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 V_max is the rate when the enzyme is fully saturated with substrate, and K_M, 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 K_M = [S] at 1/2 V_max (as discussed below). That is, when S = K_M then K_M / S = 1 and V = V_max / (1 + 1) = V_max / 2.

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

(e) The kinetic constant k_3, 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, K_M.

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

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

(k) K_M 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 K_M, [S] (substrate concentration), and [E_T] (total amount of enzyme). Substituting V_max, 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 K_M, the velocity of the reaction is half the maximum velocity. Put another way, K_M is the substrate concentration required for the reaction to reach half its maximum velocity.

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

· A high K_M means that [S] must be relatively high to achieve 1/2 V_max, 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 K_M means that [S] need only be relatively low to achieve 1/2 V_max, 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), K_M reduces to k₂/k₁, the dissociation constant of the enzyme-substrate complex. Under these conditions, a low value for K_M indicates a high affinity of E for S and a high K_M indicates a low affinity-thus under certain conditions the value of K_M 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 V_max = k₃[E_T] (equation 27), so if we know the concentration of enzyme and determine V_max 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 V_max = k₃[E_T] 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 ([E_T]).

· Here E_T 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 K_M (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) k_cat/K_M ratio. For more complex reactions, the limit on reaction velocity is determined by the ratio of several rate constants (denoted k_cat) to K_M.

· 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 k_cat/K_M 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 K_M and V_max 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 V_max 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/V_max and a slope equal to K_M/V_max.

· 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 K_M 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 V_max) is the same in the presence or absence of inhibitor. The slope increases as the amount of inhibitor increases, showing that the apparent K_M increases: more substrate is required to bring the reaction to half V_max.

· 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 V_max 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.

· K_M 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, V_max is lowered as the concentration of inhibitor increases. K_M is not affected.

· See Figure 8-21, p. 198. Note that this is saying that the absolute rate of catalysis, V_max, has actually been reduced (Y intercept is higher which, since this is a reciprocal, means that V_max 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 P_i 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)