Two requirements for life: Self-replication and appropriate catalysis. A. Most enzymes (def.: biological catalysts) are proteins

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1 Enzymes We must be able to enhance the rates of many physical and chemical processes to remain alive and healthy. Support for that assertion: Maladies of genetic origin. Examples: Sickle-cell anemia (physical) & PKU and hemophilia (chemical). Two requirements for life: Self-replication and appropriate catalysis. I. An Introduction to Enzymes (History, 1700 now) A. Most enzymes (def.: biological catalysts) are proteins 1. RNA (ribosomes, hammerhead ribozymes) 2. Protein based enzymes vs. ribozymes? 1

2 3. Cofactors (a.k.a.: prosthetic group) a) inorganic (Table 6-1, p. 190) b) organic/organometallic (Table 6-2) 4. Apoenzyme + cofactor = holoenzyme In most cases, an apoenzyme is devoid of activity. i.e., The cofactor must be present for the enzyme to have any function. B. Enzymes are classified by type of rxn. catalyzed 2

3 II. How Enzymes Work (substrate, active site) A. Enzymes affect rxn. rates, not equilibria (Fig. 6-2, 3) 1. ÄG relation to K (K = )? How does the graph below (right) illustrate that a catalyst does not alter K eq? How does the graph below (right) illustrate that a catalyst decreases k f and k r equally? 3

4 2. ÄG (a.k.a. ) relation to k (k = )? 3. Think back to organic chem. reaction mechanisms a) intermediates b) rate limiting step B. Rates & equilibria have unambiguous (quantitative) definitions. 1. Re. K under standard conditions a) K = [P] [S] means ref to ph 7 b) ÄG = RT ln K See Table Re. k a) from CHEM. 112 for A + B C + D: rate = k [A] x [B] y b) for enzyme catalyzed rxns.: rate = k [S] 4

5 k = (kt h) e ÄG RT k = Boltzmann constant h = Plank s constant T = What happened to ñ? Do you remember ñ? We will find out that some enzymes have major effects on ñ. 5

6 C. A Few Principles Explain the Catalytic Power and Specificity of Enzymes. 1. Binding energy (weak interactions between S and E) creates much of the catalytic efficiency. 2. Enzymes tend to bind (stabilize) transition state species (more than they do reactants or products). D. Weak Interactions Between Enzyme & Substrate Are Optimized In the Transition State. Look at stickase. Fig Reaction: 2. Energetic effect of binding straight stick? 3. Energetic effect of binding bent stick? 6

7 Fig 6-4 Binding pocket- substrate complimentarity pdb: 1ra2 See the Ligands tab for both ligands. 7

8 E. Binding Energy Contributes to Reaction Specificity and Catalysis. What does that mean? 1. Specificity: a) Charging trna Ile w/ Ile, not Val (see p. 75) 8

9 b) Hydroxylation at 11â, not 11á (or 17) site of steroids: 9

10 Rate enhancements 2. Entropy reduction: holding substrates: enhances rate of favorable collisions, giving: a) Regiospecificity b) Stereospecificity See Fig. 6-7, p. 198 Check with models? c) Arrhenius ideas i. Collision frequency ii. ñ 10

11 3. Desolvation- often less obvious, less easy to understand. Having groups on the enzyme sub for water in interactions with substrate. This creates a non-aqueous environment inside the enzyme. (Imagine a circumstance where water could engage in a competing side rxn.) 4. Distortion of substrates (stickase analogy, reaching transition state) 5. Proper alignment of catalytic groups is a must. Chymotrypsin will demonstrate this nicely. 11

12 F. Specific Catalytic Groups Contribute to Catalysis (That sounds a little cyclic???) 1. Acid-base catalysis a) Specific (H + and OH from water solvent) b) General (source other than water) c) Competing rxns. Outcome is determined by their relative rates. Fig. 6-8, p Amino acid side chains that are employed in general acid-base catalysis. 12

13 2. Covalent catalysis H 2 O uncat: A B A + B H 2 O cat: A B + X: A X + B A + X: + B Ser 195 of chymotrypsin is an archetypal example of covalent catalysis. Can you imagine another amino acid residue that might engage in this sort of activity? 3. Metal ion catalysis a) Charge stabilization (example: enolase) b) Redox activity eg.: Fe 2+ Fe 3+ + e 13

14 G. Nice summary 1. Enzymes: remarkably effective catalysts ( rate enhancements) 2. Mechanism involves formation of ES complex 3. Catalysts function by providing an alternative rxn. pathway with a lower ÄG than uncatalyzed rxn. 4. Weak interactions, binding energy (ÄG B ), induced fit 5. Additional tricks: acid-base, covalent, metal ion 14

15 III. Enzyme Kinetics as an Approach to Understanding Mechanism Back to CHEM 112? A. [S] affects the rate of enzyme-catalyzed rxns. 1. Graphically, Figs. 6-10, 11 This is a standard enzyme catalytic isotherm???? 15

16 2. I hope that looks familiar & you are pondering (have figured out?) why! k 1 k 2 E + S ES ES E + P k 1 k 2 3. Steady state vs. pre-steady state kinetics. 4. Comment on initial rate, V 0. 16

17 B. The rate vs. [S] relationship can be expressed quantitatively. Michaelis-Menton derivation. 1. Getting started: V 0 = k 2 [ES] Why? 2. Is [ES] easy to determine? 3. Steady state assumption. ES formation rate = ES breakdown rate. Depends on relative values of k s. 4. Outcome: V max [S] Definition of K m V 0 = Km + [S] 5. Comment: What happens when [S] = K m? Fig

