than in the oxy state.

(e)    Models of allosteric interactions.

·        In the sequential model, a conformational change from the T to the R form, induced in one subunit by substrate binding, makes it easier for the substrate to bind to another subunit and for that subunit to change from the T to the R form. Hybrid tetramers containing both T and R forms are possible.

·        The concerted model allows no hybrid forms. Each substrate-binding event increases the chance that all subunits will assume the R form and thus bind substrate more easily.

·        Study figures 7-37 and 7-39, which summarize the two models.

(f)     My understanding of these models is that in the sequential model the protein can consist of a mixture of T and R forms with O2 bound only to the R form. The more O2 bound, the more R forms, the less salt bridges holding the rest of the subunits in the T form, so the more readily O2 can bind [to additional subunits]. In the concerted model the protein can be either be all R or all T. O2 can bind to either form, though most readily to the R form. The more O2 bound, the more likely it will be all R. Hence, in both cases, the more O2 bound, the greater the affinity of the protein for additional O2 molecules (i.e., with more O2 bound, subsequently binding O2s do so more readily).

(g)    Allosteric interactions in hemoglobin. The binding of O2 to hemoglobin probably involves a combination of the two models.

(h)    Review the information on O2, CO2, H+, and BPG binding in hemoglobin. Make sure you understand the interplay between the various types of binding and the deoxy-oxy transition of the protein molecule. For example, you should be able to relate the graph in figure 7-26 to the structural differences between the oxy and deoxy forms of fetal and adult hemoglobin.

(P)            Sickle Cell Anemia

(i)                  Discovery of Defect

(a)    Sickle-cell disease and Hb S. Stryer describes James Herrick's dramatic clinical findings in 1904 of sickle-cell disease. Later studies revealed the existence of a mutant hemoglobin, hemoglobin S, in individuals with this disease.

(ii)                Effect of Defect

(a)    A comparison of the primary sequences of the b chains of Hb S and Hb A revealed only one difference. Hb S contains a Val residue at position 6, rather than the Glu found in Hb A. This single change results in a hydrophobic area on the b chain, which in deoxyhemoglobin is the site of interaction between the b chains of hemoglobin S molecules (I assume that this means interactions between hemoglobin proteins). The interaction produces long, insoluble polymers of deoxyhemoglobin. Formation of these fibers in red cells causes the cells' characteristic sickle shape.

(b)   This substitution of a nonpolar side chain for a polar one drastically reduces the solubility of deoxyhemoglobin S, which leads to the formation of fibrous precipitates that deform the red cell and give it a sickle shape. (summary)

(c)    Figures 7-47, 7-48, and 7-50 depict the characteristics of Hb S that make it prone to forming insoluble fibers.

(d)   Protection against malaria. An interesting consequence of the occurrence of the Hb S gene in some African populations is that heterozygotes (people with sickle-cell trait) appear to be protected against malaria, perhaps by destruction of infected cells.

·        Make sure you understand the difference between the heterozygous and homozygous conditions in sickle-cell disease.

·        Sickle-cell anemia arises when a person is homozygous for the mutant sickle gene. The heterozygous condition, called sickle-cell trait, is relatively asymptomatic. (summary)

(iii)               Treatments

(Q)           Fetal Hemoglobin

(a)    Fetal hemoglobin. Hemoglobin F in fetal blood has special properties that enable it to load O2 from circulating maternal blood. At the same partial pressure of O2, hemoglobin F binds O2 more strongly that does hemoglobin A (figure 7-26). This stronger binding is the result of the weaker binding of BPG to fetal hemoglobin.

(b)   Fetal hemoglobin consists of subunits coded by different genes that are similar but not identical to the subunits employed in adults. These fetal subunits are given different names (e.g., g rather than b). (p. 154)

(4)               Topic 5 (Introduction to Enzymes) [Bio 113 chapter 6]

(A)           Enzymes (Introduction)

(i)                  General Characteristics

(a)    Enzymes catalyze virtually all reactions in living cells. Chapter 8 begins with an introduction to the catalytic power, specificity, and regulation of enzymes. Stryer introduces some basic thermodynamic principles relevant to biochemical reactions, then discusses enzyme catalysis, kinetics, and inhibition.

(b)   This chapter draws on what you've learned so far about protein structure (chapter 2) and interactions between biomolecules (chapters 1 and 7). And it sets the stage for the many biochemical reactions discussed in the remaining chapters of the book.

(c)    Enzyme specificity. Each enzyme catalyzes a particular reaction and is specific for a particular substrate (or substrates). Each biochemical pathway consists of a series of reactions with a distinct set of enzymes-each enzyme catalyzing one specific step.

(d)   Stryer uses several types of proteolytic enzymes and DNA polymerase I to illustrate differences in degree of enzyme specificity.

(ii)                Kinetics

(a)    Enzyme kinetics is a description of enzyme activity.

(b)   The catalytic activity of many enzymes is regulated in vivo. Allosteric interactions, which are defined as interactions between spatially distinct sites, are particularly important in this regard. The enzyme catalyzing the first step in a biosynthetic pathway is usually inhibited by the final product. Enzymes are controlled by regulatory proteins... Covalent modificiations such as phosphorylation... are a third means of modulating enzymatic activity. The conversion of an inactive precursor protein into an active enzyme by peptide-bond cleavage, a process termed proteolytic activation, is another recurring devise. (summary)

(c)    An enzyme is a catalyst, and consequently it cannot alter the equilibrium of a chemical reaction. This means that an enzyme accelerates the forward and reverse reaction by precisely the same factor... Enzymes accelerate the attainment of equilibria but do not shift their position. (p. 188)

·        Which is not to say, by the way, that the forward and reverse reactions will have the same rate, only that the increase in rate will be by the same factor.

(B)            Activation Energy

(a)    Enzymes accelerate reactions by stabilizing the transition state and lowering the required activation energy (note how these two ideas are related).

(b)   The catalysts in biological systems are enzymes, and nearly all of them are proteins. Enzymes are highly specific and have great catalytic power. They enhance reaction rates by factors of at least 106. Enzymes do not alter reaction equilibrium. Rather, they serve as catalysts by reducing the free energy of activation of chemical reactions. Enzymes accelerate chemical reactions by providing a new reaction pathway in which the transition state (the highest-energy species) has a lower free energy and hence is more accessible than in the uncatalyzed reactions. (summary)

(c)    Transition state. The transition state is a rare form of a reactant, existing during a biochemical reaction, that has a higher free energy than any other intermediate along the reaction pathway (page 188, figure 6-7). The rate of a biochemical reaction depends on the concentration of substrate molecules in the transition state. Enzymes increase that concentration by specific binding to the substrate and by decreasing the free energy of activation for a particular reaction (figure 8-7). Another way to express this is that when enzymes lower the free energy of activation, more reactant molecules have sufficient thermal energy to reach the transition state.

(d)   Be sure that you understand the difference between the free-energy change of a reaction (pages 185-187) [which is discussed in Topic 6] and the free energy of activation (page 188).

(e)    The essence of catalysis is the stabilization of the transition state. Hence, enzymes bind the transition state more tightly than the substrate. Transition state analogs are stable compounds that mimic key features of the highest energy species. They are potent and specific inhibitors of enzymes. (summary) (emphasis mine)

(C)           Active Site

(a)    The first step in catalysis is the formation of an enzyme-substrate complex. Substrates are bound to enzymes at active-site clefts from which water is largely excluded when the substrate is bound. The specificity of enzyme-substrate interactions arises mainly from hydrogen bonding, which is directional, and the shape of the active site, which rejects molecules that do not have a sufficiently complementary shape. (summary)

(b)   Enzyme-substrate complex. In order to carry out catalysis (i.e., reduce the free energy of activation for a chemical reaction), an enzyme must bind specifically to its substrate. Recognition of this requirement led to the concept of the enzyme-substrate (ES) complex.

(c)    Evidence for the ES complex came initially from kinetic studies (figure 8-8): plotting velocity of an enzyme reaction against substrate concentration produces a saturation curve (how does this support the idea of an ES complex?[8]). More direct evidence came from visualization of ES complexes (figure 8-9) and from observation of changes in the absorption spectrum of enzymes and substrates during formation of the complex (figure 8-10).

(d)   Active site. Subsequent studies led to the concept of the active site, the region of an enzyme to which substrates and other essential molecules (cofactors or prosthetic groups) bind. Some important features of the active site are:

·        It is very small relative to the overall size of the enzyme molecule.

·        At the active site, reactive groups that may be a considerable distance apart in the primary structure are brought together by protein folding.

·        Interactions among groups in the active site and the substrate usually involve the noncovalent interactions described in chapter 1: hydrogen bonds, and electrostatic, van der Waals, and hydrophobic interactions.

·        These interactions often occur in a cleft or crevice in the folded polypeptide, from which water and other molecules are excluded.

(ii)                Lock and Key; Induced Fit; Response to Substrate

(a)    The most important feature [of the active site] is the highly specific recognition and complementary interaction among the functional groups of the active site and the substrate.

(b)   Fischer's "lock and key" concept of enzyme-substrate interaction has been modified by the finding that substrate binding can induce changes in the shape of the active site.

(c)    Compare Figure 8-13 with Figure 8-14 (p. 191).

(D)           Enzyme Kinetics

(i)                  Michaelis-Menton

(a)    The Michaelis-Menten model accounts for the kinetic properties of some enzymes. In this model, an enzyme (E) combines with a substrate (S) to form an enzyme-substrate complex, which can proceed to form a product (P) or to dissociate into E and S. (summary).

·        E + S ¾ k1 à ES

·        ES ¾ k2 à E + S (which is equivalent to E + S ß k2 ¾ ES)

·        ES ¾ k3 à E + P

(b)  

(c)    The rate V of formation of product is given by the Michaelis-Menten equation in which Vmax is the rate when the enzyme is fully saturated with substrate, and KM, the Michaelis constant, is the substrate at which the reaction rate is half maximal. (summary)

·        Note that the form of the above equation is slightly different from how it is presented in the book (it is, however, the same equation and is easily converted into the form found in the book). However, this form of the equation is, I believe, more useful since it is easier to see that KM = [S] at 1/2 Vmax (as discussed below). That is, when S = KM then KM / S = 1 and V = Vmax / (1 + 1) = Vmax / 2.

(d)   The maximal rate, Vmax, is equal to the product of k3 and the total concentration of enzyme. (summary)

(e)    The kinetic constant k3, called the turnover number, is the number of substrate molecules converted into product per unit time at a single catalytic site when the enzyme is fully saturated with substate. Turnover numbers for most enzymes are between 1 and 104 per second (summary)

(f)     See Figure 8-15 (p. 192). Make sure you understand this figure and how it relates to the Michaelis-Menten equation.

(g)    Substrate concentration and reaction velocity. Figure 8-15, showing how the velocity of an enzyme reaction varies with substrate concentration, is a typical plot for many types of enzymes. Michaelis and Menten developed an expression that models this kinetic behavior. They postulated that an enzyme binds to a substrate to form ES, an enzyme-substrate complex. Because they could not measure the concentration of ES, they worked toward an expression that contains measurable quantities: substrate concentration and reaction velocity.

(h)    Be conscious of what that last sentence means, particularly as one would attempt to determine these quantities in the laboratory.

·        Substrate concentration is easily measured, particularly under conditions where substrate concentration is depleted only very slowly (i.e., substrate concentration doesn't change very quickly from initial substrate concentration as when enzyme concentration is relatively low) or is otherwise easily measured.

·        Reaction velocity might sound like an exotic term, but it refers only to the rate of product formation (which, ideally, is also easily measured). If substrate formation stays more or less constant, then the rate of product formation also should stay more or less constant, and therefore should be easily determined.

(i)      Among the key assumptions used to develop the model are,

·        the product, P, of the ES complex seldom converts back to the initial substrate, S [this keeps things simple and is easily achieved in practice by running reactions in the presence of relatively little product-i.e., initiate reaction with no product present and run reactions for not very long]; and

·        the rates of formation and dissociation of ES are equal. [this is a steady-state idea; so long as the rate of a reaction is neither increasing nor decreasing, then the rate of formation of the ES must be exactly balanced by the rate of dissociation of the ES]

(j)     Michaelis constant. From equation 18, which expresses the key assumptions of the model, rearrangement gives an equation that includes the three rate constants for the pathway from E +S to E + P (equation 14). The ratio (k2 + k3)/k1 is defined as the Michaelis constant, KM.

·        That is, KM equals the rate of dissociation of individual ES complexes divided by the rate constant for the formation of ES

·        if the rate of formation is high, but the rate of dissociation is low, under steady-state conditions there will be more ES present at any given moment than if the rate of formation is low and the rate of dissociation is high

·        This is another way of saying that easily produced things that are durable tend to accumulate while difficult to produce things that are fragile don't tend to accumulate-and that accumulation will occur until there are so many of the things that their rate of loss, if proportional to their absolute number, will eventually come to equal their rate of formation

·        A high KM means that the ES is either difficult to produce (small k1) or is fragile (large k2 or k3), or both.

·        A low KM means that the ES is either easy to produce (large k1) or is durable once produced (low k2 and k3), or both.

(k)   KM is a useful parameter for any enzyme with the hyperbolic kinetics shown in figure 8-15.

·        Keep in mind that hyperbolic kinetics is the simplest case. It means that reaction velocity increases until it approaches a limiting velocity (a limit, by the way, which is a function of enzyme number and enzyme turnover number).

(l)      Michaelis-Menten equation. Stryer shows how to derive equation 26, which expresses the velocity of an enzyme reaction in terms of KM, [S] (substrate concentration), and [ET] (total amount of enzyme). Substituting Vmax, the maximum velocity of the reaction, into this equation gives equation 28, the Michaelis-Menten equation.

·        See above or check text (equation 28, p. 193).

(m)  The Michaelis-Menten equation shows that when the substrate concentration equals KM, the velocity of the reaction is half the maximum velocity. Put another way, KM is the substrate concentration required for the reaction to reach half its maximum velocity.

·        This is a second way to think about what KM means. KM = [S] at 1/2 Vmax where Vmax is the maximum rate that a reaction can proceed for a given enzyme concentration (i.e., under S-saturating conditions).

·        A high KM means that [S] must be relatively high to achieve 1/2 Vmax, which means that k1 can't keep up with k2 and k3 unless sufficient substrate is present such that [S]*k1 is relatively large.

·        A low KM means that [S] need only be relatively low to achieve 1/2 Vmax, which means that k1 easily keeps up with k2 and k3 such that [S]*k1 is relatively large even when [S] is not.

(n)    Dissociation constant and affinity. When k2 is much greater than k3 (ES dissociates to E and S more rapidly than it forms E and P), KM reduces to k2/k1, the dissociation constant of the enzyme-substrate complex. Under these conditions, a low value for KM indicates a high affinity of E for S and a high KM indicates a low affinity-thus under certain conditions the value of KM provides information about how strongly the enzyme can bind its substrate.

·        Note that k2/k1 does not consider the rate of catalysis, just the relative rates of dissociation of E and S (k2) and association of E and S (k1).

·        If this number is small, then that means that E and S associate more readily than they dissociate.

·        If this number is larger, then that means that E and S associate less readily than they dissociate.

·        Keep in mind that these are arguments, though generally valid, are measurable solely in terms of k2/k1 only when k3, the rate of the catalysis step, a.k.a., the turnover number, is relatively small.

(o)   Turnover number. Another useful characteristic of an enzyme is its turnover number, the kinetic constant k3. Recall that Vmax = k3[ET] (equation 27), so if we know the concentration of enzyme and determine Vmax we can calculate the turnover number-the number of catalytic events carried out per second. The reciprocal of k3 is the time required for a single catalytic event to occur.

·        Note that Vmax = k3[ET] means simply that the reaction at substrate saturation is equal simply to the intrinsic rate of catalysis given ES (i.e., the turnover number) times the concentration of the enzyme ([ET]).

·        Here ET is used rather than ES because it is assumed that at saturation the total amount of enzyme in the system is continually present as ES.

·        Note that the smaller k3 is, the longer it takes for a catalytic step to proceed even at saturating substrate concentration.

·        As you can see from table 8-3, enzymes vary greatly in the time it takes to carry out a single catalytic event. Can you see any correlation between the complexity of the substrate and the maximum turnover number?

(i)                  The more complex the substrate (and/or reaction) the smaller turnover number.

·        Different enzymes have different turnover numbers.

(p)   Make sure you understand (a) the difference between Michaelis constant and dissociation constant, (b) the meaning of affinity and turnover number, and (c) what these various parameters tell you about an enzyme.

·        (a) The Michaelis constant relates the dissociation (and catalysis) constant(s) (k2 and k3) to the association constant (k1).

·        (b) The meaning of affinity is how k1 (the rate of association of E and S) relates to k2 (the reverse reaction whereby E and S are reformed from ES). If affinity is low, then ES tends to fall apart and therefore k2 is relatively high. If affinity is high, then ES tends to form from E and S so k1 is relatively high.

·        (c) The higher the affinity an enzyme has for a substrate combined with its rate of catalysis given ES together describe the kinetics of catalysis.

(q)   Reaction rate (velocity). In most enzyme reactions in cells, the concentration of substrate is considerably less than that required for maximum velocity, so the reaction rate is less than k3, the turnover number. Under these conditions, the reaction rate depends on substrate concentration and on the ratio of k3 to KM (equation 35).

·        In other words, if the rate of catalysis given ES is higher than the rate of dissociation to E and S, then the reaction will proceed with a higher rate than if this is not true.

·        Furthermore, the rate of a reaction given limited substrate is also highly dependent on the rate with which E and S form ES (a function of k1)

(r)     Stryer shows that the rate of the reaction cannot be faster than k1, the rate at which the ES complex is formed. In other words, reaction velocity is limited by the time it takes for a substrate molecule to diffuse into the active site. In solution, the limit on k1 is between 108 and 109 M-1 sec-1.

·        An enzyme can catalyze a reaction only so long as substrate is available in the enzyme's active site, so under substrate-limiting concentrations the rate of a reaction is limited by the rate of formation of ES which is a function of k1 (as well as, of course, a function of S).

(s)    kcat/KM ratio. For more complex reactions, the limit on reaction velocity is determined by the ratio of several rate constants (denoted kcat) to KM.

·        That is, for complicated reactions things are more complex than the simple Michaelis-Menton equation can easily deal with.

(t)     A few enzymes have a kcat/KM ratio between 108 and 109 M-1 sec-1. This means that these enzymes can catalyze reaction of the substrate as fast as they encounter it-Stryer calls this situation "kinetic perfection."

·        That is, the rate of some enzymes is limited only by diffusion rates, the inherent rate at which a given chemical reaction could proceed given otherwise perfect conditions.

(u)    Only a faster diffusion rate could further increase the rate of catalysis. One way to accomplish this is to organize a group of enzymes into a multienzyme complex (which you'll encounter in later chapters), in which the product of one enzyme reaction passes to the active site of the next enzyme without diffusing into the medium.

(ii)                Lineweaver-Bark Plots

(a)    Lineweaver-Burk plot. The KM and Vmax of an enzyme can be determined by measuring reaction velocities at various substrate concentrations. Note that for plots of the type shown in figure 8-15, the exact value of Vmax is difficult to determine. The Lineweaver-Burk double-reciprocal plot is a useful alternative in that it yields a relatively straight line with an intercept equal to 1/Vmax and a slope equal to KM/Vmax.

·        1 / S (x axis) vs. 1 / V (y axis)

(b)  

(c)    See Figure 8-16, p. 194. Make sure you understand this figure and how it relates to the Michaelis-Menten equation.

(d)   The KM values for most enzymes are between 10-1 and 10-7 moles/liter.

(E)            Reversible Inhibitors

(a)    One outcome of the concept of the active site has been the ability to use compounds that resemble natural substrates [particularly transition state analogs] to inhibit specific enzyme reactions. The molecular basis of the action of drugs (and poisons) can often be understood in terms of interactions of small molecules with enzymes, either at active sites or at other sites that influence the rate of catalysis.

(b)   Reversible enzyme inhibition. In this type of inhibition, the inhibitor interferes with the formation of, or the dissociation of, the ES complex. Lineweaver-Burk plots can help you distinguish between two forms of reversible inhibition.

·        Enzymes can be inhibited by specific small molecules or ions. (summary)

·        ...reversible inhibition is characterized by a rapid equilibrium between enzyme and inhibitor. (summary)

·        A competitive inhibitor prevents the substrate from binding to the active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to substrate. (summary)

·        Competitive inhibition can be distinguished from noncompetitive inhibition by determining whether the inhibition can be overcome by raising the substrate concentration. (summary)

·        See Figure 8-19 (p. 197).

