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Chapt. 8 Enzymes as catalysts

Chapt. 8 Enzymes as catalysts

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Chapt. 8 Enzymes as catalysts

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  1. Chapt. 8 Enzymes as catalysts • Ch. 8 Enzymes as catalysts • Student Learning Outcomes: • Explain general features of enzymes as catalysts: Substrate -> Product • Describe nature of catalytic sites • general mechanisms • Describe how enzymes lower activation energy of reaction • Explain how drugs and toxins inhibit enzymes • Describe 6 categories of enzymes

  2. Catalytic power of enzymes • Enzymes do not invent new reactions • Enzymes do not change possibility of reaction to occur (energetics) • Enzymes increase the rate of reaction by factor of 1011 or higher Fig. 8.1 box of golfballs, effect of browning enzyme

  3. Enzymes catalyze reactions • Enzymes provide speed, specificity and regulatory control to reactions • Enzymes are highly specific for biochemical reaction catalyzed (and often particular substrate) • Enzymes are usually proteins • (also some RNAs = ribozymes) • E + S ↔ ES binding substrate • ES ↔ EP substrate converted to bound product • EP ↔ E + P release of product

  4. Glucokinase is a typical enzyme • Glucokinase is typical enzyme: • ATP: D-glucose 6-phosphotransferase • Very specific for glucose • Not phosphorylate other hexoses • Only uses ATP, not other NTP • 3D shape of enzyme critical for its function (derived from aa sequence) Fig. 8.2 glucokinase

  5. A. Active site of enzyme • Enzyme active site does catalysis • Substrate binds cleft formed by aa of enzyme • Functional groups of enzyme, also cofactors bond to substrate, perform the catalysis; Fig. 8.4

  6. B. Binding site specificity • Substrate binding site is highly specific • ‘Lock-and-key’ model: 3D shape ‘recognizes substrate (hydrophobic, electrostatic, hydrogen bonds) • ‘Induced-fit’ model: enzyme conformational change after binding substrate • galactose differs from • glucose, needs separate • galactokinase Fig. 8.5 glucokinase

  7. Glucokinase conformational change • Conformation change of glucokinase on binding glucose • Binding positions substrate to promote reactions • Large conformational change adjusts actin fold, and facilitates ATP binding • Actin fold named for G-actin (where first described; Fig. 7.8) Fig. 8.6 glucokinase (Yeast hexokinase)

  8. Transition state complex • Energy Diagram: substrates are activated to react: • Activation energy: barrier to spontaneous reaction • Enzyme lowers activation energy • Transition-state complex is stabilized by diverse interactions Fig. 8.7

  9. Transition-state complex • Transition-state complex binds enzyme tightly: • transition-state analogs are potent inhibitors of enzymes (more than substrate analogs) • make prodrugs that convert to active analogs at site of action • Abzymes: catalytic antibodies that have aa in variable region like active site of transition enzyme: • Artificial enzymes: catalyze reaction • Ex. Abzyme to Cocaine esterase destroys cocaine in body

  10. II. Catalytic mechanism of chymotrypsin - example enzyme • Chymotrypsin, serine protease, digestive enzyme: • Hydrolyzes peptide bond (no reaction without enzyme) • Serine forms covalent intermediate • Unstable oxyanion (O-) intermediate • Cleaved bond is • scissile bond Fig. 8.8

  11. B. Catalytic mechanism of chymotrypsin • 1. Specificity of binding: • Tyr, Phe, Trp on denatured proteins • Oxyanion tetrahedral intermediate • His57, Ser195, Asp • 2. acyl-enzyme intermediate • 3. Hydrolysis of acyl-enzyme intermediate Fig. 8.9

  12. Mechanism of chymotrypsin, cont. • 3. Hydrolysis of acyl-enzyme intermediate • Released peptide product • Restores enzyme Fig. 8.9

  13. Energy diagram revisited with detail • Chymotrypsin reaction has several transitions: • See several steps • Lower energy barrier to uncatalyzed Fig. 8.10

  14. III. Functional groups in catalysis • Functional groups in catalysis: • All enzymes stabilize transition state by electrostatic • Not all enzymes form covalent intermediates • Some enzymes use aa of active site (Table 1): • Ser, Lys, His - covalent links • His - acid-base catalysis • peptide backbone – NH stabilize anion • Others use cofactors (nonprotein): • Coenzymes (assist, not active on own) • Metal ions (Mg2+, Zn2+, Fe2+) • Metallocoenzymes (Fe2+-heme)