18 6. Lineweaver-Burk, Box 6-1. C. Kinetic parameters (constants) are used to compare enzyme activities. (My enzyme s better than yours?) 1. Interpreting V max and K m a) K m is sometimes like a K d, and sometimes not. Table 6-6 b) k cat = V max [E T ] the turnover number 2. M-M becomes: k cat [E T ] [S] V 0 = Km + [S] 18

19 3. Comparing catalytic mechanisms and efficiencies: k cat K m specificity constant 4. At teeny [S], M-M becomes V 0 = (k cat K m ) [E T ][S] k cat K m looks like a 2nd order rate constant. 5. Think about diffusion limitations. Table 6-8. This means the enzyme has become so talented, it s functions are no longer rate limiting in the process. 19

20 D. Many enzymes catalyze rxns. with 2 substrates. Order of addition: required vs random? E. Pre-steady state kinetics can provide evidence for specific reaction steps. 1. Measuring parameters like k 1 directly. 2. Often requires expensive equipment and lots of E. F. Enzymes are subject to Reversible or Irreversible inhibition. 1. Reversible: a) Competitive: 20

21 V 0 = V max [S] ákm + [S] Where á = 1 + [I] K I & K I = [E] [I] [EI] i) What occurs re. á when [I] = K I? ii) What does that mean regarding [E]? iii) See Fig & Fig 1 in Box 1 re. double recip. iv) Fig re. interpretation of intersecting lines v) What does this mean when [S]? b) Uncompetitive (allosteric?) i) Fig (b) for model ii) Fig. 2 in Box 6-2 for double reciprocal plot iii) Why don t these lines intersect? iv) What does that mean in terms of the activity of different E species in the system? 21

22 2. Irreversible: a) Highly reactive compounds that (covalently) modify key groups at the active site. i) Nerve gas modifies. ii) DIFP, Fig b) Compounds that become highly reactive only when activated by key residues at the E active site: i) Names: mechanism-based inhibitors, suicide inactivators ii) Comment re. pharmaceuticals (antibiotics) Box 6-3!!!!!!!! G. Enzyme activity depends on ph. See Fig What is your interpretation in terms of changes occurring at the active site? 22

23 IV. Examples of Enzymatic Reactions Chymostrypsin: The E. coli of enzymes? A. Chymostrypsin: Acylation phase, deacylation phase Write reaction, view animation, think. See How to Read... on p (What does chymotrypsin do?) 1. Substrate binds to active site: E + S ES 2. Non-bonding e pair on hydroxyl O of Ser 195 attacks carbonyl C of peptide bond: ES E interm am 23

24 a) O of Ser 195 is (electrophilic or nucleophilic?) And what does that mean? b) Note oxyanion pocket c) This step is an example of catalysis. 3. H of His 57 forms covalent bond with amide N to form and release amine component of substrate: E interm am E acyl interm + H amine prod a) An example of catalysis. b) Comment on catalytic triad (Ser 195 -His 57 -Asp 102 ) 24

25 4. H 2 O is deprotonated by His 57, H 2 O + E HE + + OH (localized ph is?) a) An example of general (?) catalysis. b) Note proximity, OH release from E unlikely (entropy?) 5. OH attacks acyl C: E acyl interm + OH E short-lived interm a) Note: This is the 2nd time we have gone tetrahedral b) C with 3 bonds to O should look unstable 6. Short-lived intermediate collapses to form carboxyl: E short-lived interm E carboxyl prod 25

26 a) Note transfer of H from His 57 to Ser Carboxyl product dissociates to re-generate free E: E carboxyl prod E + carboxyl prod B. Hexokinase (What does hexokinase do?) C. Enolase (What does it do?) (What s an enol?) Write reaction, view animation, think. A somewhat simpler mechanism than chymostrypsin. 1. Lys 345 abstracts H + from C-2 of 2-phosphoglycerate. See Fig

27 An example of: catalysis. Is proton extraction from C normally facile? 2. This generates enolic intermediate: Is charge accumulation generally a stable arrangement? The 2 Mg 2+ ions present at the active site: a) Increase acidity of H at C 2 b) Stabilize charge associate with enolic intermediate 3. H from carboxylic acid group of Glu 211 (Does that bother you, particularly relative to the initial protonation state of Lys 345? Why? ) attacks OH on 27

28 C 3 of enolic intermediate to release H 2 O and phosphoenolpyruvate (PEP). An example of: catalysis. D. Lysozyme (What does lysozyme do?) (Where can you find it?) E. Enzymology & medicine/pharmacology. Fig 6-29 to

29 V. Regulatory Enzymes A. Allosteric (other site) enzymes change conformation when a modulator molecule binds. See Fig & 34. Some are complex. B. Allosteric regulation is common in enzymes catalyzing steps of a biochemical (synthetic) pathway. Fig Think for a moment about how your amino acid synthesis requirements change when you adopt (or disinherit) the Atkins Diet. Natural selection? 29

30 C. Kinetic properties of allosteric enzymes differ from simple Michaelis-Menton behavior. See Fig This should look familiar. D. Some enzymes are regulated by covalent modification. Most of the modifying substrates have other important functions in the cell (eg. ATP). See Fig

31 E. Addition/removal of phosphoryl groups alters the structure and activity of some proteins. 1. Classic example is the glycogen phosphorylase/glycogen synthetase system. 2. Most often phosphorylation occurs at S, T, and Y. 3. Protein kinases recognize specific sequences. Table F. Regulation by phosphorylation is sometimes not just on or off, but gradual change in speed: 1, or 2, or 3, 4, 5,... etc. Fig

32 G. Some enzymes are regulated by proteolytic cleavage. 1. Chymotrypsin, trypsin (zymogens) Fig Many others (clotting system: prothrombin) H. Blood Clotting I. Some enzymes are regulated by multiple mechanisms. Consider physiological and biochemical systems you are familiar with. How can regulation of these systems provide you with a selective advantage? 32