·        In competitive inhibition. an inhibitor competes with the substrate for binding at the active site. Study Stryer's example of malonate inhibition of succinate dehydrogenase. The inhibition can be overcome by increasing the concentration of the substrate, succinate. In the double-reciprocal plot (figure 8-20), the 1/V intercept (and thus Vmax) is the same in the presence or absence of inhibitor. The slope increases as the amount of inhibitor increases, showing that the apparent KM increases: more substrate is required to bring the reaction to half Vmax.

·        See Figure 8-20, p. 197. Note that this is saying simply that more substrate must be present to allow the same level of catalysis since the inhibitor is competing for the enzyme's active site.

·        Note that Vmax does not change: If you add enough substrate you will completely eliminate the effect of having competitive inhibitor present.

·        This is shown by the Y intercept not changing in the plot following addition of inhibitor.

·        KM increases because in the presence of the inhibitor the enzyme is either less-readily adhering to the substrate (reduced k1) or the rate of dissociation of ES to E and S is increased (increased k2)-that is, enzyme rejects S when I is already present in the active site.

·        In noncompetitive inhibition, the inhibitor decreases the turnover number.

·        In noncompetitive inhibition, an inhibitor binds to a site on the enzyme that is remote from the active site, and this causes a change in enzyme conformation that impairs the binding of the normal substrate. In the Lineweaver-Burk plot (figure 8-21), the 1/V intercept is increased in the presence of inhibitor, as is the slope of the line. In this case, Vmax is lowered as the concentration of inhibitor increases. KM is not affected.

·        See Figure 8-21, p. 198. Note that this is saying that the absolute rate of catalysis, Vmax, has actually been reduced (Y intercept is higher which, since this is a reciprocal, means that Vmax is lower). This means that no matter how much substrate is added, the rate of catalysis found in the absence of inhibitor cannot be matched.

·        The enzyme that catalyzes the first step in a biosynthetic pathway is usually inhibited by the ultimate product. (p. 183)

·        Note that the following are animated gifs:

·       

(F)            Irreversible Inhibitors

(a)    In irreversible inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. (summary)

(b)   Irreversible inhibition. This occurs when a molecule forms a covalent bond with an essential residue at the active site and thus inactivates the enzyme. Stryer discusses the example of the action of a nerve gas on the enzyme acetylcholinesterase. Covalent modifications of other side chains that alter the overall conformation of an enzyme can also lead to irreversible inhibition.

(G)           Note that quite a bit of stuff that is covered in Chapter 8 of Stryer is discussed as Topic 9 of this outline. Therefore proceed to Topic 9. Note that I still think that we ultimate should read chapter 17 (Metabolism, Basic Concepts and Design) but other than the discussion of coenzymes there may not be all that much from it that we will explicitly include from that chapter in this outline.

(5)               Topic 6 (Mechanisms of Enzyme Action-Serine Proteases)

(a)    This chapter showed how several well-understood hydrolytic enzymes bind substrates and facilitate the formation of transition states. (summay)

(B)            Chymotrypsin-a Serine Protease

(i)                  Nature of Chymotripsin

(a)    The biological role of chymotrypsin is to catalyze the hydrolysis of proteins in the small intestine. (p. 222)

(b)    …chymotrypsin is synthesized as a single-chain inactive precursor called chymotrypsinogen. (p. 222)

(c)    All charged groups are on the surface of the molecule except for three that play a critical role in catalysis. (p. 222)

(ii)                Its Catalytic Action as a Protease

(a)    It is selective for peptide bonds on the carboxyl side of the aromatic side chains of tyrosine, tryptophan, and phenylalanine, and of large hydrophobic residues such as methionine. (p. 222)

(iii)               Its Catalytic Action as an Esterase

(a)    Chymotrypsin, like many proteases, hydrolyzes ester bonds in addition to peptide bonds. Although unimportant physiologically, ester-bond hydrolysis is of interest because of its close relationship to peptide-bond hydrolysis. Inded, much of our knowledge of the catalytic mechanism of chymotrypsin comes from studies of the hydrolysis of simple esters. (p. 222)

(iv)              Kinetics

(a)    …kinetics of hydrolysis of p-nitrophenyl acetate. When large amounts of enzyme are used, there is an initial rapid burst of p-nitrophenol product, followed by its formation at a much slower steady-state rate. (p. 223)

(b)   See Figure 9-29 (p. 223)

(c)    The initial rapid burst of p-nitrophenol production corresponds to the formation of the acetyl-enzyme complex. This step is called acylation. The slower steady-state production of p-nitrophenol corresponds to the hydrolysis of the acetyl-enzyme complex to regenerate the free enzyme. This second step is called deacylation, is much slower than the first, so that it determines the overall rate of hydrolysis of esters by chymotripsin.

(v)                Detection of Important Functional Groups

(a)    Serine 195

·        Highly reactive serine detected via its reaction (and specifically labeling) with DIPF.

·        This is the only serine in chymotripsin that is labeled by DIPF.

·        All serine proteases may be labeled via DIPF.

(b)   Histidine 57

·        The importance of a second residue in catalysis was shown by affinity labeling. The strategy was to react chymotrypsin with a molecule that (1) specifically binds to the active site because it resembles a substrate and that (2) forms a stable covalent bond with a group on the enzyme that is in close proximity. (p. 224)

·        Three lines of evidence indicated that histidine 57 is part of the active site. First, the affinity-labeling reaction was highly stereospecific; the D isomer of TPCK [the reactive substrate analog] was totally ineffective. Second, the reaction was inhibited when a competitive inhibitor of chymotrypsin… was present. Third, the rate of inactivation by TPCK varied with pH in nearly the same way as did the rate of catalysis. (p. 224)

(c)    Aspartate 102

·        The catalytic activity of chymotrypsin depends on the unusual properties of serine 195. A –CH2OH group is ordinarily quite unreactive under physiological conditions. What makes it so reactive in the active site of chymotrypsin?  As was forseen by affinity-labeling studies, histidine 57 is adjacent to serine 195. The carxylate of aspartate 102, buried in the protein, also is next to histidine 57. (p. 225)

(d)   These three residues for a catalytic triad. (p. 225)

(vi)              The ???

(vii)             Mechanisms of Action

(a)    In chymotrypsin and other serine proteases, a highly reactive serine 195 plays a critical role in catalysis. The first stage in the hydrolysis of a peptide substrate is acylation, the formation of a covalent acyl-enzyme intermediate, in which the carboxyl component of the substrate is eterified to the hydroxyl group of serine 195. The nucleophilicity of the serine –OH is markedly enhanced by histidine 57, which accepts a proton from serine as serine attacks the carbonyl carbon atom of the substrate. The resulting positively charged histidine is stabilized by electrostatic interactions with negatively charged aspartate 102. Serine, histidine, and aspartate form a catalytic triad that is at the heart of the catalytic action of all serine proteases. The negative charge on the tetrahedral transition state is also stabilized by hydrogen bonding to two main-chain NH groups in the oxyanion hole. The second stage, deacylation, is in essence a reverse of the first, with H2O substituting for the amine component. Chymotrypsin, trypsin, elastase, several clotting factors, and other vertebrate serine proteases probably arose from a common ancestral gene. (summary)

(b)  

(c)    Chymotrypsin is a serine protease that, as part of their catalytic mechanism, become covalently attached to their substrate.

·        This binding of the enzyme to the substrate is at the carbonyl-bound carbon of the peptide chain. The peptide bond is thus cleaved.

·        This enzyme-bound group is know as an acyl group and the process of its formation is known as acylation.

(d)   The second step of catalysis is the separation of enzyme from substrate. This occurs via hydrolysis (addition of water) between the carbonyl-carbon and the enzyme.

·        This hydrolysis step is known as deacylation.

(e)    The acyl group is attached to an unusually reactive serine residue (p. 224)

·        This residue is serine 195

·        Proteolytic enzymes containing a highly reactive serine… are known as serine proteases. (p. 224)

(f)     See Figure 9-34 (p. 225) for an indication of how Aspartate 102, Histidine 57, and Serine 195 together conspire to make Serine 195 highly reactive.

·        Note how the negative charge of Aspartate 102 stabilizes the positive charge of Histidine 57.

·        Note that Histidine 57 becomes charged upon accepting a proton from Serine 195 during the binding of serine to substrate.

·        Serine 195 thus easily loses its proton (is highly reactive) because Histidine 57 easily accepts this proton.

(g)    Note in Figure 9-35 (p. 225) how aromatic and large hydrophobic side chains are accommodated by a nonpolar pocket associated with the active site.

(h)    Hydrolysis of the peptide bond starts with an attack by the oxygen atom of the hydroxyl group of serine 195 on the carbonyl carbon atom of the susceptible peptide bond. The carbon-oxygen bond of this carbonyl group becomes a single bond, and the oxygen atom acquires a net negative charge. The four atoms now bonded to the carbonyl carbon are arranged as in a tetrahedron. (p. 225)

(i)      The formation of this transient tetrahedral intermediate from a planar amide group is made possible by hyrdrogen bonds between the negatively charged carbonyl oxygen atom (called an oxyanion) and two main-chain NH groups (Figure 9-36). This site called the oxyanion hole. (pp. 225-226)

(j)     The other essential event in the formation of this tetrahedral transition state is the transfer of a proton from serine 195 to histidine 57 (Figure 9-37). This proton transfer is markedly facilitated by the presence of the catalytic triad. Asparatate 102 precisely orients the imidazole ring of histidine 57 and partly neutralizes the charge that develops on it during the transition state. The proton held by the protonated form of histidine 57 is then donated to the nitrogen atom of the susceptible peptide bond, which thus is cleaved. At this stage, the amine component is hydrogen bonded to histidine 57, whereas the acid component of the substrate is esterified to serine 195. The amine component diffuses away, completing the acylation stage of the hydrolytic reaction. (p. 226)

·        See Figure 9-37 (p. 226) and note how the proton is first transferred from Serine 195 to Histidine 57 and then to the other side of the to-be-cleaved peptide bond. Note how it is the donation of the proton that does the actual cleavage of the bond.

(k)   The next stage, deacylation (Figure 9-38), begins when a water molecule takes the place occupied earlier by the amine component of the substrate. In essence, deacylation is the reverse of acylation, with H2O substituting for the amine component. First, histidine 57 draws a proton away from water. The resulting OH ion immediately attacks the carbonyl carbon atom of the acyl group that is attached to serine 195. As in acylation, a transient tetrahedral intermediate is formed. Histidine 57 then donates the proton to the oxygen atom of serine 195, which then releases the acid component of the substrate. This acid component diffuses away and the enzyme is ready for another round of catalysis. (p. 227)

·        Note in Figure 9-38 (p. 226) that histidine 57 grabs an H+ from water. The resulting OH then attacks the carbonyl carbon. The H+ attached to histidine 57 is then donated to serine 195, thereby cleaving the bond between serine 195 and the substrate.

(C)           Trypsin as Serine Protease

(a)    See p. 227.

(D)           Elastase as Serine Protease

(a)    See p. 227.

(E)            Factors Important in the Catalytic Action(???)

(6)               Topic 7 (Coenzymes)

(a)    Why no discussion of ATP????

(b)   This is ATP (dots are electrons):

(B)            Role of Coenzymes

(a)    Many of the central molecules of metabolism in all forms of life are ribonucleotides. Why do activated carriers such as ATP, NADH, FADH2, and coenzyme A contain adenosine phosphate units? A likely explanation is that RNA came before proteins and Dna in evolution. The earliest catalysts most probably were RNA molecules, termed ribozymes… When proteins replaced RNA as the major catalysts to achieve greater versatility, the ribonucleotide coenzymes stayed essentially unchanged because they were already well suited to their metabolic roles. The nicotinamide unit of NADH, for example, can readily transfer electrons irrespective of whether the adenine unit interacts with a base in a ribozyme or with amino acid residues in a protein enzyme. That molecules and motifs of metabolism are common to all forms of life testifies to their common origin and to the retention of functioning modules over billions of years. (p. 459)

(b)   Note: Is it important to know these various structures? I am taking the approach that unless learning a structure gives significant insight into the function of that molecule, then the structure need not be memorized. Although the various coenzymes listed below may serve as recognizable carriers of electrons (etc.) as well as donors and receivers of these things, understanding the subtleties of why these molecules can function as they do is beyond the level of understanding I believe is necessary for a non-major’s, one-quarter biochemistry course. On the other hand, if memorization of these structures is required in Columbus, then I suppose that we have no choice but to memorize them…

(C)           Specific Examples of Coenzymes (and Their Action)

(i)                  NAD+

(a)    Nicotinamide adenine dinucleotide (NAD+) is a major electron acceptor in the oxidation of fuel molecules. (p. 449)

(b)   In the oxidation of a substrate, the nicotinamide ring of NAD+ accepts a hydrogen ion and two electrons, which are equivalent to a hydride ion. (p. 449)

(c)    NAD+ is the electron acceptor in many reactions of the type [see second reaction from top on p. 450, i.e., H-C-OH à C=O]. (p. 450)

(d)   In this dehydration, one hydrogen atom of the substrate is directly transferred to NAD+, whereas the other appears in the solvent as a proton. Both electrons lost by the substrate are transferred to the nicotinamide ring. (p. 450)

(ii)                NADP+

(a)    In most biosyntheses, the precursors are more oxidized than the products. (p. 450)

(b)   The electron donor in most reductive biosyntheses is NADPH, the reduced form of nicotinamide dinucleotide phosphate (NADP+). (p. 451)

(c)    NADPH carries electrons in the same way as NADH. However, NADPH is used almost exclusively for reductive biosynthesis, whereas NADH is used primarily for the generation of ATP. The extra phosphate group on NADPH is a tag that directs this reducing agent to discerning biosynthetic enzymes. (p. 451)

(iii)               FAD

(a)    The other major electron carrier in the oxidation of fuel molecules is flavin adenine dinucleotide. The abbreviation for the oxidized and reduced forms of this carrier are FAD and FADH2, respectively.

(b)   FAD is the electron acceptor in reactions of the type [see third reaction from top on p. 450, i.e., 2HC-CH2 à HC=CH]. (p. 450)

(iv)              FMN

(a)    [FAD] consists of a flavin mononucleotide (FMN) unit and an AMP unit [see Figure 17-8, p. 450]. (p. 450)

(v)                CoA

(a)    Coeynzyme A is a universal carrier of acyl groups. (p. 451)

(b)   See abbreviated structures at the lower-right corner of p. 451 of Acyl CoA and Acetyl CoA.

(7)               Topic 8 (Membranes) [Bio 113 chapter 8]

(A)           Importance of Membranes

(a)    Biological membranes are sheetlike structures, typically 75 Å thick, that are composed of protein and lipid molecules held together by noncovalent interactions. Membranes are highly selective permeability barriers. They create closed compartments, which may be entire cells or organelles within a cell. Pumps and gates in membranes regulate the molecular and ionic compositions of these compartments. Membranes also control the flow of information between cells. For example, many membranes contain receptors for hormones such as insulin. Furthermore, membranes are intimately involved in such energy conversion processes as photosynthesis and oxidative phosphorylation. (summary)

(B)            General Composition of Membranes

(a)    The major classes of membrane lipids are phospholipids, glycolipids, and cholesterol. Phosphoglycerides, a type of phospholipid, conist of a glycerol backbone, two fatty acid chains, and a phosphorylated alcohol. The fatty acid chains usually contain between 14 and 24 carbon atoms; they may be saturated or unsaturated. Phosphatidyl choline, phosphatidyl serine, and phosphatidyl enthanolamine are major phosphoglycerides. (summary)

(C)           Amphipathic Lipids

(a)    A common feature of these membrane lipids is that they are amphipathic molecules. They spontaneously form extensive bimolecular sheets in aqueous solutions because they contain both a hydrophilic and a hydrophobic moiety. (summary)

(b)   These lipid bilayers are highly impermeable to ions and most polar molecules, yet they are quite fluid, which enables them to act as a solvent for membrane proteins. (summary)

(c)   

(D)           Lecithins (is this lectins??)

(E)            Sphingosine Lipids

(a)    Sphingomyelin, a different type of phospholipid, contains a sphingosine backbone instead of glycerol. Glycolipids are sugar-containing lipids derived from sphingosine. (summary)

(F)            Cholesterol

(a)    See bottom of p. 267.

(b)   Please memorize this structure. (New: 5/6/01)

(G)           Micelle Formation

(a)    Now let us consider the arrangement of phospholipids and glycolipids within an aqueous medium… How can this be done inside water? One way is to form a micelle, a globular structure in which polar head groups are surrounded by water and hydrocarbon tails are sequestered inside, facing one another. (p. 269)

(b)   See Figure 11-11. (p. 269)

(c)    [However,] two fatty acyl chains are too bulky to fit into the interior of a micelle. In contrast, salts of fatty acids (such as sodium palmitate, a constituent of soap), which contain only one chain, readily form micelles. (p. 270)

(H)           Lipid Bilayer Formation

(a)      Alternatively, the strongly opposed tastes of the hydrophilic and hydrophobic moieties of membrane lipids can be satisfied by forming a bimolecular sheet, which is also called a lipid bilayer (Figure 11-12). (p. 270)

(b)   The favored structure for most phospholipid and glycolipids in acqueous media is a bimolecular sheet rather than a micelle. (p. 270)

(c)    The formation of lipid bilayers is a self-assembly process. In other words, the structure of a bimolecular sheet is inherent in the structure of the constituent lipid molecules. (p. 270)

(d)   Another imporent feature of lipid bilayers is that they are cooperative structures. They are held together by many reinforcing, noncovalent interactions. Phospholipids and glycolipids cluster together in water to minimize the number of exposed hydrocarbon chains. A pertinent analogy is the huddling together of sheep in the cold to minimize the area of exposed body surface. Clustering is also favored by the van der Waals attractive forces between adjacent hydrocarbon chains. These energetic factors have three significant biological consequences: (p. 271)

(i)                  Lipid bilayers have an inherent tendency to be extensive,

(ii)                Lipid bilayers will tend to close on themselves so that there are no edges with exposed hydrocarbon chains, which results in the formation of a compartment, and

(iii)               Lipid bilayers are self-sealing because a hole in a bilayer is energetically unfavorable.

(e)    Note also liposomes which are small, artificial vesicles consisting of lipid bilayer enclosing a small aqueous volume. (p. 271)

(f)     Lipid bilayers are highly impermeable to ions and most polar molecules. (p. 272)

(g)    …the permeability coefficients of small molecules are correlated with their solubility in a nonpolar solvent relative to their solubility in water. This relationship suggests that a small molecule might traverse a lipid bilayer membrane in the following way: (p. 272)

·        first, it sheds its solvation shell of water;

·        then, it becomes dissolved in the hydrocarbon core of the membrane;

·        finally, it diffuses through this core to the other side of the membrane, where it becomes resolvated by water.

(h)    An ion such as Na+ traverses membranes very slowly because the removal of its coordination shell of water molecules is highly energetically unfavored. (p. 272)

(I)              Role of Cholesterol in Membrane Liquidity

(a)    Cholesterol prevents the crystallization of fatty acyl chains by fitting between them. In fact, high concentrations of cholesterol abolish phase transitions of bilayers. An opposite effect of cholesterol is to sterically block large motions of fatty acyl chains, which makes membranes less fluid. Thus, cholesterol moderates the fluidity of membranes. (p. 280)

(J)              Role of Unsaturated Fatty Acids in Membrane Liquidity

(a)    A phosphoglyceride consist of a glycerol backbone, two fatty acids, and a phosphorylated alcohol. (p. 265)

(b)   See in column below Figure 11-3 for the simplest phosphoglyceride, phosphatidate. (p. 265)

(c)    The major phosphoglycerides are derivatives of phosphatidate. The phosphate group of phosphatidate becomes esterified to the hydroxyl groups of one of several alcohols. (p. 266)

(d)   Of those derivatives, learn the complete structure of phosphatidyl choline (Figure 11-5, p. 266).

(e)    Also learn the structure of glycerol, fatty acids, trans vs. cis double bonds, and dehydration synthesis forming ester linkage between fatty acid and glycerol residues. (New: 5/5/01)

(f)     The degree of fluidity of a membrane partly depends on the chain length of its lipids and the extent to which their constituent fatty acids are unsaturated. (summary)

(g)    The configuration of a double bond in unsaturated fatty acids is nearly always cis. (p. 265)

(K)           Freeze Fractioning

(a)    Three-dimensional images of membrane protein can be reconstructed from electron micrographs of two-dimensional crystalline arrays. (summary)

(b)   Freeze-fracture elelctron microscopy is a valuable technique for ascertaining whether proteins are located in the interior of biological membranes. Cells or membrane fragments are rapidly frozen to the temperature of liquid nitrogen. The frozen membrane is then fractured by the impact of a microtome knife. Cleavage usually occurs along a plane in the middle of the bilayer, between its leaflets (Figure 11-27). Hence, extensive regions within the lipid bilayer are exposed. These exposed regions can then be shadowed with carbon and platinum, which produces a replica of the interior of the bilayer. (p. 276)

(L)            Fluid Mosaic Model

(a)    Distinctive membrane functions such as transport, communication, and energy transduction are mediated by specific proteins. Integeral membrane proteins span the lipid bilayer, whereas peripheral membrane proteins are bound to membrane surfaces by electrostatic and hydrogen bond interactions. Membranes are structurally and functionally asymmetric, as exemplified by the directionality of ion transport systems and the restriction of sugar residues to the external surface of mammalian plasma membranes. Membranes are dynamic structures in which proteins and lipids diffuse rapidly in the plane of the membrane (lateral diffusion), unless restricted by special interactions. In contrast, the rotation of lipids from one face of a membrane to the other (transverse diffusion, or flip-flop) is usually very slow. Proteins do not rotate across bilayers; hence, membrane asymmetry can be preserved. (summary)

(b)  

(M)          Red Blood Cell Membranes

(a)    The erythrocyte membrane, one of the most intensively studied and best-understood membrane systems, contains two abundant transmembrane proteins. Glycophorin A, which bears many covalently attached sugar units, gives red cells a negatively charged carbohydrate coat. The anion channel mediates the exchange of bicarbonate and chloride ions. These integral membrane proteins are linked by protein 4.1 and ankyrin skeleton enables erythrocytes to resist strong shearing forces. (summary)

(b)   Note: need to work on above. See p. 285-on.

(N)           Hydropathy Plots

(a)    Studies of synthetic polypeptides had shown that a helices are more stable in nonpolar media than in water, which competes for hydrogen bonding with main-chain NH an CO groups. One approach to identifying transmembrane helices is to ask whether a postulated helical segment prefers to be in a hydrocarbon milieu or in water. (p. 264)

(b)   The hydrocarbon core of membranes is typically 30 Å wide, which can be traversed by an a helix consisting of 20 residues. We can take the amino acid sequence of a protein and calculate the free-energy change in transferring a hypothetical a helix of residues 1 to 20 from the membrane interior to water. The same calculation can be made for residues 2 to 21, 3 to 22, and so forth until we reach the end of the sequence. The span of 20 residues chosen for this calculation is called the window. (p. 284)

(c)    See Figure 11-44, p. 284. Note that the hydrophobicity plot yields a hydrophobicity index value for each amino acid (plus 20) of the protein.

(d)   See also table 11-2 (p. 284) for an idea of how the different amino acid R-chains compare in terms of their willingness to be transferred from a hydrophobic to hydrophilic solvent.

(8)               Topic 9 (Introduction to Metabolism) [Bio 113 chapter 6]

(A)           Note that quite a bit of stuff that is covered in Chapter 8 of Stryer, which is also covered, in part, in Topic 5 of this outline. Note that I still think that we ultimate should read chapter 17 (Metabolism, Basic Concepts and Design) but there may not be all that much from it that we will explicitly include from that chapter in this outline.

(B)            First Law of Thermodynamics

(i)                  ...the total energy of a system and its surroundings is constant. (p. 185)

(C)           Second Law of Thermodynamics

(i)                  ...a process can occur spontaneously only if the sum of the entropies of the system and its surroundings increases. (p. 185)

(D)           Entropy

(i)                  ...entropy... is a measure of the degree of randomness or disorder of a system. (p. 185)

(E)            Spontaneity of Reactions

(a)    Thermodynamics and biochemical reactions. Only the second of the two laws of thermodynamics can be used to predict whether a biochemical reaction can occur spontaneously. Make sure you understand why this is so. You should also recognize why difficulties in obtaining information about entropy changes limit the use of DS in predicting spontaneity.

(F)            Free Energy

(a)    Free energy is the most valuable thermodynamic function for determining whether a reaction can occur and for understanding the energetics of catalysis. Free energy is a measure of the capacity of a system to do useful work at a constant temperature and pressure. (summary)

(b)   Free energy. Gibbs developed the useful concept of free energy, G, providing a way to predict the direction of reactions from the equilibrium constant and the concentrations of substrates and products.

(c)    If the free-energy change of a reaction, DG, is negative, the reaction is spontaneous. If DG is positive, an input of free energy is required to drive the reaction. If DG is zero, the reaction is at equilibrium, with no net change in the concentration of reactants and products.

(d)   Two other principles are important in considering free energy:

·        The change in free energy as reactants are converted to products is independent of the path taken during the reaction.

·        For any reaction, the value of DG provides no information about the rate at which the reaction proceeds.

(G)           Gibbs Equation

(a)    DG = DH - TDS (equation 3, p. 185)

(b)   Note that the entropy term is subtracted. Hence, an increase in entropy will result in a small DG and consequently a greater likelihood that DG will be negative (and therefore the reaction/process spontaneous).

(H)           Difference Between Kinetic and Thermodynamic Parameters

(a)    ...DG of a reaction is independent of the path (or the molecular mechanisms) of the transformation. (p. 186)

(b)   ...DG provides no information about the rate of a reaction. (p. 186)

(I)              DG

(a)    Standard free-energy change and equilibrium constant. Equation 6 on page 186 shows that DG depends on two factors: the standard free-energy change, reflecting the nature (chemical activity) of the components of the reaction, and the relative concentration of the components.

(b)   It's now possible to go back to equation 6 and determine the value of DG, the actual free-energy change of a reaction, from the sum of the standard free-energy change and the concentration term.

·        Stryer shows how this is done for the triosephosphate isomerase reaction (page 187). As you'll see in chapter 19, in glycolysis the reaction is in the direction of net formation of glyceraldehyde 3-phosphate.

(c)    A reaction can occur spontaneously only if the change in free energy (DG) is negative. The DG of a reaction is independent of path and depends only on the nature of the reactants and their activities (which can sometimes be approximated by their concentrations). The free-energy change of a reaction that occurs when reactants and products are at unit activity is called the standard free-energy change (DG°). Biochemists usually use DG°', the standard free-energy change at pH 7. (summary)

(J)              DG°'

(a)    Take special note of the correction for standard free-energy changes at pH 7, as denoted by DG°', and how the standard free energy can be determined from K'eq, the equilibrium constant (equation 10).

(b)   The free-energy change of a reaction that occurs when reactants and products are at unit activity is called the standard free-energy change (DG°). Biochemists usually use DG°', the standard free-energy change at pH 7. (summary)

(c)    It is important to stress that whether the DG for a given reaction is larger, smaller, or the same as DG°' depends on the concentrations of the reactions [meaning both reactants and products for reversible reactions] (p. 187)

(K)           Relation to Equilibrium Constant

(a)    Every biochemical reaction has a characteristic equilibrium constant, and the enzyme that catalyzes the reaction does not affect that constant. The enzyme can, however, tremendously increase the rate at which the reaction reaches equilibrium.

(b)   4/13/01 new stuff: An important thermodymanic fact is that the overall free-energy charge for a chemically coupled series of reactions is equal to the sum of the free-energy changes of the individuals steps. (p. 444)

(c)    4/13/01 new stuff: a thermodynamically unfavorable reaction can be driven by a thermodynamically favorable reaction that is coupled to it. (p. 444)

·        The reactions are coupled by a shared intermediate.

·        An activated protein conformation can store free energy, which can then be used to drive a thermodynamically unfavorable reaction.

·        Ionic gradients across membranes also serve as versatile means of coupling uphill reactions to downhill reactions.

(L)            Method of Determining DG°'

(a)    These may be covered on page 187...

(M)          Overview of Metabolic Pathways

(a)    All cells extract energy from their environment and convert foodstuffs into cellular components by a highly integrated network of chemical reactions called metabolism. Most of the central molecules of metabolism are the same in all forms of life. Ribonucleotides such as ATP and NADH are especially prominent, reflecting their ancient origins. Moreover, many metabolic patterns are essentially the same in bacteria, plants, and animals. (summary, p. 460, chapter 17)

(b)   The most valuable thermodynamic concept for understanding bioenergetics is free energy. A reaction can occur spontaneously only if the change in free energy (DG) is negative. A thermodynamically unfavorable reaction can be driven by a thermodyanically favorable one. ATP, the universal currency of energy in biological systems, is an energy-rich molecule because it contains two phosphoanhydride bonds. The repulsion between the negatively charged phosphate groups is reduced when ATP is hydrolyzed. Also, ADP and Pi are stabilized by a resonance more than is ATP. The hydrolysis of ATP shifts the equilibrium of a coupled reaction by a factor of about 108. (summary, p. 460, chapter 17)

(c)    The basic strategy of metabolism is to form ATP, NADPH, and building blocks for biosynthesis. ATP is consumed in muscle contraction and other motions of cells, active transport, signal transduction processes, and biosyntheses. NADPH, which carries two electrons at a high potential, provides reducing power in the biosynthesis of cell components from more-oxidized precursors. ATP and NADPH are continuously generated and consumed. (summary, p. 460, chapter 17)

(9)               Topic 10 (Carbohydrates) [Bio 113 chapter 5]

(a)    What structures will we be memorizing?

·        D-Glyceraldehyde

·        L-Glyceraldehyde

·        Dihydroxyacetone

·        D-Glucose (Fisher and Haworth representations, see pp. 464-465; note how rings are formed, p. 467)

·        D-Fructose (ditto)

·        D-Galactose (ditto)

·        Sucrose (figure 18-10) (note how fructose is bonded “backward”)

·        Lactose (ditto)

·        Maltose (ditto)

·        Starch (figure 18-13)

·        Cellulose (figure 18-14)

(b)  

(B)            Carbohydrate Structure

(a)    Carbohydrates are aldehyde or ketone compounds with multiple hydroxyl groups. (p. 462)

(b)   Monosaccharides, the simplest carbohydrates, are aldehydes or ketones that have two or more hydroxyl groups; the empirical formula of many is (CH2O)n. (p. 464)

·        The smallest ones, for which n = 3, are glyceraldehydes and dihydroxyacetone. They are trioses.

·        Glyceraldehyde is also an aldose because it contains an aldehyde group, wherease

·        dihydroxyacetone is a ketose because it contains a keto group.

(c)    See and know the structures of D-Glyceraldehyde, L-Glyceraldehyde, and Dihidroxyacetone on p. 464 and be able to distinguish these.

(d)   Aldoses with 4, 5, 6, and 7 carbon atoms are called tetroses, pentoses, hexoses, and heptoses. (p. 464)

(e)    Two common hexoses are D-glucose (an aldose) and D-fructose (a ketose). (p. 464)

(f)     D-fructose is the most abundant ketohexose. (p. 466)

(C)           D and L Configuration

(a)    For sugars with more than one asymmetric carbon atom, the symbols D and L refer to the absolute configuration of the asymmetric carbon farthest from the aldehyde or keto group. These hexoses [D-glucose and D-fructose] belong to the D series because their configuration at C-5 is the same as that in D-glyceraldehyde. (p. 464)

(b)   Note that when one compares a D-form of a sugar with an L-form of a sugar one observes mirror images. This means that switching, for example, the orientation on the C-5 of glucose or fructose alone (e.g., in the Fisher projection) is not sufficient to have successfully changed D-glucose to L-glucose. (New: 5/5/01)

(c)    Most naturally occurring ring sugars belong to the D series. (summary)

(D)           Parent ???

(E)            Fisher Representation

(a)    The representation of sugars in a linear form is commonly presented as a Fisher representation (e.g., Figures 18-3 and 18-4, pp. 465 and 466).

(b)   See Figure 18-1 (p. 464) for an overview of a Fisher representation.

(c)    Note that in a Fisher representation the substitutions placed to the left and right of the carbon are considered to be projecting, on an angle, out of the page and towards the reader, while the vertical substitutes project into the page, away from the reader. Hence, in the Fisher representation of a sugar what is being presented is the backbone curving away from the reader with the –Hs and –OHs projecting outward from the backbone.

(F)            Hemiacetal

(a)    The prominent forms of glucose and fructose in solution are not open chains. Rather, the open-chain forms of these sugars cyclize into rings. In general, an aldehyde can react with an alcohol to form a hemiacetal. (p. 466)

(b)   A hemiacetal is a carbon bound to an –OH, an R group, an O-R’ group, and an –R’’ group (or –H). See bottom of p. 466. Note how hemiacetals form from the reaction of an alcohol with a carbonyl carbon.

(c)    Sugars, especially pentoses and above, tend to form intramolecular hemiacetals. See reactions and structures on p. 467.

(G)           Ring Nomenclature

(a)    The six-membered pyranose ring, like cyclohexane, cannot be planar because of the tetrahedral geometry of its saturated carbon atoms. Instead, pyranose rings abopt chair and boat conformations (Figure 18-7). (p. 468)

(b)   The substituents on the ring carbon atoms have two orientations: axial and equatorial… Axial substitutions sterically hinder each other if they emerge on the same side of the ring. In contrast, equatorial substituents are less crowded. (p. 469)

(c)    The chair form of b-D-glucopyranose predominates because all axial positions are occupied by hydrogen atoms. (p. 469)

(d)   …pyranose rings usually adopt the chair conformation. (summary)

(e)    Furanoses take on an envelope form with the three carbons and the oxygen planar and the fourth ring carbon found above the plane, giving the rings a shape that resembles and envelope. See Figure 18-8, p. 468.

(f)     An additional asymmetric center is formed at the anomeric carbon atom (C-1 in aldoses and C-2 in ketoses). (p. 480)

(H)           Mirror Images

(a)    A sugar in the D configuration can have a mirror-image sugar that is found in the L configuration.

(b)   The D and L configurations are not interconvertible without extensive chemical modification, but may be visualized in the linear representations found in Figure 18-3 (p. 465), for example, by reversing the –H and –OH around every asymmetric carbon.

(c)    Thus, the D sugar has the –OH on the asymmetric carbon farthest from the carbonyl group projected to the right, whereas the mirror-image L sugar has all –OH groups in the reversed orientation including the farthest –OH, which would be found on the left of the farthest asymmetric carbon.

(I)              Diastereoisomers

(a)    Diasterioisomers are sugars with the same formula and structure but differ in the configuration of groups around asymmetric carbons other than that carbon that is farthest from the carbonyl group.

(b)   See, for example, Figure 18-3 (p. 465) where all of the sugars in a given row are diastereoisomers, though all are D sugars since the hydroxyl group on the carbon farthest from the carbonyl group are found to the right of the carbon chain. 

(J)              Epimers

(a)    Sugars differing in configuration at a single asymmetric center are epimers. (p. 465)

(b)   Thus, D-glucose and D-galactose are epimers at C-4. (p. 465)

(c)    Note how this nomenclature eases structure memorization of at least some sugars, e.g., galactose. Simply memorize glucose and then memorize glucoses various epimers as differing at only a single carbon.

(K)           Anomers

(a)    The C-1 carbon [in aldoses] is called the anomeric carbon atom, and so the a and b forms are anomers. (p. 467)

(b)   Note that the anomeric carbon, in general, is that holding the carbonyl group in the linear-chain form of the sugar.

(L)            Pyranose Ring

(a)    …six-membered ring is called pyranose because of its similarity to pyran. (p. 467)

(b)   See structures on p. 467.

(M)          Furanose Ring

(a)    …five-membered ring is called furanose because of its similarity to furan. (p. 467)

(b)   See structures on p. 467.

(N)           Haworth Representations

(a)    The depictions of glycopyranose and fructopyranose on [page 467] are Haworth projections. In such a projection, the carbon atoms in the ring are not explicitly shown. The approximate plane of the ring is perpendicular to the plane of the paper, with the heavy line on the ring projecting toward the reader. (p. 467)

(b)   See structures on p. 467.

(c)    For D sugars drawn as Haworth projections, the designation a means that it is above the plane of the ring. (p. 468)

(O)           [Note how there is no explicit discussion of O- and N-glycosidic bonds (pp. 469-470), phosphosugars (p. 470), the sugar reducing group (p. 471), a-1,4 and a-1,6 O-glycoside bonds (as found in glycogen and starch, pp. 472-473), b-1,4 O-glycosidic bonds (as found in cellulose, pp. 473-474), etc. Oh well… See Topic 14 for a discussion of glycogen.]

(10)           Topic 11 (Glycolysis) [Bio 113 chapter 9]

(A)           Reaction Overview

(a)    See Table 19-2, p. 491.

(b)   Note order of enzymes:

·        Hexokinase

·        Phosphoglucose Isomerase

·        Phosphofructo Kinase

·        Aldolase

·        Triose Phosphate Isomerase

·        Glyceraldehyde 3-Phosphate Dehydrogenase

·        Phosphoglycerate Kinase

·        Phosphoglycerate Mutase

·        Enolase

·        Pyruvate Kinase

(c)    Note order of participates (substrates/products):

·        Glucose

·        Glucose 6-phosphate

·        Fructose 6-phosphate

·        Fructose 1,6-biphosphate

·        Dihydroxyacetone phosphate

·        Glyceraldehyde 3-phosphate (G3P)

·        1,3-biphosphoglycerate + NADH + H+ - Pi

·        3-phosphoglycerate

·        2-phosphoglycerate

·        Phosphoenolpyruvate + H2O

·        Pyruvate

(d)   Make sure you know the structures of the above.

(e)    Note that the easiest way to memorize the above (in my humble opinion and if you are a visual learner) is to memorize the reactions, then enzyme names, then the structures, then the structure names. This way you can walk through the chemistry of the reaction and then, more or less, derive the names from the chemistry.

(B)            Yeast and Muscle

(a)    Again, make sure you know names, structures, and enzymes

(b)   Yeast Alcoholic (ethanol) fermentation (p. 497)

·        Pyruvate – (Pyruvate Decarboxylase) à Acetaldehyde + CO2

·        Acetaldehyde + NADH + H+ – (Alcohol Dehydrogenase) à Ethanol + NAD+

·        Overall: Glucose + 2 Pi + 2ADP + 2H+ à 2 Ethanol + 2CO2 + 2ATP + 2H2O

(c)    Muscle lactic acid fermentation

·        Pyruvate + NADH – (Lactate Dehydrogenase) à L-Lactate + NAD+

·        Overall: Glucose + 2 Pi + 2ADP + 2H+ à 2 Lactate + 2ATP + 2H2O

(C)           Mechanism for Glyceraldehyde-3-Phosphate Dehydrogenase

(a)    This is the most complex reaction in glycolysis

(b)   The overall reaction is:

·        G3P + NAD+ – (G3P dehydrogenase) à 1,3-Biophosphoglycerate + NADH + H+.

(c)    In overview I like to imagine the reaction occurring as the removal of a hydrogen atom from the 1 carbon of G3P along with a hydrogen from phosphoric acid (Pi) resulting in the formation of an ester linkage between the 1 carbon and phosphate.

(d)   Note that in this reaction the removed hydrogens are transferred to NAD+ to form NADH + H+.

(e)    The actual reaction is a bit more complicated (see Figure 19-18, p. 502):

·        The 1 carbon of G3P reacts with the sulfhydril group of a cysteine residue.

·        This nuclephilic attack results in the 1 carbon being bound to four members: (i) the 2 carbon of G3P (as prior to the attack), (ii) an H (as prior to the attach), (iii) a hydroxyl group (instead of the carbonyl group present prior to the attack, with the H on the oxygen essentially formerly found on the –SH of the cysteine), and (iv) the sulfur atom of the cysteine: C-, H-, HO-, and S- where formerly it was C-, H-, O=.

·        The two H’s are then transferred as one hydrogen atom and one electron to an NAD+ coenzyme “tightly bound to the enzyme” (p. 502).

·        These two H’s are then transferred as one hydrogen atom and one electron to an NAD+ not so tightly bound to the enzyme.

·        Note that the 1 carbon is now bound to C-, O=, and S~, where the S~C bond replaced the S-C bond present prior to the removal of the hydrogens.

·        The S~C bond is energy rich and that energy is used to drive its replacement with a less-energetic ester linkage between the 1 carbon and a phosphate.

·        The 1 carbon is thus phosphorylated, forming 1,3-Biophosphoglycerate.

(D)           Substrate Level Phosphorylation

(a)    Substrate level phosphorylation is the transfer of a phosphate group from a substrate (e.g., 1,3-biophosphoglycerate or phosphoenolpyruvate) to ADP to form ATP.

(b)   Note that substrate level phosphorylation is different particularly from oxidative phosphorylation.

(c)    Note that there is one substrate level phosphorylation step in the Kreb’s cycle along with the two in glycolysis.

(d)   Note that substrate level phosphorylation demands that the phosphate transfer be energetically favorable meaning that the loss of the phosphate by the substrate is more favorable than the loss of phosphate by ATP to form ADP.

(E)            Irreversible Steps

(a)    In metabolic pathways, enzymes catalyzing essentially irreversible reactions are potential sites of [metabolic] control. (p. 493)

(b)   Three kinase-catalyzed steps in glycolysis are sufficiently energetically favorable (overall) that they are essentially irreversible under physiological conditions (i.e., without the input of energy from an additional source).

(c)    These are the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.

(d)   Note that the last one (pyruvate kinase), involving the formation of ATP, is driven by the highly energetically favorable conversion of the enolpyruvate to pyruvate (once the phosphate is gone) (p. 504)

(e)    In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are virtually irreversible; hence, they would be expected to have regulatory as well as catalytic roles. In fact, each of them serves as a control site. Their activities are regulated by the reversible binding of allosteric effectors or by covalent modificiation. Also, the amounts of these key enzymes are varied by transcriptional control to meet changing metabolic needs. Reversible allosteric control, regulation by phosphorylation, and transcriptional control typically occur in times of milliseconds, seconds, and hours, respectively. (p. 493)

(F)            Committed Step

(a)    Phosphofructokinase is the most important control element in the glycolytic pathway of animals. (p. 493)

(b)   Why is phosphorfructokinase rather than hexokinase the pacemaker of glycolysis? The reason becomes evident on noting that glucose 6-phosphate is not solely a glycolytic intermediate. Glucose 6-phosphate can also be converted into glycogen or it can be oxidized by the pentose phosphate pathway to form NADPH. The first irreversible reaction unique to the glycolytic pathway, called the committed step, is the phosphorylation of fructose 6-phosphate to fructose 1,6-biphosphate. Thus, it is highly appropriate for phosphofructokinase to be the primary control site in glycolysis. In general, the enzyme catalyzing the committed step in a metabolic sequence is the most important control element in the pathway. (pp. 495-496)

(11)           Topic 12 (Citric Acid Cycle) [Bio 113 chapter 9]

(A)           Aerobic Metabolism

(a)    The citric acid cycle is the final common pathway for the oxidation of fuel molecules—amino acids, fatty acids, and carbohydrates. Most fuel molecules enter the cycle as acetyl CoA. The cycle also provides intermediates for biosynthesis. In eukaryotes, the reactions of the citric acid cycle occur inside mitochondria, in contrast with those of glycolysis which occur in the cytosol. (p. 509)

(b)   The overall pattern of the citric acid cycle is shown in Figure 20-2. A four-carbon compound (oxaloacetate) condenses with a two-carbon acetyl unit to yield a six-carbon tricarboxylic acid (citrate). An isomer of citrate is then oxidatively decarboxylated to yield a four-carbon compound (succinate). Oxaloacetate is then regenerated from succinate. Two carbon atoms enter the cycle as an acetyl unit and two carbon atoms leave the cycle in the form of two molecules of CO2. An acetyl group is more reduced than CO2, and so oxidation-reduction reactions must take place in the citric acid cycle. In fact, there are four such reactions. Three hydride ions (hence, six electrons) are transferred to three NAD+ molecules, whereas one pair of hydrogen atoms (hence, two electrons) is transferred to a flavin adenine dinucleotide (FAD) molecule. These electron carriers yield nine molecules of adenosine triphosphate (ATP) when they are oxidized by O2 in the electron transport chain. In addition, one high-energy phosphate bond is formed in each round of the citric acid cycle itself. (p. 510)

(B)            Membrane Transport of Pyruvate

(a)    Note that this topic is covered in Chapter 21 and we will delay our discussion of it until then.

(C)           Formation of Acetyl CoA from Pyruvate (Pyruvate Oxidation)

(a)    The oxidative carboxylation of pyruvate to form acetyl CoA, which occurs in the mitochondrial matrix, is the link between glycolysis and the citric acid cycle. (p. 509)

(b)   Pyruvate + CoA + NAD+  (Pyruvate Dehydrogenase Complex) à Acetyl CoA + CO2 + NADH

(D)           Steps of TCA Cycle

(a)    See Table 20-1, p. 515.

(b)   Note order of enzymes:

·        Citrate Synthetase

·        Aconitase

·        Isocitrate dehydrogenase

·        a-Ketoglutarate dehydrogenase complex

·        Succinyl CoA Synthetase

·        Succinate dehydrogenase

·        Fumarase

·        Malate Dehydrogenase

(c)    Note order of participants (substrates/products)

·        Acetyl CoA

·        Oxaloacetate

·        Citrate + CoA + H+ (where does the H+ come from?)

·        cis-Aconiate + H2O

·        Isocitrate – H2O

·        a-Ketoglutarate – NAD+ + NADH + CO2

·        Succinyl CoA – NAD+ – CoA + NADH + CO2

·        Succinate – FAD – Pi – GDP + FADH2 + GTP

·        Fumarate

·        Malate – H2O

·        Oxaloacetate – NAD+ + NADH + H+ + CO2

(d)   Make sure you know the structures of the above.

·        Note that especially helpful for learning structures may be Figure 20-5 (p. 513).

(e)    Note that, as with Glycolysis, the easiest way to memorize the above (in my humble opinion and if you are a visual learner) is to memorize the reactions, then enzyme names, then the structures, then the structure names. This way you can walk through the chemistry of the reaction and the, more or less, derive the names from the chemistry.

(f)     Dicarboxylic acid mnemonic devise (which is helpful for learning/remembering the Kreb’s cycle):

 

oh

oxalic acid

HOOC-COOH

my

malonic acid

HOOC-CH2-COOH

such

succinic acid

HOOC-(CH2)2-COOH

good

glutaric acid

HOOC-(CH2)3-COOH

apple

adipic acid

HOOC-(CH2)4-COOH

pie

pimelic acid

HOOC-(CH2)5-COOH

 

(E)            Mitochondrial Structure

(a)    See Figure 20-1 (p. 509) for a quicky overview of the structure of a mitochondrion. Note:

·        The matrix.

·        The Inner mitochondrial membrane.

·        The outer mitochondrial membrane.

(b)   Note that the citric acid cycle occurs within the matrix of the mitochondrion, with some enzymes found within inner membrane of the mitochondrion (though the reactions occur within the matrix).

(c)    Electron transport and proton pumping (next topic) occurs within and across the inner membrane while ATP synthetase, the reverse-acting proton pump that is responsible for oxidative phosphorylation, is also located in the inner membrane.

(F)            Generation of NADH

(a)    Generation of NADH occurs three times per TCA cycle/per acetyl CoA entering the cycle.

(b)   Be sure to be aware of where the removed electrons come from.

(c)    The enzymes involved are:

·        Isocitrate dehydrogenase

·        a-Ketoglutarate dehydrogenase complex

·        Malate dehydrogenase

(d)   Note that the electrons removed in the Isocitrate dehydrogenase step are removed from the lone hydroxyl group of isocitrate (to form oxalosuccinate).

·        The removal of a carboxyl group to form CO2 is also associated with this reaction.

(e)    Note that the reactions catalyzed by a-Ketoglutarate dehydrogenase complex are very similar to those involved in the formation of Acetyl CoA from pyruvate.

·        The removal of a carboxyl group to form CO2 is also associated with this reaction.

(f)     Note that the electrons removed in the Malate dehydrogenase  step are removed from the lone hydroxyl group of fumarate.

(G)           Generation of FADH2

(a)    Generation of FADH2 occurs during the Succinate dehydrogenase step.

(b)   Note, as with the NADH formation steps, where the electrons are removed from in the formation of fumarate (which is fairly obvious once one knows fumarate’s structure).

(c)    FAD is the hydrogen acceptor in this reaction because the free-energy change is insufficient to reduce NAD+. (p. 512)

(d)   …succinate dehydrogenase is directly linked to the electron transport chain. The FADH2 produced by the oxidation of succinate does note dissociate from the enzyme, in contrast with NADH produced in other oxidation-reduction reactions. (p. 512)

(H)           Generation of GTP

(a)    The succinyl thioester of CoA has an energy-rich bond… The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of guanosine diphosphate (GDP). (p. 510)

(b)   Note that despite the phosphorylation of GDP in this reaction, the reaction is readily reversed (since the free energy of the substrates and products is very similar.

(c)    As a consequence of the ready reversibility of the reaction, the enzyme catalyzing the reaction is actually named for the reverse reaction, the formation of succinyl CoA, i.e., Succinyl CoA syntase.

(12)           Topic 13 (Oxidative Phosphorylation) [Bio 113 chapter 9]

(A)           These are adapted from Jennifer’s notes:

(i)                  Oxidative Phosphorylation = Name of the process whereby we oxidize NADH and FADH2, transfer those electrons to O2, and generate ATP.

(ii)                Mitochondria anatomy (going from in to out):

(a)    Matrix

·        Krebs cycle occurs here.

·        This is where the hydrogen ions are pumped from.

(b)   Inner membrane

·        Very selective barrier.

·        Permeable to pyruvate but not much else.

·        Electron transport chain found there.

·        ATP synthetase found here

(c)    Lumen = intermembrane space

·        This is where the hydrogen ions are pumped to.

(d)   Outer membrane

·        relatively permeable

(iii)               NADH + H+ + ½O2 ßà NAD+ + H2O

(a)    produces ~2.5 ATP from 10 pumped protons.

(iv)              FADH2 + ½O2 ßà NAD+ + H2O

(a)    produces ~1.5 ATP from 6 pumped protons.

(v)                This is the electron transport chain, inner membrane, ATP synthetase:

(a)    Note oxidation of NADH.

(b)   Note flow of electrons.

(c)    Note pumping of protons from matrix to intermembrane space.

·        This pumping raises the pH within the matrix and lowers the pH in the intermembrane space.

(d)   Note chemical reduction of O2 in matrix.

(e)    Note flow of electrons.

(f)     Note movement of protons back through inner membrane via ATP synthetase.

(g)    Note phosphorylation of ADP to make ATP.

(vi)              Electron Transport Chain, Electron Transport System (ETS):

(a)    NADH

(b)   NADH-Q Reductase

·        Pumps 4 protons

(c)    Ubiquinone

·        Lipid soluble

·        Carries electrons from NADH-Q Reductase to Cytochrome Reductase

·       

·        Top and bottom carbonyl groups are converted to hydroxyl groups upon addition of 2H.

·        Q ßà QH2.

·        Note long-chain hydrocarbon tail.

·        In humans n = 10.

·        FADH2 + Q ßà FAD + QH2, which is where FADH2 adds its electrons to ETS and why FADH2 produces fewer ATPs (4 fewer protons pumped).

(d)   Cytochrome reductase

·        Pumps 2 protons.

(e)    Cytochrome C

·        Small, water-soluble protein.

·        Holds single heme.

·        Carries single electron from Cytochrome Reductase to Cytochrome Oxidase.

·        During electron carriage Fe3+ is converted to Fe2+.

(f)     Cytochrome oxidase

·        Pumps 4 protons.

·        O2 reduction occurs here.

·        It is hazardous if the intermediate, O2- = superoxide, escapes from enzyme.

·        Superoxide dismutase prevents this from happening by catalyzing:

(i)                  2 O2- + 2H+ — (superoxide dismutase) à H2O2 + O2 – catalase à H2O + ½O2

(vii)             ATP Synthetase

(a)    ATP Synthetase catalyzes the hydrolysis of ATP.

(b)   ATP Synthetase is a reverse-running proton pump.

(c)    In the absence of a proton gradient across the inner membrane, ATP Synthetase will attempt to create such a gradient at the expense of ATP hydrolysis.

(d)   ATP Synthetase is thought to have three ADP-binding sites, at least one of which is always occupied.

(e)    ATP Synthetase is thought to spin, change conformation, and phosphorylate ADP as driven by protons crossing the inner membrane via ATP Synthetase (which spans membrane).

(f)     Incoming protons are only energetic enough to spin ATP Synthetase (“spinning” domain) if proton-motive force is strong enough.

(g)    A physically damaged inner membrane (porous) prevents ATP synthesis by eliminating the proton-motive force.

(viii)           ATP transport out of mitochondria.

(a)    ATP is made within the matrix of the mitochondria.

(b)   ATP is not transported across inner membrane but instead the energy associated with it’s last phosphate is.

(c)    The reaction involves ATP on one side of inner membrane (matrix) and ADP + Pi on other side (intermembrane space).

(d)   Products of reaction are ADP + Pi in matrix and ATP in intermembrane space.

(ix)              NADH transport into mitochondria.

(a)    NADH is generated during glycolysis.

(b)   NADH also is not transported across inner membrane but instead its electrons are exchanged with FAD.

(c)    NADH in intermembrane space transfers its electrons to FAD found in the matrix to product NAD in the intermembrane space and FADH2 in the matrix.

(d)   Keep in mind that there is no transportation barrier to NADH across the outer membrane.

(x)                Poisoning ATP synthesis

(a)    In addition to damaging the inner membrane, various substances that bind to the metal ions employed in the ETS inhibit oxidative phosphorylation.

(b)   Cyanide (CN-) is perhaps the most famous of these poisons.

(c)    Proton shuttles also exist that can short circuit the proton-motive force by shuttling protons across (dissolving in while carrying a proton) an otherwise intact inner membrane.

(B)            Overview

(a)    In oxidative phosphorylation, the synthesis of ATP is coupled to the flow of electrons from NADH or FADH2 to O2 by a proton gradient across the inner mitochondrial membrane. Electron flow through three asymmetrically oriented transmembrane complexes results in the pumping of protons out of the mitochondrial matrix and the generation of a membrane potential. ATP is synthesized when protons flow back to the matrix through a channel in an ATP-synthesizing complex, called ATP synthetase. Oxidative phosphorylation exemplifies a fundamental theme of bioenergetics: the transmission of free energy by proton gradients. (summary)

(C)           Membrane Transport of Pyruvate

(a)    Even here (p. 551 of text) pyruvate transport isn’t covered well; isn’t it covered well anywhere in the text? Where?

(D)           Reducing Potential as an Electrical Concept

(a)    The reduction potential of a compound can be measured by employing an apparatus such as that pictured in Figure 21-4 (p. 531).

(b)   Note that the idea is that a reduced substance can reduce an oxidized substance by sending its electrons through a wire.

(c)    This reduction will occur, however, only to the extent that the redox potential of the reduced substance is greater than the redox potential of the to-be-reduced substance.

(E)            Standard Hydrogen Electrode and Measurement of E0' and DG°'

(a)    Eo' denotes the standard reduction potential (in volts) associated with a substance (as measured at 1 M concentrations of reduced and oxidized forms relative to pH 7 H+ concentration).

(b)   See Table 21-1 (p. 532) for a list or reduction potentials.

·        Note that the values of E0' represent the ability of the oxidant (left-most column) to take electrons from H2 gas (under standard conditions of 1 M water solution and 1 atmosphere H2).

·        The higher the number (more positive) the better able to the oxidant is to take electrons from H2.

·        Therefore, the stronger an oxidizer the substance is.

·        Note that the converse is true for the Reductant; the higher the number in the far-right column, the less able the substance is to give away electrons in the reverse reaction.

(c)    Note that in redox reactions if the E0' of the reactions add up to >0 (i.e., DE0' > 0) then the reduction will occur spontaneously.

(F)            Conversion of DEo' to DG°'

(a)    The DE0' and DG°' of a reaction can be interconverted.

(b)   DG°' = -nFDEo'

(c)    Note that n refers to the number of electrons transferred within a reaction.

(d)   See Table 21-1 (p. 532) for a listing of n values for various electron donors (and acceptors).

(e)    F is a constant called a faraday (23.06 kcal V-1 mol-1).

(f)     Note in particular that only a positive DEo' will give rise to a spontaneous reaction and that the amount of free energy lost or gained in a reaction is also dependent on the number of electrons transferred.

(G)           Overall Energy Possible for Electron Transport from Reduced Coenzymes to O2

·        See p. 533 for the book’s presentation of this discussion.

(b)   ½ O2 + 2 H+ = 2 e- à H2O

·        Eo' = +0.82 V

(c)    NAD+ + H+ + 2 e- à NADH

·        Eo' = -0.32 V

(d)   ½ O2 + NADH + H+ ßà H2O + NAD+

·        DEo' = +1.14 V (meaning that it occurs spontaneously)

(e)    Conversion to DG°' looks like this:

·        DG°' = -2 ´ 23.06 ´ 1.14 = -52.6 kcal/mol

(f)     Compare this DG°' with that associated with the hydrolysis of ATP (ATP + H2O à ADP + Pi):

·        DG°' = -7.3 kcal/mol

·        Thus, there is more than enough free energy associated with the transfer of two electrons from NADH to O2 to generate the 2.5 ATP associated with NADH in oxidative phosphorylation.

(H)           Electron Transport System Steps

(a)    The electron transport steps are

·        NADH oxidation.

·        NADH-Q reductase reduction and proton pumping (4).

·        Ubiquinone (Q) reduction (electron transport within membrane).

·        Cytochrome oxidase reduction and proton pumping (2).

·        Cytocrhome c reduction (electron transport external to inner membrane).

·        Cytochrome reductase reduction and proton pumping (4).

·        O2 reduction.

(I)              Nature and Structure of Electron Transport System

(i)                  Components

(a)    The electron carriers in the respiratory assembly of the inner mitochondrial membrane are flavins, iron-sulfur complexes, quinines, heme groups of cytochromes, and copper ions. Electrons from NADH are transferred to the FMN prosthetic group of NADH-Q reductase, the first of three complexes. This reductase also contains Fe-S centers. The electrons emerge in QH2, the reduced form of ubiquinone (Q). This highly mobile hydrophobic carrier transfers its electrons to cytochrome reductase, a complex that contains cytochromes b and c1 and an Fe-S center. This second complex reduces cytochrome c, a water-soluble peripheral membrane protein. Cytochrome c, like Q, is a mobile carrier of electrons, which it then transfers to cytochrome oxidase. This third complex contains cytochromes a and a3 and two copper ions. A heme iron and a copper ion in this oxidase transfer electrons to O2, the ultimate acceptor, to form H2O. (summary)

(ii)                Placement in Mitochondrial Inner Membrane

(a)    Oxidative phosphorylation takes place in the inner mitochondrial membrane, in contrast with most the reactions of the citric acid cycle and fatty acid oxidation, which occur in the matrix. (p. 530)

(iii)               Complex I

(a)    NADH-Q reductase

(iv)              Complex II

(a)    Succinate-Q reductase

(b)   Note that this is the Kreb’s cycle enzyme that generates the FADH2.

(c)    This enzyme enzyme is found in the mitochondrial inner membrane and directly hands off the FADH2 enzymes to the ETS.

(v)                Complex III

(a)    Cytochrome reductase

(vi)              Complex IV

(a)    Cytochrome oxidase

(vii)             Generation of 2.5 ATP's per Electron Pair

(a)    The flow of two electrons through NADH-Q reductase, cytochrome reductase, and cytochrome oxidase generates a gradient sufficient to synthesize 1, 0.5, and 1 molecules of ATP, respectively. Hence, 2.5 ATP are formed per NADH oxidized in the mitochondrial matrix, whereas only 1.5 ATP are made per FADH2 oxidized because its electrons enter the chain at QH2, after the first proton pumping site. (summary)

(J)              Shuttle Systems for Cytosolic NADH

(a)    …only 1.5 ATP are generated by the oxidation of cytosilic NADH because its electrons enter the respiratory chain at the second site owing to the action of the glycerol phosphate shuttle. (summary)

(K)           Glycerol Phosphate

(a)    Glycerol 3-phosphate serves as a shuttle for electrons across the mitochondrial inner membrane.

(b)   Dihydroxyacetone phosphate can receive two electrons and two hydrogens (from NADH + H+), converting it to glycerol 3-phosphate (which is simply dihydroxyacetone phosphate with its carbonyl group converted to a hydroxyl group, or glycerol with one of its end hydroxyl groups phosphorylated).

(c)    Glycerol 3-phosphate can diffuse across the mitochondrial inner membrane.

(d)   Once in the matrix it can reduce FAD to form FADH2.

(e)    See Figure 21-30 (p. 549).

(f)     The reduced flavin inside the mitochondria transfers its electrons to the electron carrier Q, which then enters the respiratory chain as QH2. (p. 549)

(L)            Malate Aspartate

(a)    This is an alternative carrier of NADH’s electrons into mitochondria that is found in heart and liver tissue. (see p. 549)

(M)          DNP as an Uncoupler

(a)    Electron transport is normally tightly coupled to phosphorylation. NADH and FADH2 are oxidized only if ADP is simultaneously phosphorylated to ATP. This coupling, called respiratory control, can be disrupted by uncouplers such as DNP, which dissipate the proton gradient by carrying protons across the inner membrane. (summary)

(N)           Mitchel's Proton Gradient as Energy Source

(a)    The flow of electrons through each of these complexes leads to the pumping of protons from the matrix side to the cytosolic side of the inner mitochondrial membrane. A proton-motive force consisting of a pH gradient (cytosolic side acidic) and a membrane potential (cytosolic side positive) is generated. The flow of protons back to the matrix side of a hydrophobic F0 unit that conducts protons through the membrane and a hydrophilic F1 unit that catalyzes ATP synthesis sequentially at three sites. Protons flowing through ATP synthetase relesase tightly bound ATP. (summary)

(O)           Charge Across Membrane as Energy Source

(a)    …the primary energy-conserving event induced by electron transport  is the generation of a proton-motive force across the inner mitochondrial membrane. (p. 544)

(b)   Electron transport generates a proton gradient across the inner mitochondrial membrane. The pH outside is 1.4 units lower than inside, and the membrane potential is 0.14 V, the outside being positive. The proton-motive force Dp (in volts) consists of a membrane-potential contribution (Em) and a chemical gradient contribution (DpH). (p. 545)

(c)    See Figure 21-37 (p. 555).

(P)            Structures of F1 and Fo Particles

(a)    ATP synthetase spans the mitochondrial inner membrane

(b)   It has two parts,

·        The F0 unit which spans the membrane and is involved in the channeling of protons into the matrix (note, in figure below, the alpha helices spanning the membrane).

·        The F1 unit which is found in the matrix, attached to the F0 unit and which contains the ADP/ATP-binding domains (see http://www.cse.ucsc.edu/~hongwang/ATP_synthetase.html, or below, for cartoons of this rotation as well as an idea of how the movement of protons can be converted into the mechanical work that drives the rotation of the F1 subunit).

(c)    The F1 unit is thought to rotate, driving by incoming protons, in the course of catalysis.

(d)    = ATP synthetase

(e)     = model of F0 unit (converts proton-motive force to mechanical work)

(f)      = model of F1 unit (converts mechanical work to ATP synthesis)

(Q)           Boyer's Concept of ATP Formation

(a)    ATP does not leave the catalytic site unless protons flow through the enzyme. Paul Boyer showed that the role of the proton gradient is not to form ATP but to release it from the synthetase. He also found that the nucleotide-binding sites of this enzyme interact with each other. The binding of ADP and Pi to one site promotes the release of ATP from another. In other words, ATP synthetase exhibits catalytic cooperativity. (p. 547)

(R)            Three Conformational Changes Drive by H+ Gradient

(a)    See Figure 21-28 (p. 548).

(b)   The three catalytic sites cycle through three conformational states: O (open), L (loose binding), and T (tight binding). (p. 548)

·        L binds ADP + Pi

·        T catalyzes ATP synthesis

·        O releases ADP

(c)    The translocation of three H+ through the synthetase leads to the formation of one ATP. (p. 548?)

(13)           Topic 14 (Additional Carbohydrate Metabolism)

(A)          

(B)            Gluconeogenesis

(a)    Gluconeogenesis is the synthesis of glucose from noncarbohydrate sources, such as lactate, amino acids, and glycerol. (summary)

(b)   Several of the reactions that convert pyruvate into glucose are common to glycolysis. (summary)

(C)           Reversal of Irreversible Step

(a)    Gluconeogenesis… requires four new reactions to bypass the essential irreversibility of the corresponding reactions in glycolysis.

·        Pyruvate is carboxylated in mitochondria to oxaloacetate, which in turn is decarboxylated and phosphorylated in the cytosol to phosphoenolpyruvate.

·        Two high-energy phosphate bonds are consumed in these reactions, which are catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase. (summary)

(b)   The other distinctive reactions of glyconeogenesis are the hydrolysis of fructose 1,6-biphosphate and glucose 6-phosphate, which are catalyzed by specific phosphatases. (summary)

(c)    Here is an overview of the novel reactions:

·        Pyruvate + CO2 + ATP + H2O – (pyruvate carboxylase in mitochondria) à oxaloacetate + ADP + Pi + 2H+

·        (important to remember is pyruvate plus CO2 + ATP gives you oxaloacetate in the liver)

·        Oxaloacetate + GTP – (phosphoenolpyruvate carboxykinase) à phophoenolpyruvate + CO2

·        (important to remember is that oxaloacetate is phosphorylated at the expense of a GTP and stripped of a CO2 to generate phosphoenolpyruvate)

·        fructose 1,6-biphosphase – (fructose 1,6-biphosphatase) à fructose 6-phosphate

·        glucose 6-phosphate – (glucose 6-phosphatase) à glucose

(d)   Thus, the order of the substrates in gluconeogenesis is:

·        Pyrvate

·        Oxaloacetate

·        Phosphoenolpyruvate

·        2-phosphoglycerate

·        3-phosphoglycerate

·        1,3-biphosphoglycerate

·        Glyceraldehyde 3-phosphate

·        Dihydroxyacetone phosphate

·        Fructose 1,6-biphosphate

·        Fructose 6-phosphate

·        Glucose 6-phosphate

·        Glucose

(e)    For comparison, the order of substrates in glycolysis is:

·        Glucose

·        Glucose 6-phosphate

·        Fructose 6-phosphate

·        Fructose 1,6-biphosphate

·        Dihydroxyacetone phosphate

·        Glyceraldehyde 3-phosphate

·        1,3-biphosphoglycerate

·        3-phosphoglycerate

·        2-phosphoglycerate

·        Phosphoenolpyruvate

·        Pyrvate

(f)     The major raw materials for gluconeogenesis by the liver are lactate and alanine produced from pyruvate by active skeletal muscle. The formation of lactate during intense muscular activity buys time and shifts part of the metabolic burden from muscle to liver. (summary)

(g)    Gluconeogenesis and glycolysis are reciprocally regulated so that one pathway is relatively inactive while the other is highly active. (summary)

(D)           Glycogen

(a)    Glycogen, a readily mobilized fuel store, is a branched polymer of glucose residues. (summary)

·        Most of the glucose units in glycogen are linked by a-1,4 glycocidic bonds. (summary)

·        At about every tenth residue, a branch is created by an a-1,6 glycocidic bond. (summary)

·        Glycogen is present in large amounts in muscle and liver, where it is stored in the cytoplasm in the form of hydrated granules. (summary)

(b)  

(E)            Degradation and Synthesis of Glycogen

(a)    The primary degradative enzyme is called phosphorylase.

(b)   The other enzymes involved are oligosaccharide transferase and an a-1,6-glucosidase.

(c)    Most of the glycogen molecule is degraded to glucose 1-phosphate by the action of phosphorylase. The glycosidic linkage between C-1 of a terminal residue and C-4 of the adjacent one is split by orthophosphate to give glucose 1-phosphate, which can be reversibly converted into glucose 6-phosphate. (summary)

(d)  

(e)    Note that the above figure is, in fact, incorrect since it shows glucose 4-phosphate labeled as glucose 6-phosphate (glucose 6-phosphate is the correct end product—the structure is what is wrong).

(f)     Branch points are degraded by the concerted action of an oligosaccharide transferase and an a-1,6-glucosidase. (summary)

·        See Figure 23-3 (p. 584).

·        The latter enzyme (also known as debranching enzyme) catalyzes the hydrolysis of a-1,6 linkages, yielding free glucose. (summary)

·        The former enzyme transfers three glucose residues from a highly shortened branch (four residues remaining) to a greatly shortened non-reducing (non anomeric carbon) end of glycogen; there the residues may be further removed by phosphorylase.

·        Note that a-1,6-glucosidase yields free glucose, which is in contrast with phosphorylase, which yields Glucose 1-phosphate.

(g)    Glycogen is synthesized by a different pathway. (summary)

·        The enzymes involved are glycogen synthetase (a residue-adding enzyme) plus a branching enzyme (which creates a-1,6 linkages from a-1,4 linkages) plus glycogenin (a priming enzyme)

·        UDP-glucose, the activated intermediate in glycogen synthesis, is formed from glucose 1-phosphate and UTP. (summary)

·        UDP is uridine diphosphate, i.e., the ribonucleic acid and the structure of UDP-glucose is shown in the upper left-hand corner of p. 586.

·        Note the loss of two phosphates in this reaction as pyrophosphate (PPi); these are all of the phosphates (2) employed to add a glucose residue to glycogen.

·        Glycogen synthetase catalyzes the transfer of glucose from UDP-glucose to the C-4 hydroxyl group of a terminal residue in the growing glycogen molecule. (summary)

·        A branching enzyme converts some of the a-1,4 linkages into a-1,6 linkages to increase the number of ends so that glycogen can be made and degraded more rapidly. (summary)

(h)   

(i)      Note (sigh) once again that the above structure is not quite correct. Though misleading that the glucose is being added as the second residue on a branch (it works just like that when being added to a non-branch or farther out on the branch), it is the structure of UDP that is incorrect; it is shown as a deoxyribonucleotide but, in fact, (at least according to Stryer) it is a ribonucleotide. Interesting…

(j)     Synthesis is primed by glycogenin, an autoglycosylating protein that contains a covalently attached oligosaccharide unit on a specific tyrosine residue. Glycogen synthetase is active only when associated with glycogenin, which serves to limit the size of glycogen granules. (summary)

(F)            Metabolic Controls

(a)    Phosphorylase is regulated by several allosteric effectors that signal the energy state of the cell and by reversible phosphorylation… (p. 590)

(b)   Phosphorylase… exists in two interconvertible forms: an active [phosphorylated] phosphorylase a and a usually inactive [not phosphorylated] phosphorylase b. (p. 590)

·        Remember that active phosphorylase results in glycogen degradation (glucose 1-phosphate formation).

·        The protein that phosphorylates phosphorylase is called phosphorylase kinase.

·        Therefore, an active phosphorylase kinase results in an active phosphorylase (glucose 1-phosphate formation).

·        Protein phosphatase I, on the other hand, dephosphorylates phosphorylase (glucose 1-phosphate not necessarily formed form of phosphorylase).

·        My guess is that protein phosphatase I is always (more or less) active thereby leaving phosphatase inactive by default while phosphorylase kinase is only transiently active, thereby fully activating phosphorylase only when glucose is needed.

(c)    Muscle phosphorylase b is active only in the presence of high concentrations of AMP… ATP acts as a negative allosteric effector by competing with AMP. Glucose 6-phosphate also inhibits phosphorylase b, primarily by binding to the AMP site… Phosphorylase b is active only when the energy charge of the muscle cell is low. In contrast, phosphorylase a  is fully active, irrespective of the levels of AMP, ATP, and glucose 6-phosphate. In resting muscle, nearly all the enzyme is in the inactive b form. During exercise, the elevated level of AMP leads to the activation of phosphorylase b. (p. 590)

·        So glycogen is degraded in muscle only as energy stores are depleted and, therefore, glucose is needed.

·        There are various indicators for a decline in energy stores and therefore a need for glucose: little ATP, much AMP, little phosphorylated glucose (glucose 6-phosphate).

(d)   The purpose of glycogen degradation in liver is to form glucose for export to other tissues when the blood glucose level is low. Hence, liver phosphorylase is responsive to glucose but not to AMP [the latter which is] an indicator of [the liver’s] own metabolic status. [p. 591]

·        Thus, the liver’s phosphatase is less responsive to the needs of its own cell than it is to the needs of the cells of the rest of the body, which it infers via the blood glucose level.

(e)    Generally, those things that activate phosphorylase inactivate glycogen synthesis (though note that the signals indicating activation of degradation and inactivation synthesis may be the same, the specific mechanisms of activation and inactivation are not identical but, rather, are specific for the specific proteins involved—which are not identical between the two pathways).

(f)     We will concentrate on the regulation of phosphorylase; the following considers more than phosphorylase and you will not be held responsble for these details (which, by the way, are also relevant to the next topic) unless they are presented elsewhere.

·        Glycogen synthesis and degradation are coordinated by several amplifying reaction cascades. Glycogen synthetase is inactive when phosphorylase is active, and vice versa. Epiniphrine and glucagons stimulate glycogen breakdown and inhibit its synthesis by increasing the cytosolic level of cyclic AMP, which activates protein kinase A (PKA). Phosphorylase kinase becomes more active, wheras glycogen synthetase becomes less active when phosphorylated by by PKA. Elevated cyhtosolic Ca2+ levels directly activate phosphorylase kinase, which contains calmodulin as one of its subunits. Hence, muscle contraction and calcium-mobilizing hormones promote glycogen breakdown. (summary)

·        The glycogen-mobilizing actions of PKA are reversed by protein phosphatase I, which is regulated by several hormones. Epinephrine inhibits this phosphatase by blocking its attachment to glycogen granules and by turning on an inhibitor; both effects are mediated by PKA-catalyzed phosphorylation. Insulin, by contrast, activates this phosphatase by triggering a cascade that phosphorylates the glycogen-targetting subunit of this enzyme. Hence, glycogen synthesis and phosphorylase are also regulated by noncovalent allosteric interactions. In fact, phosphorylase is a key part of the glucose-sensing system of liver cells. Glycogen metabolism exemplifies the power and precision of reversible phosphorylation in regulating biological processes. (summary)

(14)           Topic 15 (Hormone Action) [Bio 113 chapter 11]

(A)           Hormonal Control of Carbohydrate Metabolism

(a)    Phosphorylase… reversible phosphorylation… is responsive to hormones such as insulin, epinephrine, and glucagons. (p. 590)

(b)   Phosphorylase kinase is activated by phosphorylation and calcium ion (p. 593)

·        Recall that phosphorylase kinase activates phosphorylase by converting the usually inactive phosphorylase b into the always active phosphorylase a.

·        Calcium is released in muscles as a contraction-triggering signal.

·        Thus, muscle contraction activates the kinase that activates phosphorylase that leads to glycogen degradation.

(B)            Epinephrine

(a)    Epinephrine induces the breakdown of glycogen.

(b)   Muscular activity or its anticipation leads to the release of epinephrine… Epinephrine markedly stimulates glycogen breakdown in muscle and, to a lesser extent, in liver. (p. 594)

(c)    Thus, epinephrine, via a breakdown of glycogen in the muscles as well, to some extent, in the liver, leads to an increase in the amount of glucose available to muscle cells.

(C)           Glucagon

(a)    Glucagon induces the breakdown of glycogen.

(b)   The liver is more responsive to glucagons [than epinephrine]… a… hormone… secreted… when the blood sugar level is low. (p. 594)

(c)    Thus, glucagon, via breakdown of glycogen in the liver, leads to an increase in blood glucose levels.

(D)           Insulin

(a)    Insulin induces the synthesis of glycogen

(b)   High levels of insulin in the blood signal the fed state, whereas low levels signal a fasted state. (p. 594)

(c)    Thus, insulin, via the synthesis of glycogen in the liver and muscles, leads to a decrease in blood glucose levels.

(d)   (of course, insulin, as well as the rest of these hormones, do more than just affect glycogen synthesis and degradation)

(E)            Role of cAMP Cascade

(a)    This is an abridged form of the cascade described on p. 595:

·        Epinephrine and glucagons bind to… receptors in the plasma membrane of target cells and trigger the activation of the stimulatory G protein.

·        This results in an activation of adenylate cyclase.

·        Adenylate cyclase activity results in the production of cyclic AMP (cAMP).

·        cAMP activates protein kinase A.

·        Protein kinase A phosphorylates phosphorylase kinase.

·        Phosphorylase kinase converts phosphorylase b into phosphorylase a.

(b)   Each round of activation/synthesis of cAMP results in the further amplification of the cascade.

(c)    …the binding of a small number of hormone molecules to cell-surface receptors leads to the release of a very large number of sugar units [via glycogen degradation]. (p. 595)’

(d)   Note that in addition to the activation of glycogen degradation, the above leads to an inhibition of glycogen synthesis.

(F)            Role of Phosphofructokinase II and Fructose-2,6-Bisphosphate in Different Tissues

(a)    ???

(15)           Topic 16 (Fatty Acid Metabolism)

(A)           Overview

(a)    Fatty acids are physiologically important as (summary)

·        Components of phospholipids and glycolipids [membranes]

·        Lipophilic modifiers of proteins [lipoproteins]

·        Fuel molecules [fuel]

·        Hormones and intracellular messengers [messages] (summary)

·        Antimicrobial chemical defenses (soap)

(b)   They can be stored in adipose tissue as triacyglycerols (neutral fat), which can be mobilized by the hydrolytic action of lipases that are under hormonal control. (summary)

(B)            Oxidation of Even, Odd, Saturated, and Unsaturated Fatty Acids

(i)                  Oxidation of even-number fatty acids

(a)    Triacylglycerols are hydrolyzed by cyclic AMP-regulated lipases. (p. 605)

(b)   Fatty acids are… degraded in the mitochondrial matrix by a recurring sequence of four reactions: (summary)

·        Oxidation linked to FAD

·        Hydration

·        Oxidation linked to NAD+

·        Thiolysis by CoA (summary)

(c)    See Figure 24-5 (p. 609): Reaction sequence in the degradation of fatty acids: oxidation, hydration, oxidation, and thiolysis. Know/understand this figure (don't worry about nomenclature or enzyme names). Note the various products (i.e., FADH2, NADH, Acetyl CoA, shortened Acyl CoA).

(d)   These reactions essential cause the creation of a new acyl group by adding a water, converting the resulting hydroxyl group to a carbonyl group, and then cleave the resulting structure just prior to the carbonyl group leaving the carbonyl carbon linked to coenzyme A.

(e)    Note the starting molecule, an Acyl CoA. This is generated on the outer mitochondrial member, as discussed on page 607, at the expense of one of high-energy (pyrophosphate) bond.

(ii)                Oxidation of saturated fatty acids

(a)    Even-number saturated fatty acids are oxidized as described above.

(b)   Odd-number saturated fatty acids are oxidized as described below.

(iii)               Oxidation of odd-number fatty acids

(a)    Fatty acids having an odd number of carbon atoms are minor species. They are oxidized in the same way as fatty acids having an even number, except that propionyl CoA and acetyl CoA, rather than two molecules of acetyl CoA, are produced in the final round of degradation. (p. 612)

(iv)              Oxidation of unsaturated fatty acids

(a)    The oxidation of unsaturated fatty acids gets into trouble in the above schemes because in fatty-acid metabolism a single carbon cannot simultaneously be twice double bonded (C=C=C) and the FAD reaction would have to create such a bond if the third and fourth carbon already shared a double bond:

·        -C=C-C-CO-S-CoA cannot be made into -C=C=C-CO-S-CoA.

·        Instead an isomerase converts -C=C-C-CO-S-CoA to -C-C=C-CO-S-CoA.

·        Note that this bypasses the FAD step thereby reducing the amount of ATP produced in the oxidation of a fatty acid at a double bond by 1.5 (i.e., equivalent to amount generated by FADH2 in oxidative phosphorylation).

(b)   Alternatively, at some point in the oxidation of a fatty acid the FAD reaction can give rise to two double bonds separated by two carbons (rather than just one which cannot occur).

·        -C=C-C=C-COS-CoA is converted to -C-C=C-C-COS-CoA at the expense of one NADPH.

·        Note that -C-C=C-C-COS-CoA you have just seen (in the previous scheme) and that this structure, as before, is resolved employing isomerase.

·        Note that here the FAD step is not bypassed since it required an FAD to form -C=C-C=C-COS-CoA.

·        However, what has been lost is the NADPH required to reduce the fatty acid before isomerase could act.

(C)           Acidosis and Ketone Bodies

(a)    The acetyl CoA formed in fatty acid oxidation enters the citric acid cycle only if fat and carbohydrate degradation are appropriately balanced. The reason is that the entry of acetyl CoA into the citric acid cycle depends on the availability of oxaloacetate for the formation of citrate, but the concentration of oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized. Recall that oxaloacetate is normally formed from pyruvate, the product of glycolysis. The molecular basis of the adage that fats burn in the flame of carbohydrates is now evident. (p. 612)

(b)   In fasting or diabetes, oxaloacetate is consumed to form glucose by the gluconeogenic pathway and hence is unavailable for condensation with acetyl CoA. Under these conditions, acetyl CoA is diverted to the formation of acetoacetate and D-3-hydroxybutyrate. Acetoacetate, D-3-hydroxybutyrate, and acetone [don't worry about specific molecule names] are sometimes referred to as ketone bodies. Abnormally high levels of ketone bodies are present in the blood of untreated diabetics. (p. 612)

(D)           Fatty Acid Synthetase Complex

(a)    Fatty acid synthesis (in higher organisms) is catalyzed by what is essentially a single polypeptide chain called fatty acid synthase.

(b)   Seven different catalytic sites are present on a single polypeptide chain. (p. 618)

(c)    This complex is found in the cytosol (in contrast with the fatty acid degradation which takes place in the matrix of the mitochondria).

(E)            Role of ACP

(a)    Instead of the Coenzyme A employed as an acyl carrier in fatty acid degradation, Intermediates in fatty acid synthesis are covalently linked to… an acyl carrier protein (ACP). (p. 614)

(b)   Nevertheless, all the carbon atoms of fatty acids containing an even number are derived [ultimately] from acetyl CoA. (p. 616)

(F)            Acetate Transport

(a)    Acetyl CoA must be transported from the matrix of the mitochondria if it is to participate in fatty acid synthesis.

(b)    The mitochondria inner membrane is impervious to acetyl CoA, however.

(c)    The barrier to acetyl CoA is bypassed by citrate, which carries acetyl groups across the inner mitochondrial membrane. (p. 620)

(d)   The citrate is produced within the mitochondrial matrix by the reaction of acetyl CoA with oxaloacetate (i.e., the "first" step of the Kreb's cycle)

(e)    In the cytosol the citrate is then converted back to oxaloacetate and acetyl CoA.

(f)     The oxaloacetate is converted to pyruvate and CO2 with a gain of one NADPH, the latter which may then be employed in fatty acid synthesis.

(g)    Pyruvate may then reenter the mitochondrial matrix.

(h)    See Figure 24-17 (p. 620)

(G)           Essential and Non-Essential Fatty Acids

(a)    Mammals lack the enzymes to introduce double bonds at carbon atoms beyond C-9 in the fatty acid chain. Hence, mammals cannot synthesis linoleate (18:2 cis-D9, D12) and linolenate (18:3 cis-D9, D12, D15). Linoleate and linolenate are the two essential fatty acids. The term essential means that they must be supplied in the diet because they are required by the organism and cannot be endogenously synthesized. Linoleate and Linolenate furnished by the diet are the starting points for the synthesis of a variety of other unsaturated fatty acids. (p. 623)

(16)           Topic 17 (Nitrogen Metabolism)

(A)           Connection of Specific Non-Essential Amino Acids to Metabolic Pathways

(a)    Amino acids in excess of those needed for the synthesis of proteins and other biomolecules cannot be stored, in constrast with fatty acids and glucose, nor are they excreted. Rather, surplus amino acids are used as metabolic fuel. The a-amino group is removed and the resulting carbon skeleton is converted into a major metabolic intermediate. Most of the amino groups of surplus amino acids are converted into urea, whereas their carbon skeletons are transformed into acetyl CoA, acetoacetyl CoA, pyruvate, or one of the interemediates of the citric acid cycle. Hence, fatty acids, ketone bodies, and glucose can be formed from amino acids. (p. 629)

·        This is acetyl CoA:  

·        This is acetoacetyl CoA:

·        Notice how Acetoacetyl CoA is essentially acetyl CoA with a second acetyl group attached to a first acetyl group.

(b)   The major site of amino acid degradation in mammals is the liver. (p. 630)

(c)    One can divide up the degradation of amino acids into three categories:

·        Glucogenic, which means that the amino acids are converted only into intermediates that can then be converted into glucose.

·        Ketogenic, which means that the amino acids are converted only into intermediates that cannot be converted into glucose.

·        Glucogenic and Ketogenic, which means that portions of the amino acids are converted into intermediates that can be converted into glucose and other portions are converted into intermediates that cannot be converted into glucose.

(d)   Recall that acetyl CoA (as well as acetoacetyl CoA) cannot be converted into glucose but, instead, give rise to ketone bodies.

(e)    On the other hand, pyruvate, a-ketoglutarate, succinyl CoA, fumarate, and oxaloacetate can be converted into glucose (since both pyruvate and oxaloacetate are on the gluconeogenesis pathway).

(f)     See Figure 25-10 (p. 638) for an overview of the breakdown of amino acids into non-amino acid intermediates. Don’t worry about memorizing this figure, however.

(B)            Introduction of Inorganic Nitrogen

(C)           Glutamate Dehydrogenase

(a)    Glutamate dehydrogenase restores a-ketoglutarate by oxidatively deaminating glutamate.

(b)   This is how ammonium ion is formed.

(c)    This reaction is shown on p. 630 or your text:

·        Glutamate + NAD+ (or NADP+) + H2O ßà NH4+ + a-ketoglutarate + NADH (or NADPH) + H+

(d)  

(e)   (note that the “NH3+” should be NH4+ or NH3)

(f)     The removed NH4+ is then converted into urea, which the kidneys excrete.

(D)           Transaminases

(a)    The a-amino group of many amino acids is transferred to a-ketoglutarate to form glutamate, which is then oxidatively deaminated to yield NH4+ [and a-keotglutarate]. (p. 630)

(b)   Recall that a-ketoglutarate looks like this: -OOC-CO-C-C-COO- while glutamate looks like this -OOC-CNH3+-C-C-COO-, that is, the same except that a carbonyl group is swapped for an amino group.

(c)    Note that your text uses the synonym “aminotransferase”.

(d)   Transaminases transfer amino groups from one amino acid to a second amino acid.

(e)   

(f)     The mechanism of transfer involves leaving the first amino acid with an a-carbonyl group, e.g., a-ketoglutarate, in place of its a-amino group.

·        That is, –C=O is replaced with –C-NH3+.

(g)    The amino group is then swapped with an a-carbonyl group on carbon skeleton from which the second amino acid is to be made via the addition of an a-amino group.

·        That is, –C-NH3+ is replaced with –C=O.

(h)    During the catalysis of these reactions the amino acid is bound to an enzyme prosthetic group via its amino group, which is then hydrolyzed off of the amino acid, leaving behind a carbonyl group (and, hence, an a-carbonyl carboxylic acid is formed).

(i)      A second a-carbonyl carboxylic acid then binds this bound nitrogen via a dehydration reaction.

(j)     The second amino acid is formed via its replacement on the prosthetic group by the amino group found on a specific lysine residue associated with the transaminase.

(k)   These reactions, in greater detail, are presented on pp. 631-632.

(E)            Toxicity of NH3 and Removal by Urea Cycle

(a)    In most terrestrial vertebrates, NH4+ is converted into urea by the urea cycle. (summary)

(b)   Urea is formed by the hydrolysis of arginine. The subsequent reactions of the urea cycle synthesize arginine from ornithine, the other product of the hyrolysis reaction… The carbon atom and one nitrogen atom of urea come from cabamoyl phosphate, which is synthesized from CO2, NH4+, and ATP. The other nitrogen atom of urea comes from aspartate. (summary)

(c)   

(d)   Don’t worry about learning all of the names or enzymes involve in the above figure. However,

·        Certainly you should know the structure of urea.

·        Note how urea resembles the end of the arginine side chain.

·        Note how urea is generated by the hydrolytic removal of this side chain (the H2O added is not explicitly shown in the above figure, but it is added).

·        Note how ornithine is simply arginine with urea hydrolyzed off.

·        Note how the rest of the cycle involves the reformation of arginine.

·        Note how much carbamoyl phosphate looks like it was formed by combining ammonia, carbon dioxide, and a phosphate.

·        Note how carbamoyl phosphate is a charged molecule (i.e., possesses that phosphate) and how the removal of the phosphate allows the carbamoyl group (?) to attach to the ornithine amino side chain.

·        Note how aspartate enters the reaction essentially by a dehydration reaction between aspartate’s amino group and citruline’s ketone (?) group. (this reaction involves the hydrolysis of an ATP to AMP and PPi and note that arginosuccinate’s structure is drawn differently here than it is in your book, i.e., see p. 635; in fact, I believe that the above structure is incorrect since there is a carbon that only has three bonds around it; in fact, one of the nitrogens should be double bonded to this carbon)

(17)           Topic 18 (Photosynthesis) [Bio 113 chapter 10]

(A)           Chloroplast and Component Parts

(a)   

(b)   Like a mitochondrion, a chloroplast has an outer membrane and an inner membrane, with an intervening intermembrane space. The inner membrane surrounds a stroma containing soluble enzymes and membranous structures called thylakoids, which are flattened sacs. A pile of these sacs is called a granum. Different grana are connected by regions of thylakoid membrane called stroma lamellae. The thylakoid membranes separate the thylakoid space from the stroma space. Thus, chloroplasts have three different membranes (outer, inner, and thylakoid membranes) and three separate spaces (intermembrane, stroma, and thylakoid spaces). In developing chloroplasts, thylakoids arise from invaginations of the inner membrane, and so they are analogous to mitochondrial cristae. (p. 654)

(B)            Light Gathering in Thylakoid Membrane

(a)    Photosynthesis in green plants takes place in chloroplasts. The energy conversion apparatus is an integral part of the thylakoid membrane system of these organelles. The first step in photosynthesis is the absorption of light by chlorophyll, a porphyrin with a coordinated magnesium ion. The resulting electronic excitation passes from one chlorophyll molecule to another in a light-harvesting complex until the excitation is trapped by a chlorophyll pair with special properties. At such a reaction center, the energy of the excited electron is converted into a separation of charge. In essense, light is used to create reducing potential. (p. 653)

(C)           Light and Dark Reactions

(a)    Photosynthesis in green plants is mediated by two kinds of light reactions. Photosystem I generates reducing power in the form of NADPH. Photosystem II transfers the electrons of water to a quinone and concomitantly evolves O2. Electron flow within each photosystem and between them generates a transmembrane proton gradient that drives the synthesis of ATP, as in oxidative phosphorylation. Indeed, photosynthesis closely resembles oxidative phosphorylation. The principal difference between these energy transduction processes is the source of high-potential electrons. In oxidative phosphorylation, they come from the oxidation of fuels; in photosynthesis, they are produced by photoexcitation of chlorophyll. (pp. 653-654)

(b)   NADPH and ATP formed by the action of light then reduce CO2 and convert it into 3-phosphoglycerate by a series of dark reactions called the Calvin cycle, which occurs in the stroma of chloroplasts. Hexoses are formed from 3-phosphoglycerate by the gluconeogenesis pathway. (p. 654)

(D)           Special Reaction Centers

(a)    …most chlorophyll molecules in the photosynthetic unit absorb light, but only a small portion of them, those at reaction centers, mediate the transformation of light into chemical energy. The energy level of chlorophylls at the reaction center is lower than that of other chlorophylls, which enables the reaction center to trap the excitation. The transfer of energy by direct electromagnetic interactions between chlorophylls and then to the reaction center is very rapid, occurring in picoseconds. (p. 657)

(b)   See Figures 26-7 and 26-8 (p. 657)

(E)            Components of Z Scheme and Their Association with Thylakoid Membrane

(a)    Figure 26-15: Pathway of electron flow from H2O to NADP+ in photosynthesis. This endergonic reaction is made possible by the absorption of light by photosystem II (P680) and photosystem I (P700). Reduced plastoquinone (QH2) formed by photosystem II feeds electrons into the cytochrome bf complex. Reduced plastocyanin (PC) carries electrons to photosystem I, which generated reduced ferredoxin (Fd). This powerful reductant transferes its electrons to NADP+ to form NADPH. A proton gradient across the thylakoid membrane (inside acidic) is formed when electrons flow through the cytochrome bf complex. The splitting of water and the reduction of NADP+ one opposite sides of the thylakoid membrane also contributes to a proton gradient. (p. 662).

(b)  

(c)    You should understand the Z scheme and these components:

·        H2O à 2 H+ + ½ O2 + 2 e-

·        Photosystem II

·        Plastoquinone

·        Cytochrome bf complex (pumps 2 H+)

·        Plastocyanine

·        Photosytem I

·        Ferrodoxin

·        NADP+ à NADPH (contributes to pH gradient)

(F)            Stationary and Mobile Carriers

(a)    Photosystem II… catalyzes the light-driven transfer of electrons from water to plastiquinone. This electron acceptor closely resembles ubiquinone, a component of the electron transport chain of mitochondria. (p. 660)

(b)   Thylakoid membranes of most plants are differentiated into stacked (appressed) and unstacked (nonappressed) regions. Stacking increases the amount of thylakoid membrane in a given chloroplast volume. Both regions surround a common internal thylakoid space, but only unstacked regions make direct contact with the chloroplast stroma. Stacked and unstacked regions differ  in the nature of their photosynthetic assemblies. Photosystem I and ATP synthase are located almost exclusively in unstacked regions. The cytochrome bf complex is found in both regions. Indeed, this complex rapidly moves back and forth between the stacked and unstacked regions. Plastoquinone and plastocyanin are the mobile carriers of electrons between assemblies located in different regions of the thylakoid membrane. A common internal thylakoid space enables protons liberated by photosystem II in stacked membranes to be utilized by ATP synthase molecules that are located far away in unstacked membranes. (pp. 666-667)

(c)    What is the functional signficance of this lateral differentiation of the thylakoid membrane system? The positionintg of photosystem I in the unstacked membranes also gives it direct access to the stroma for the reduction of NADP+. ATP synthase, too, is located in the unstacked region to provide space for its large CF1 globule and to give access to ADP. In contrast, the tight quarters of the appressed region pose no problem for photosystem II, which interacts with a small polar electron carrier (plastoquinone). (p. 667)

(G)           Proton Gradient

(a)    The thylakoid space becomes markedly acidic, with the pH approaching 4. The light-induced transmembrane proton gradient is about 3.5 pH units… In chloroplasts, nearly all of the Dp arises from the pH gradient, whereas in mitochondria the contribution from the membrane potential is larger. The reason for this difference is that the thylakoid membrane is quite permeable to Cl- and Mg2+. The light-induced transfer of H+ into the thylakoid space is accompanied by the transfer of either Cl- in the same direction or Mg2+ (1 per 2 H+) in the opposite direction. Consequently, electrical neutrality is maintained and no membrane potential is generated. (pp. 665-666)

(b)   About three protons flow through the [ATP synthase] per ATP synthesized, which corresponds to a free-energy input of 14.4 kcal per mole of ATP. No ATP is synthesized if the pH gradient is less than two units, because the driving force is then too small. (p. 666)

(H)           Energy Considerations

(a)    ???

(I)              ATP and NADPH in Light Reaction

(a)    Needless to say, these are the end products of the light reaction with the ATP generated via a proton motive force through an ATP synthase that is very similar to that employed in mitochondria.

(J)              Cyclic Photophosphorylation

(a)    An alternative pathway for electrons arising from P700, the reaction center of photosystem I, contributes to the versatility of photosynthesis. The high-potential electron in ferredoxin can be transferred to the cytochrome bf complex rather than to NADP+. This electron then flows back to the oxidized form of P700 through plastocyanin. The net outcome of this cyclic flow of electrons is the pumping of protons by the cytochrome bf complex. The resulting proton gradient then drives the synthesis of ATP. In this process, called cyclic photophosphorylation, ATP is generated without the concomitant formation of NADPH. Photosystem II does not participate in cyclic photophosphorylation, and so O2 is not formed from H2O. Cyclic photophosphorylation takes place when NADP+ is unavailable to accept electrons from reduced ferrodoxin because of a very high ratio of NADPH to NADP+. (p. 664)

(K)           Calvin and Dark Reactions

(a)   What happened to the notes I had on this subtopic?

(L)            Rubisco

(a)   What happened to the notes I had on this subtopic?

(M)          Calvin Reductive Cycle

(a)    What happened to the notes I had on this subtopic?

(18)           Topic 19 (RNA and DNA Structure and Function) [Bio 113 chapter 5] [Bio 113 chapter 6]

(A)           Polymeric Forms

(B)            Important Differences in Structure and Function

(a)    Nucleosides are composed of a base in b-glycosidic linkage with a sugar, either ribose or deoxyribose. When the sugar is deoxyribose, the structure is called a deoxynucleoside. When one or more phosphates are esterified to a deoxynucleoside, the molecule is called a deoxynucleotide.

(b)   A nucleoside consists of a purine or pyrimidine base bonded to a sugar. The four nucleosides are deoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine. (p. 76) [All of the structures on page 76 should be memorized including the numbering]

(c)    Corresponding ribonucleosides contain a base along with ribose as their sugar moiety. When one or more phosphates are esterified to a ribonucleoside, the molecule is called a ribonucleotide.

(d)   A nucleotide is a phosphate ester of a nucleoside. (p. 76)

(e)    Study the structures (page 76) of the four bases and of deoxyadenosine and deoxyadenosine 5'-triphosphate (dATP) (a representative nucleoside and nucleotide). Make sure you can draw and name the nucleoside and nucleotide containing any of the four bases in DNA. Note the numbering convention for the two cyclic portions of a nucleoside or nucleotide: a primed number (such as 3' or 5') refers to an atom in the sugar ring (top of page 76); an unprimed number refers to an atom in the purine or pyrimidine ring.

(f)     DNA sequence. Individual deoxynucleotides can be linked to form a strand of DNA. The 3'-hydroxyl of the deoxyribose in one deoxynucleotide is linked to the 5'-hydroxyl of the next by a phosphodiester bridge. The alternating sugars and phosphates form the invariable part, or backbone, of the DNA strand. Each nucleotide unit can contain any one of the purine or pyrimidine bases. The linear order of the bases in a DNA strand is referred to as its sequence. [see Figure 4.2, p. 77]

(g)    Polarity of DNA strands. There are a number of ways to depict a sequence of nucleotides, as shown on page 77. Even the simplest description, such as ACG, contains information about the polarity of the chain. In this case, the nucleotide containing adenine has an unlinked 5'-hydroxyl group, whereas the nucleotide containing guanine has an unlinked 3'-hydroxyl group. Once you grasp the polar nature of a DNA chain, you'll see why ACG is different from CGA.

(h)    DNA is the molecule of heredity in all prokaryotic and eukaryotic organisms. In viruses, the genetic material is either DNA or RNA. All cellular DNA consists of two very long, helical polynucleotide chains coiled around a common axis. The two strands of the double helix run in opposite directions [they are arranged antiparallelly]. The sugar-phosophage backbone of each strand is on the outside of the double helix, whereas the purine and pyrimidine bases are inside. The two chains are held together by hydrogen bonds between pairs of bases. Adenine (A) is always paired with thymine (T), and guanine (G) is always paired with cytosine (C). Hence, one strand of a double helix is the complement of the other. Genetic information is encoded in the precise sequence of bases along a strand. Most DNA molecules are circular. (summary)

(C)           Watson and Crick

(i)                  Semiconservative Replication Concept

(a)    Semiconservative replication. In experiments using bacteria with labeled chromosomes, Meselson and Stahl demonstrated that DNA synthesis is semiconservative. At each generation, one strand in a daughter DNA molecule is newly synthesized, the other is transmitted from the parent molecule.

(b)   Study the text that describes these experiments. Make sure that you can relate the results to Watson and Crick's proposal for replication of DNA.

(c)    In the replication of DNA, the two strands of a double helix unwind and separate as new chains are synthesized. Each parent strand acts as a template for the formation of a new complementary strand. Thus, the replication of DNA is semiconservative-each daughter molecule receives one strand from the parent DNA molecule. The replication of DNA is a complex process carried out by many proteins, including several DNA polymerases. The activated precursors in the synthesis of DNA are the four deoxyribonucleoside 5' triphosphates. The new strand is synthesized in the 5' à 3' direction by a nucleophilic attack by the 3'-hydroxyl terminus of the primer strand on the innermost phosphorus atom of the incoming deoxyribonucleoside triphosphate [see Figure 4-21, p. 89]. Most important, DNA polymerases catalyze the formation of a phosphodiester bond only if the base on the incoming nucleotide is complementart to the base on the template strand. In other words, DNA polymerases are template-directed enzymes. (summary)

(ii)                Base Pairing

(a)    The Watson-Crick model. Study the characteristics of DNA structure (pages 80 and 81). The two helical chains of a DNA molecule are held together by hydrogen bonds between purine-pyrimidine base pairs. Adenine in one strand forms two hydrogen bonds with thymine in the other strand; guanine forms three hydrogen bonds with cytosine. The base pairs are located inside the helix, the deoxyribose and phosphate moieties on the outside.

(b)   The base-pairing scheme proposed by Watson and Crick provided a clue to how genetic material could be exactly replicated. Given that one chain is the complement of the other, enzymes catalyzing replication could use base pairing to ensure faithful copying.

(c)    The Watson-Crick model also clearly explained the earlier observations by Chargaff that, in DNA from a variety of organisms, the ratios of adenine to thymine and of guanine to cytosine are approximately 1.0.

(d)   Note that you should be able to draw the nucleotides (nitrogenous bases) in their Watson-Crick base pairing. See Figure 4.9 and Figure 4.10, both on page 81. Note that it may be (very) helpful to you if you draw these base-pairs out yourself with the double bonds explicitly shown

(e)   

(D)           DNA Replication (but see below)

(i)                  DNA Polymerases

(a)    Catalytic activity of DNA polymerase I. This enzyme catalyzes the step-wise addition of deoxynucleotides to a DNA chain, using any of the four deoxynucleotide triphosphates as precursors. Besides these precursors, the enzyme requires

·        a template DNA chain; and

·        a preexisting DNA chain that acts as a primer.

(b)   The enzyme attaches deoxynucleotides to the free 3'-hydroxyl terminus of the primer, extending it in the 5' to 3' direction. The base to be inserted at each elongation step is determined by the sequence in the template chain: the enzyme inserts a nucleotide containing the base complementary to the base in the corresponding position in the template.

(c)    Replication produces a DNA strand with a sequence complementary to that of the template. This semiconservative process produces a double-stranded DNA with a newly synthesized strand (extended from the primer) and a parental strand (which was used as the template). Make sure you understand how hydrogen bonding is the basis for faithful copying of a DNA molecule.

(d)   DNA polymerase I can also correct mistakes. It can remove a mismatched nucleotide and insert the correct one.

(e)    This correction step involves proofreading.

(19)           The following material is covered in chapter 31 and we will cover it then. See especially pages 793 (DNA ligase) and 799-810 (DNA replication)

(A)           DNA replication

(i)                  DNA polymerases

(a)    DNA polymerases are template-directed enzymes that catalyze the formation of phosphodiester bonds by the nucleophilic attack of a 3’-OH on the innermost phosphorous atom of a deoxyribonucleoside 5’-triphostate. They cannot start chains de novo; a primer with a free 3’-OH is required.  (summary)

(b)    Polymerization is catalyzed by a single-active site that can bind any of the four dNTPs. Which one binds depends on the corresponding base on the template strand. The likelihood of binding and of making a phosphodiester bond is very low unless the incoming nucleotide forms a Watson-Crick base pair with the opposing nucleotide on the template. (p. 800)

(c)    DNA polymerases proofread the nascent product; their 3’ à 5’ exonuclease activity examines the outcome of each polymerization step. A mispaired nucleotide is excised before the next polymerization step. (summary)

(d)   the 3’ à 5’ nuclease activity has an editing function in polymerization. (p. 800)

(e)    In general, DNA polymerase I removes mismatched residues at the primer terminus before proceeding with polymerization. (p. 800)

(f)     In effect, DNA polymerase I examines the result of each polymerization it catalyzes before proceeding to the next. (p. 801)

(g)    …the exonuclease site is distinct from the polymerase site. (p. 800)

(h)    DNA is precipitated by trichloroacetic acid, whereas precursor nucleotides stay in solution. (p. 799)

(ii)                Ligase

(a)    Nascent DNA fragments are joined by DNA ligase in a reaction driven by ATP or NAD+. (summary)

(b)   It makes sense that some form of energy would be required because this reaction simply is the formation of a phosphodiester linkage between adjacent but not linked nucleotides found on one strand of a double helix.

(c)    …ligase seals breaks in double-stranded DNA molecules. (p. 793)

(d)   DNA Ligase links together Okazaki fragments.

(iii)               Replication Fork

(a)    …the synthesis of new DNA is closely coupled to the unwinding of parental DNA. (p. 804)

(b)   DNA replication in E. coli starts at a unique origin (oriC) and proceeds sequentially in opposite directions. More than 20 proteins are required for replication. DNAB protein, an ATP-driven helicase, unwinds the oriC region to create a replication fork. At this fork, both strands of parental DNA serve as templates for the synthesis of new DNA. (summary)

(c)    See Figure 31-36 (p. 808).

(iv)              Primase

(a)    Recall that all known DNA polymerases require a primer with a free 3’-OH group for DNA synthesis. (p. 805)

(b)   DNA synthesis is primed by a short stretch of RNA formed by primase, an RNA polymerase. (summary)

(c)    Primase synthesizes a short stretch of RNA (~5 nucleotides) that is complementary to one of the template DNA strands. (p. 805-806)

(d)   An RNA primer would be unnecessary if DNA polymerases could start chains de novo. However, such a property would be incompatible with the very high fidelity of DNA polymerases… (p. 806)

(v)                Replisomes

(a)    Not covered by text?

(vi)              Single Strand Binding Protein

(a)    Single-strand binding protein keeps the unwound DNA extended and accessible so that both strands can serve as templates. (p. 808)

(b)   See Figure 31-36 (p. 808).

(vii)             Leading and Lagging Strands

(a)    One strand (the leading strand) is synthesized continuously, whereas the other strand (the lagging strand) is synthesized discontinuously, in the form of 1-kb [1000 nucleotide] fragments (Okazaki fragments). Both new strands are formed simultaneously by the concerted actions of DNA polymerase… The discontinuous assembly of the lagging strand enables 5’ à 3’ polymerization at the atomic level to give rise to overall growth of this strand in the 3’ à 5’ direction. (summary)

(b)   See Figures 31-30 and 31-31 on page 804.

(c)    See Figure 31-36 (p. 808).

(viii)           Okazaki Fragments

(a)    Immediately above.

(b)   All known DNA polymerases synthesize DNA in the 5’ à 3’ direction but not in the 3’ à 5’ direction. (p. 804)

(B)            Retrovirus and Role of AZT

(a)    Not covered by text?

(20)           Topic 20 (RNA Synthesis) [Bio 113 chapter 17]

(a)    Most RNA molecules are single-stranded, but many contain extensive double-helical regions that arise from the folding of the chain into hairpins... All cellular RNA is synthesized by RNA polymerase according to instructions given by DNA templates. The activated intermediates are ribonucleoside triphosphates. The direction of RNA synthesis is 5' à 3', like that of DNA synthesis. RNA polymerase differs from DNA polymerase in not requiring a primer... Many RNA molecules are cleaved and chemically modified after transcription. (summary)

(b)   Role of the messenger. Although DNA is (in almost all cells) the repository of information for making proteins, it does not serve directly as the template for protein synthesis. Information in DNA is copied to messenger RNA (mRNA), which is then the template for the exact linear sequence of amino acids in a protein. The flow of information from DNA to protein thus occurs in two stages:

·        Transcription: the synthesis of RNA molecules with a sequence complementary to that of DNA. These RNA molecules include not only mRNA (the template for a protein sequence) but transfer RNA (tRNA) and ribosomal RNA (rRNA), all of which participate in protein synthesis.

·        Translation: the synthesis of protein molecules based on instructions from mRNA.

(c)    Structure of RNAs. RNA is composed of ribonucleotides joined by 3' to 5' phosphodiester bonds. As you learned in chapter 4, the sugar unit is ribose, rather than deoxyribose as in DNA, and uracil replaces thymine as one of the pyrimidines. Thus the bases of RNA are A, U, G, and C.

·        RNA molecules are generally single-stranded, but double-helical regions can be formed through hairpin loops. In these loops, uracil usually pairs with adenine and guanine with cytosine (in the same RNA strand-not in another strand as in double-stranded DNA).

·        RNA molecules range in size from about 75 to thousands of nucleotides. (Recall that DNA molecules are much longer.)

(d)   See structures near top of p. 96.

(e)    Types of RNA. Several classes of RNA are found in prokaryotic and eukaryotic cells:

·        Messenger RNAs carry information for every gene to be expressed. The mRNA molecules vary in size, depending on the gene from which they were transcribed.

·        Transfer RNAs carry amino acids to the site of protein synthesis. Each tRNA carries a particular amino acid. The amino acid is then linked to another amino acid in a growing peptide chain during translation. Transfer RNAs range from 75 to 90 nucleotides in length.

·        Ribosomal RNA is the most abundant type of RNA in the cell. The rRNAs associate with more than 50 ribosomal proteins, and play both catalytic and structural roles in translation. Bacteria have three types of rRNA, of sedimentation size 5S, 16S, and 23S (corresponding to 120, 1500, and 2900 ribonucleotides).

·        Eukaryotes also contain additional, small RNA molecules. These have a role in processing other RNA molecules or in moving nascent (newly formed) protein molecules to specific locations inside or outside the cell

(B)            RNA Polymerase and its Characteristics (but see below)

(a)    Bacteria have a single form of RNA polymerase that synthesizes all types of RNA molecules. In eukaryotes, different types of RNA are synthesized by different RNA polymerases.

(b)   DNA-dependent RNA polymerases are in many ways similar to the polymerases that make DNA, but with some important differences.

(c)    Comparing RNA polymerase and DNA polymerase.

·        While both types use DNA as a template, only RNA polymerase can use either double-stranded or single-stranded DNA.

·        Synthesis of RNA is conservative, rather than semiconservative.

·        RNA polymerase uses ribonucleoside triphosphates as activated precursors, rather than deoxynucleotides.

·        RNA polymerase does not require a primer.

·        RNA polymerase cannot remove mismatched ribonucleotides from newly created RNA strands. (DNA polymerase I can do this as part of its repair function.)

(d)   DNA as template. All RNA polymerases use DNA as a template. In most cases, only one of the two strands of DNA is copied. Study figure 5-10. Make sure that you understand the difference between the DNA template strand and the DNA coding strand. As you can see, the sequence of the RNA transcript and the DNA coding strand are identical, except that the transcript contains uracils rather than thymines.

(C)           mRNA (but see below)

(a)    See types of RNA, above.

(D)           tRNA (but see below)

(a)    See types of RNA, above.

(E)            rRNA (but see below)

(a)    See types of RNA, above.

(F)            Aminoacyl tRNA sythetases (but see below)

(a)    Each tRNA molecule contains a site to which a particular amino acid (in an activated form) is attached, to form an aminoacyl-tRNA. This reaction is catalyzed by a specific aminoacyl-tRNA synthetase.

(21)           The following material is covered in chapter 33 and we will cover it then..

(A)           RNA Polymerase and its Characteristics

(i)                  Oh, to heck with it. Let’s just considered RNA polymerase essentially already covered.

(B)            mRNA

(i)                  This we will cover in the context that it is presented in chapter 34 (topic 21).

(C)           tRNA

(i)                  This we will cover in the context that it is presented in chapter 34 (topic 21).

(D)           rRNA

(i)                  This we will cover in the context that it is presented in chapter 34 (topic 21).

(E)            Aminoacyl tRNA sythetases

(i)                  This we will cover in the context that it is presented in chapter 34 (topic 21).

(22)           Topic 21 (Protein Synthesis) [Bio 113 chapter 17]

(A)           Components required for Protein Synthesis

(B)            Nature of Code

(a)    The base sequence of a gene is collinear with the amino acid sequence of its polypeptide product. The genetic code is the relationship between the sequence of bases in DNA (or its RNA transcript) and the sequence of amino acids in proteins. Amino acids are coded by groups of three bases (called codons) starting from a fixed point. (summary)

(C)           Redundancy

(a)    For most amino acids there is more than one code word. In other words, the code is degenerate. (summary)

(b)   Codons specificying the same amino acid are called synonyms. (summary)

(c)    Most synonyms differ only in the last base of the triplet. (summary)

(d)   Degeneracy of the code. For the 20 L-amino acids found in proteins the genetic code has 61 codons. The code is thus said to be degenerate: most amino acids are encoded by two or more codons, called synonyms. Only Trp and Met have one codon; Arg, Leu, and Ser each have six. Generally, the higher the overall frequency of an amino acid in proteins, the more codons it has. Synonyms often have identical bases in the first and second positions, with variation in the third.

(e)    Stryer discusses the significance of degeneracy in the genetic code (pages 109-110). One reason for degeneracy may be that it minimizes the effects of mutations (changes in DNA and thus mRNA sequence) on the structure and function of the encoded protein.

(D)           Wobble (but see below)

(E)            Initiation (but see below)

(a)    Start signals. In prokaryotes, start signals include the AUG codon, read by a tRNA that carries the modified amino acid formylmethionine, and an upstream sequence that pairs with part of the ribosomal complex to initiate transcription. In eukaryotes, an AUG (methionine) codon closest to the 5' end of the message serves as a start signal.

(F)            Elongation (but see below)

(G)           Termination (but see below)

(a)    Sixty-one of the 64 codons specify particular amino acids, whereas the other three codons (UAA, UAG, and UGA) are signals for chain termination. (summary)

(H)           Importance of Aminoacyl tRNA Synthetases in Authenticity (but see below)

(I)              Action of Puromycin (but see below)

(J)              University of the Code

(a)    Universality of the code. Once the basics of the genetic code were worked out using bacterial systems, investigators began to look at the code in other organisms. Much of this work was done by analyzing spontaneous or induced mutations, or alterations in one or more bases in an mRNA.

(b)   Evidence showed that the genetic code is for the most part universal, used by organisms from the most primitive to the most complex.

(c)    mRNAs can be correctly translated by the protein-synthesizing machinery of very different species. (p. 111)

(23)           The following material is covered in chapter 34 and we will cover it then..

(A)           Overview

(a)    A protein is synthesized in the amino-to-carboxyl direction by the sequential addition of amino acids to the carboxyl end of the growing peptide chain. The activated precursors are aminoacyl-tRNAs, in which the carboxyl group of an amion group is joined to the 3’-OH of a transfer RNA (tRNA). The linking of an amino acid to its corresponding tRNA is catalyzed by an aminoacyl-tRNA synthetase. This activation reaction, which is analogous to the activation of fatty acids, is driven by ATP. For each amino acid, there is at least one kind of rRNA and activating enzyme. (pp. 875-876)

(b)   Protein synthesis takes place in three stages: initiation, elongation, and termination. (p. 876)

·        Initiation results in the binding of the initiator tRNA to the start signal of mRNA. The initiator tRNA occupies the P (peptidyl) site on an ribosome.

·        Elongation starts with the binding of an aminoacyl-tRNA to the A (aminoacyl) site, a distinct tRNA-binding site on the ribosome. A peptide bond then forms between the amino group of the incoming aminoacyl-tRNA and the carboxyl group of the formylmethionine carried by the initiator tRNA. The resulting dipeptidyl-tRNA then moves from the A site before leaving the ribosome. The binding of aminoacyl-tRNA, the movement of the peptidyl-tRNA, and the associated movement of the ribosome to the next codon are powered by the hydrolysis of GTP. An aminoacyl-tRNA then binds to the vacant A site to start another round of elongation, which proceeds as described above.

·        Termination occurs when a stop signal on the mRNA is read by a protein release factor, which leads to the release of the completed polypeptide chain from the ribosome.

(c)    Ribosomes, in essense, are enzymes that catalyze mRNA-directed formation of peptide bonds. (p. 876)

(B)            tRNA

(a)    More than 100 sequences [of tRNAs] are now known [in fact, by this writing, far          more than that must now be known]. The striking finding is that all of them can be written in a cloverleaf pattern in which about half the residues are base-paired. Hence, tRNA molecules have many common structural features. This finding is not unexpected, because all tRNA molecules must be able to interact in nearly the same way with ribosomes, mRNAs, and elongation factors. For example, they must fit into the A, P, and E sites on the ribosome and interact with the enzymatic site that catalyzes peptide-bond formation. (pp. 876-877)

(b)   The activated amino acid and anticodon of tRNA are at opposite ends of the L-shaped molecule. (p. 878)

(C)           Aminoacyl tRNA synthetases

(a)    An amino acid ester of tRNA is called an aminoacyl-tRNA… it is sometimes called a charged amino acid. (p. 880)

(b)   See Figure 34-7 (p. 880)

(c)    Note that an aminoacyl group is an acetyl group with an amino group added: +3HN-C-CO-

(d)   Note that the aminoacyl group is ester linked to the 3’-OH (or 2’-OH) of the activated tRNA.

(e)    …two ~P are consumed in the synthesis of an aminoacyl-tRNA. One of them is consumed in forming the ester linkage of aminoacyl-tRNA, whereas the other is consumed in driving the reaction forward. (p. 880)

(f)     The activation and transfer steps for a particular amino acid are catalyzed by the same aminoacyl-tRNA synthetase. In fact, [during the reaction] the aminoacyl-AMP intermediate does not dissociate from the synthetase. Rather, it is tightly bound to the active site of the enzyme by noncovalent interactions. (p. 880)

(g)    At least one aminoacyl-tRNA synthetase exists for each amino acid. (p. 881)

(h)    …the anticodon on the tRNA is the recognition site for the codon on mRNA and that recognition occurs by base pairing (p. 886)

(i)      …the amino acid in aminoacyl-tRNA does not play a role in selecting a codon. (p. 886)

(D)           Wobble

(a)    What are the rules that govern the recognition of a codon by the anticodon of a tRNA? A simple hypothesis is that each of the bases of the codon forms a Watson-Crick type of base pair with a complementary base on the anticodon. The codon and anticodon would then be lined up in an antiparallel fashion… A specific prediction of this model is that a particular anticodon can recognize only one codon. (p. 886)

(b)   The facts are otherwise. Some pure tRNA molecules can recognize more than one codon. (p. 886)

(c)    The first two bases of these codons are the same, whereas the third base of a codon is different. Could it be that the recognition of the third base of a codon is sometimes less discriminating than recognition of the other two? The pattern of degeneracy of the genetic code indicates that this might be so. (p. 887)

(d)   This assumption of “steric freedom” is known as “wobble.”

(e)    Two generalization concerning the codon-anticodon interaction can be made: (pp 887-888)

·        The first two bases of a codon pair in the standard way. Recognition is precise. Hence, codons that differ in either of their first two bases must be recognized by different tRNAs…

·        The first base of an anticodon determines whether a particular tRNA molecules reads one, two, or three kinds of codons: C or A (1 codon), U or G (2 codons), or I (3 codons).

(f)     Thus, part of the degeneracy of the genetic code arises from imprecision (wobble) in the pairing of the third base of the codon with the first base of the anticodon. We see here a strong reason for the frequent appearance of inosine, one of the unusual nucleosides, in anticodons. Inosine maximizes the number of codons that can be read by a particular tRNA molecule. (p. 888)

(E)            rRNA

(a)    Ribosomal RNAs (5S, 16S, and 23S rRNA) play a central role in protein synthesis. (p. 889)

(b)   See Figure 34-18 (p. 888). Note how the 70S E. coli ribosome consists of a 30S and a 50S subunit.

·        The 30S subunit consists of a 16S rRNA plus a number of proteins (22).

·        The 50S subunit consists of a 23S and a 5S rRNA plus a number of proteins (34).

(c)    For many years, it was presumed that ribosomal proteins orchestrate protein synthesis and that ribosomal RNAs serve primarily as a structural scaffold. The current view is almost the reverse. The discovery of catalytic RNA made us receptive to the possibility of a much more active role for RNA in ribosomal function. Indeed, several lines of evidence now suggest that ribosomal RNAs have directive roles in protein synthesis and may be dominant… (p. 889)

(F)            mRNA

(a)    Messenger RNA is translated in the 5’ à 3’ direction. (p. 893)

(b)   An important feature of prokaryotic gene expression is that translation and transcription are closely coupled in space and time. (p. 893)

(c)    Many ribosomes can simultaneously translate an mRNA molecule. This parallel synthesis markedly increases the efficiency of utilization of the mRNA. The group of ribosomes bound to an mRnA molecule is called a polyribosome or a polysome. (p. 893)

(d)   Translation does not begin immediately at the 5’ terminus of mRNA. Indeed, the first translated codon is nearly always more than 25 nucleotides from the 5’ end. (p. 894)

(e)    all known mRNA molecules contain signals that define the beginning and end of each encoded polypeptide chain. (p. 894)

(f)     The initiating codon in mRNA is AUG (methionine) or, much less frequently, GUG (valine). (p. 895)

(g)    30 nucleotide-long initiator regions in mRNAs contain the start codon.

(h)    In addition, each initiator region contains a purine-rich sequence centered about 10 nucleotides on the 5’ side of the initiator codon… The role of this purine-rich region (called the Shine-Delgarno sequence) became evident when the sequence of 16S rRNA was elucidated. The 3’ end of this RNA component of the 30S subunit contains a sequence of several bases that is complementary to the purine-rich region in the initiator sites of mRNA. (p. 895)

(G)           Initiation

(a)    …two kinds of interactions determine where protein synthesis starts: the pairing of mRNA bases with the 3’ end of 16S rRNA, and the pairing of the initator codon on mRNA with the anticodon of fMet initiator tRNA. (p. 896)

(b)   How are mRNA and formylmethionyl-tRNAf brought together to initiate protein synthesis? Three protein initiation factors (IF1, IF2, and IF3) are essential. (p. 896)

(c)    …protein synthesis in bacteria starts with N-formylmethionine (p. 894)

(d)   See Figure 34-26 (p. 894).

(e)   

(f)     Note how the amino terminus essentially is tagged by the formyl group.

(H)           Elongation

(a)    …for the formation of a peptide bond… This reaction is catalyzed by peptidyl transferase, an enzymatic activity of the 50S subunit. (p. 899)

(b)   [formation of] a peptide bond is a thermodynamically favorable reaction; the free-energy cost of making a peptide bond was paid earlier in forming an aminoacyl-tRNA. (p. 900)

(c)    Peptide-bond formation is accompanied by a change in the interactions of both tRNAs with the 50S subunit but not with the 30S subunit (Figure 34-34). The deacylated tRNA now occupies the E (exit) site on the 50S subunit while staying in the P site on the 30S subunit. The new dipeptidyl-tRNA occupies the P site on the 30S subunit. The new dipeptidyl-tRNA occupies the P site on the 50S subunit while staying in the A site on the 30S subunit. The next phase of the elongation cycle is translocation. Three movements occur: the deacylated tRNA moves from the A site on the 30S subunit to the P site on the 30S subunit, and mRNA moves a distance of three nucleotides. The result is that the next codon is positioned for reading by the incoming aminoacyl-tRNA. (p. 900)

(d)   Elongation is driven forward by GTP hydrolysis.

(e)    After translocation, the A site is empty, ready to bind an aminoacyl-tRNA to start another round of elongation. The filling of the A site induces the release of deacylated tRNA from the E site; the A and E sites cannot be simultaneously occupied. (p. 900)

(f)     See Figure 34-34 (p. 901)

(I)              Termination

(a)    Protein synthesis is terminated by release factors that read stop codons. (p. 901)

(b)   Aminoacyl-tRNA does not normally bind to the A site of a ribosome if the codon is [a stop codon]. (p. 901)

(c)    The specificity of peptidyl transferase is altered by the release factor so that water rather than an amino group is the acceptor of the activated peptidyl moiety. The detached polypeptide chain leaves the ribosome, followed by tRNA and mRNA. Finally, the ribosome dissociates into 30S and 50S subunits as the prelude to the synthesis of another protein molecule. (pp. 901-902)

(J)              Importance of Aminoacyl tRNA Synthetases in Authenticity...

(a)    Some synthetases recognize their tRNA partner primarily on the basis of its anticodon. (p. 884)

(b)   Aminoacyl-tRNA synthetases are highly selective in their recognition of both the amino acid to be activated and the prospective tRNA acceptor… tRNA molecules that accept different amino acids have different base sequences, and so they can be readily distinguished by their synthetases. A much more demanding task for these enzymes is to discriminate between similar amino acids. (p. 883)

(c)    The synthetases do this by

·        complementarily binding to amino acid R groups

·        possessing pockets capable of distinguishing sterically similar amino acids, and

·        hydrolyzing off mistakenly activated amino acids.

(d)   …the synthetase corrects its own errors. (p. 883)

(K)           Action of Puromycin...

(a)    The antibiotic puromycin inhibits protein synthesis by releasing nascent polypeptide chains before their synthesis is completed. Puromycin is an analog of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA. It binds to the A site on the ribosome and inhibits the entry of aminoacyl-tRNA. Furhtermore, puromycin contains an a-amino group. This amino group, like the one on aminoacyl-tRNA, forms a peptide bond with the carboxyl group of the growing peptide chain in a reaction that is catalyzed by peptidyl transferase. The product is a peptide having a covalently attached puromycin residue at its carboxyl end. Peptidyl-puromycin then dissociates from the ribosome. (p. 902)

(24)           Topic 22 (Control of Gene Expression) [Bio 113 chapter 18]

(A)           Lactose Operon

(a)    Gene activity is regulated primarily at the level of transcription. In bacteria, many genes are clustered in units called operons. The coordinate transcription of genes in an operon is blocked by repressor proteins and activated by stimulatory proteins. (p. 949)

(b)   The presence of lactose in a culture medium induces a large increase in the amount of b-galactosidase in E. coli by eliciting the synthesis of new enzyme molecules rather than by activating a proenzyme. Hence, b-galactosidase is an inducible enzyme. Two other proteins are synthesized in concert with b-galactosidase—namely, galactoside permease and thiogalacoside transacetylase. The permease is required for the transport of lactose across the bacterial cell membrane. (p. 950)

(c)    Study of the lac operon is typically done in terms of b-galactosidase activity.

(d)   Within an E. coli cell, the physiologic inducer is allolactose, which is formed from lactose by transglycosylation. The synthesis of allolactose is catalyzed by the few b-galactosidase molecules that are present prior to induction. Studies of synthetic inducers showed that some b-galactosides are inducers without being substrates of b-galactosidase, whereas other compounds such as lactose are substrates without being inducers. For example, isopropylthiogalactoside (IPTG) is a nonmetabolizable inducer. (p. 950)

(e)    IPTG is a very commonly employed synthetic inducer.

(B)            Jacod and Monod

(a)    Francois Jacob and Jacques Monod deducted that the rate of synthesis of these three proteins [coded by the lac operon] is normally governed by a common element that is different from the genes specifying their structures. (p. 951)

(C)           Escherichia coli Constitutive Mutants

(a)      constitutive mutants synthesize large amounts of b-galactosidase, the permease, and the transacetylase whether or not inducer is present. (p. 950)

(b)   b-galactosidase, the permease, and the transacetylase are encoded by three contiguous genes, called z, y, and a. (p. 950)

(c)     Francois Jacob and Jacques Monod deducted that the rate of synthesis of these three proteins [coded by the lac operon] is normally governed by a common element that is different from the genes specifying their structures. The gene for this common regularatory element was named i. Wild-type inducible bacteria have the genotype i+ z+ y+ a+, whereas the constitutive lactose mutants have the genotype  i  z+ y+ a+. (pp. 950-951)

(D)           On-Off Switch

(a)    How does the i+ gene affect the rate of synthesis of the proteins encoded by the z, y, and a genes? The simplest hypothesis was that the i+ gene determines the synthesis of a cytoplasmic substance called a repressor, which is missing or inactive in the i mutants. (p. 951)

(b)   a diffusible repressor is specified by the i+ gene. A diffusible repressor is an example of a trans-acting factor, one that is encoded by a locus on a DNA molecule different from the one containing its target. (p. 951)

(E)            Elements of the Operon

(a)    An operon is a coordinated unit of gene expression. (p. 951)

(b)   The genetic elements of [an operon] are a regulatory gene, and operator site, and a set of structural genes.

(c)    See Figure 36-5 (p. 951)

(d)   The Lac operon - showing its genes and its binding sites:

(e)    The regulator gene produces a repressor that can interact with the operator. Subsequent work revealed that the repressor is a protein. The operator, by contrast, is a DNA segment adjacent to the structural genes it controls. The binding of the repressor to the operator prevents the transcription of these genes. The operator and its associated structural genes are called an operon. For the lactose operon, the i gene is the regulator gene, o is the operator, and the z, y, and a genes are the structural genes. The operon also contains a promoter site (denoted by p) for the binding of RNA polymerase. This site for initiation of transcription is next to the operator. An inducer such as IPTG binds to the repressor, which prevents it from interacting with the operator. The z, y, and a genes can then be transcribed to give a single mRNA molecule that codes for all three proteins. An mRNA molecule coding for more than one protein is known as a polygenic (or polycistronic) transcript. (p. 951-952)

(F)            Repressor Protein

(a)    See figure 36-6 (p. 952).

(b)   In the repressed state the “repressor” is bound to the operator:

(c)    Note that the repressor blocks the action of RNA polymerase (i.e., it blocks transcription).

(d)   The helix-turn-helix structure contained within the lac repressor protein (Figure 26-1, p. 949) is a common motif associated with DNA binding proteins. In the following image you can see four helix-turn-helix motifs (lac repressor is a homotetramer) colored pink, green, red, and yellow that  interacting with two blue DNA molecules in the upper third of the image. I’ve also blown up one of these DNA molecules interacting with these helix-turn-helix motifs for closer inspection (second figure below).

(e)     

(f)    

(G)           Allolactose Inducer

(a)    In the "induced" state, the lac repressor is NOT bound to the operator site:

(b)   Note that under normal conditions within a cell it is allolactose that serves as the inducer.

(c)    Note that it is the binding of the inducer to the repressor that results in the inactivation of the repressor.

(d)   Consequently, the action of RNA polymerase is no longer blocked, thus resulting in transcription of the operon, thus resulting in the expression of the three structural genes.

(e)    See the figures at the lower left of p. 950 for a diagram of allolactose. No need to memorize the structure.

(H)           Role of cAMP

(a)    Cyclic AMP (cAMP) also plays a role in controlling lac operon gene expression.

(b)   It has long been known that E. coli grown on glucose, a preferred energy source, have very low levels of catabolic enzymes, such as b-galactosidase, galactokinase, arabinose isomerase, and tryptophanase. Clearly, it would be wasteful to synthesize these enzymes when glucose is abundant. The molecular basis of this inhibitory effect of glucose, called catabolite repression, has been elucidated. A key clue was the observation that glucose lowers the concentration of cyclic AMP in E. coli. It was then found that exogenous cyclic AMP can relieve the repression exerted by glucose [presumably because the cyclic AMP is taken up by E. coli cells into their cytoplasm]. Subsequent biochemical and genetic studies revealed that cyclic AMP stimulates the concerted initiation of transcription of many inducible operons. (p. 953)

(I)              CAP Protein

(a)    CAMP binds to CAP (the catabolite gene activator protein). (p. 953)

(b)   The complex of CAP and cAMP, but not CAP alone, stimulates transcription by binding to certain promoter sites. (p. 953)

(c)    CAP stimulates the initiation of lac mRNA synthesis by a factor of 50. How? The contiguous and nonoverlapping arrangement of the binding sites for CAP and RNA polymerase suggested that the binding of CAP to DNA creates an additional interaction site for RNA polymerase. Indeed, the binding of RNA polymerase to the promoter is enhanced by its energetically favorable contacts with bound CAP. (p. 954)

(d)   Like the lac repressor, CAP also contains helix-turn-helix motifs.

(e)    Cyclic AMP, a hunger signal, stimulates the transcription of many catabolic operons by binding to the catabolite gene activator protein (CAP). The binding of cAMP-CAP to a specific site in the promoter region of these inducible catabolic operons enhances the binding of RNA polymerase and the initiation of transcription. Full expression of the lac operon requires both a galactoside inducer and cAMP, [the latter] which is formed when glucose is scarce. (summary)

(f)     This diagram gives an indication of the functioning of the CAP protein; note that for the CAP protein to act to increase rates of transcription at the lac operon both cAMP must be present and bound and the lac repressor must not be bound to O:  

(g)    There is an additional level of transcriptional control of gene expression for the lac operon. This involves CAP, which more recently has been renamed the cAMP receptor protein (CRP). The bacteria appear to "prefer" to use glucose as an energy source rather than lactose. So, when both are present, the lac operon does not make much messenger RNA. However, if glucose is absent, a small regulatory molecule, cyclic AMP, is made and binds to the CRP. That complex activates transcription at the lac operon. Thus the lac operon is activated by cyclic AMP-CRP and is inhibited by the repressor in the absence of lactose. There is a dual control, one positive and the second negative. Unlike the lac repressor, the CRP is active on several different operons in the bacteria. (http://www.blackwellscience.com/11thhour/wilson/about/u2ch14.html)

Alternative (and less-detailed) Syllabus, Biochemistry as taught in Columbus

(proof that not all biochemistry course are created equal?)

 

#

Lecture #

Topic

Chapter

1

1

Water, biomolecules & their intereactions

01

2

2, 3, 4, 5, 6, 7, 8

Protein structure, function & characteristics

02 & 03

3

9, 10, 11

Myoglobin, hemoglobin & O2 transport

07

4

12, 13, 14

Enyzmes: basic concepts

08

5

15, 16

Enyzyme mechanisms

09

6

17, 18

Enzyme regulation

10

7

19, 20

Carbohydrates

18

8

21, 22

RNA & DNA structures

04

9

23, 24, 25

The flow of genetic information

05

10

26, 27

Membrane structure & dynamics

11

11

28, 29, 30

Metabolism & energy

17

12

31, 32, 33

Glycolysis

19

13

34, 35, 36

Citric acid cycle

20

14

37, 38

Oxidative phosphorylation

21

15

39, 40

Pentose phosphate pathway & gluconeogenesis

22

16

41, 42, 43

Glycogen metabolism

23

17

44, 45, 46

Fatty acid metabolism

24

18

47, 48, 49, 50

Amino acid metabolism

25 & 28

 

Same as above but sorted by chapter numbers:

 

#

Lecture #

Topic

Chapter

1

1

Water, biomolecules & their intereactions

01

2

2, 3, 4, 5, 6, 7, 8

Protein structure, function & characteristics

02 & 03

8

21, 22

RNA & DNA structures

04

9

23, 24, 25

The flow of genetic information

05

3

9, 10, 11

Myoglobin, hemoglobin & O2 transport

07

4

12, 13, 14

Enyzmes: basic concepts

08

5

15, 16

Enyzyme mechanisms

09

6

17, 18

Enzyme regulation

10

10

26, 27

Membrane structure & dynamics

11

11

28, 29, 30

Metabolism & energy

17

7

19, 20

Carbohydrates

18

12

31, 32, 33

Glycolysis

19

13

34, 35, 36

Citric acid cycle

20

14

37, 38

Oxidative phosphorylation

21

15

39, 40

Pentose phosphate pathway & gluconeogenesis

22

16

41, 42, 43

Glycogen metabolism

23

17

44, 45, 46

Fatty acid metabolism

24

18

47, 48, 49, 50

Amino acid metabolism

25 & 28

 

Messages to Sta  

 

01051301

Note that we did four quizzes this quarter: The amino acids, the carbohydrates, glycolysis, and the Kreb’s cycle. By far and away the Kreb’s cycle was, for some reason, the hardest.

 

Messages to Billy Link Jr.       

 

01052001

For Tuesday’s lecture we will just consider the material found on pages 793 (DNA ligase) and 799-810 (DNA replication). (topic 19)

01052001

For Wednesday’s (or Thursday’s) lecture we will just cover the material found in chapter 34  (topic 21); we will not be reading/covering chapter 33.

01051001

Good job, re: Krebs!

01050801

We will be doing chapters 22 and 23 and Thursday (not just chapter 22). Focus on Gluconeogenesis in Chapter 22.

01042501

A slight variation on the below exam scheme: Chapter 2, 3, 8, 9, 11, 17, and 18 will all count as one-part each. Chapter 7 will count as two parts. Chapters 4 and 5 will count as a total of two parts, but I will not distinguish which of the two chapters will be placed in which part. Thus, there will be 11 parts to the exam, with 7 of those parts corresponding to a single chapter each, another two parts corresponding to only a single chapter, and then yet another two parts that will, as a whole, be associated with two chapters but without distinguishing among these two chapters. Anyway, each part will have some number of questions associated with it and that number may not be the same per part. I still haven't figured out how long an exam this is going to be.

01042201

Note to self and Billy: I really didn’t read past p. 517 of chapter 20. I’m getting lazy, I guess.

01042201

Note that I am going to divide the exam into 11 parts where each chapter covered (2, 3, 4, 5, 7, 8, 9, 11, 17, & 18) will be treated as one part (except chapters 2 and 3 which will be worth two parts together to save me from having to separate out the different parts of the topic), and an additional part will be associated with chapter 7 (thus making that chapter worth twice as much as the other chapter—this is to reflect both the importance of the chapter and the extra weight given to the chapter in the detailed syllabus). Each part will be worth the same amount, the number of questions associated with each part may vary, as likely will the types of questions asked. Thus, each question on the exam will not be worth the same amount!!!

01042101

We will be having a quiz, during week six, on glycolysis, including all enzyme names; intermediate, substrate and product names; and all structures. We’ll then probably have to have a quiz on the citric acid cycle week seven.

01041401

I’ve updated the notes through chapter 18 (the carbohydrates). There actually is not all that much that has been added (relative to our discussion of enzyme kinetics, for example). I am going to spend some time on Saturday going through Jennifer’s lecture notes to see if I have missed including anything vitally important. These will be in green font if there is anything.

 

Are you aware that as a Word document these notes are currently 54 pages long!?!

01041301

There is some new stuff added to topic 9K-on.

01041101

Chapter 11 on Tuesday, Chapter 18 on Wednesday, Chapter 18 structure "quiz" on Thursday and Chapter 17 read over weekend.

01041101

Some time next week is going to be a carbohydrate-structure memorization day. Stay tuned... D-Glyceraldehyde, L-Glyceraldehyde, Dihydroxyacetone, D-Glucose, D-Fructose, D-Galactose (see pp. 464-465), see ring forms of glucose and fructose on p. 467 and how these are formed, top of p. 470 (cellulose), figure 18-10, figure 18-13

01040401

Tuesday April 10, let's shoot for a "quiz" on the various structures associated with nucleic acids including the pyrimidine and purine basic structures; Adenine, Thymine, Guanine, Cytocine, and Uracil (all with carbons numbered); the sugar-phosphate backbone; ribose and deoxyribose with carbons numbered; H-bonding; the difference between a nucleoside and a nucleotide.

01040301

Work on having the structures on page 76 committed to memory as well as the base pairing on page 81. See the Base Pairing figure I imported (below). It may be easier to understand/memorize.

01040301

OK, so when should have the midterm? I've made one proposal below. Note that this says nothing about the date, only where we should be in the text. We should try to have the first 12 chapters covered by the end of the fourth week. Is this possible? I think if would mean be through chapter 9 by the end of next week (Egad!).

01032701

If you come up with any good mnemonics, please let me know about them.

01032501

Let us consider getting together for chapter reading/understanding sessions in my office during which you read (preferably for the second time) a given chapter while I attempt, on the computer, to reconcile the chapter with the detailed syllabus.

01031701

For Wednesday (March 27) let's shoot, minimally, for having chapter 2 read and the 20 amino acid names, structures, 3-letter abbreviations, 1-letter abbreviations, and general properties of each R group (e.g., hydrophobic) memorized. I'll see if you (we) can recite (and draw) them Wednesday afternoon from memory. Think big matrix with amino acids as rows and the various descriptions as columns. See table below.

01030001

For Tuesday (March 26) let's have chapter 1 read. We can discuss it while we clean the lab.

Grades       

 



[1] Single-letter abbreviations in bold are the ones that are other than the first letter of the amino acid name

[2] The one-letter abbreviations are useful for creating mnemonics

[3] Three-letter abbreviations in bold are the ones that are other than the first three letters of the amino acid name

[4] Need to memorize the structures as presented in the text; this is most readily accomplished by memorizing the R groups (except for Proline for which the entire structure must be memorized)

[5] "pK values depend on temperature, ionic strength, and the microenvironment of the ionizable group." (p. 23, Table 2-1)

[6] Hydrophobic = Aliphatic

[7] Yes, this is one that I made up

[8] Good question: It indicated that something is limiting in these reactions other than substrate. If we consider all interactions within biological systems to require chemical interaction, which minimally involves the intimate touching of molecules and ions, then we might posit that substrate must contact something that is in limited supply in order for reactions to mover forward at a given rate, that whatever it is that they must touch must be in limited supply, that the touching occurs for a some finite interval (not infinitely short in length), and therefore that this something must be saturable. The touching between the substrate and this something we might term a something-substrate complex. Since varying the amount of enzyme appears to change the amount of this something that is found within the reaction environment, we infer that the something is an enzyme and therefore that an enzyme-substrate complex must form for reactions to go forward at the observed rate. We might also infer, from its impact on reaction kinetics, that it is the rate formation of the enzyme-substrate limits the rate of an enzyme-catalyzed reaction below substrate saturation, and further that is the duration of contact between substrate and enzyme (turnover number) that limits the rate of reactions near and at substrate saturation.