  15. Coenzymes assist catalysis • Activation-transfer coenzymes: • Covalent bond to part of substrate; enzyme completes • Other part of coenzyme binds to the enzyme • Ex. Thiamine pyrophosphate is derived from vitamin thiamine; • works with many different enzymes • enzB takes H from TPP; carbanion attacks keto substrate, splits CO2 Fig. 8.11

  16. Other activation-transfer coenzymes • Activation-transfer coenzymes: • Specific chemical group binds enzyme • Other functional group participates directly in reaction • Depends on enzyme for specificity of substrate, catalysis Fig. 8.12 A CoA forms thioesters with many acyl groups: acetyl, succinyl, fatty acids

  17. Oxidation-reduction coenzymes • Oxidoreductase enzymes use other coenzymes: • Oxidation is loss of electrons (loss H, or gain O) • Reduction is gain electrons (gain H, loss of O) • Redox coenzymes do not form covalent bond to substrate • Unique functional groups • NAD+ (and FAD) special • role for ATP generation: • Ex. Lactate dehydrogenase • oxidizes lactate to pyruvate • transfers e- & H: to NAD+ -> NADH Fig. 8.13 lactate dehydrogenase

  18. Metal ions assist in catalysis • Positive metal ions attract electrons: contribute • Mg2+ often bind PO4, ATP; ex. DNA polymerases • Some metals bind anionic substrates • Fig. 8.14 • ADH alcohol dehydrogenase • oxidizes alcohol to acetaldehyde • and NAD+ to NADH • Zn2+ assists with NAD+ • (In Lactate dehydrogenase, a His residue assisted the reaction)

  19. pH affects enzyme activity • Each enzyme has characteristic pH optimum: • Depends on active-site amino acids • Depends on H bonds required for 3D structure • Each enzyme has optimum • temperature for activity: • Humans 37oC • Taq polymerase • for PCR: 72oC Fig. 8.15 optimal pH for enzyme

  20. V. Mechanism-based inhibitors • Inhibitors decrease rate of enzyme reaction: • Mechanism-based inhibitors mimic or participate in intermediate step of reaction; • Covalent inhibitors • Transition-state analogs • Heavy metals Fig. 8.2 organophosphate inhibitors include two insecticides, and nerve gas Sarin

  21. Covalent inhibitors • Covalent inhibitors form covalent or very tight bonds with functional groups in active site: Fig. 8.16 DFP di-isopropylfluorophosphate prevents acetylcholinesterase from degrading acetylcholine

  22. Transition state analogs • Transition-state analogs bind more tightly to enzyme than substrate or product: • Penicillin inhibits glycopeptidyl transferase, enzyme that synthesizes cross-links in bacterial cell wall. • Kills growing cells by inactivating enzyme Fig. 8.17 penicillin

  23. Allopurinol treats gout • Allopurinol is suicide inhibitor of xanthine oxidase: • Treatment for gout (decreases formation of urate) Fig. 8.18

  24. Basic reactions and classes of enzymes • 6 basic classes of enzymes: • Oxidoreductases • Oxidation-reduction reactions (one gains, one loses e-) • Transferases • Group transfer – functional group from one to another • Hydrolases cleaveC-O, C-N and C-S bonds • addition of H2O in form of OH- and H+ • Lyases diverse cleave C-C, C-O, C-N • Isomerases rearrange, create isomers of starting • Ligases synthesize C-C, C-S, C-O and C-N bonds; • Reactions often use cleavage of ATP or others

  25. Some example enzymes • Example enzymes: • Group transfer • – transamination • transfer of amino group • Isomerase • – rearranges atoms • ex. In glycolysis Fig. 8.19

  26. Key concepts • Enzymes are proteins (or RNA) that are catalysts • accelerate rate of reaction • Enzymes are very specific or substrate • Enzymes lower energy of activation – to reach high-energy intermediate state • Functional groups at active site (amino acid residues, metals, coenzymes) cause catalysis • Mechanisms of catalysis include: acid-base, formation covalent intermediates, transition state stabilization

  27. Review questions • 4. The reaction shown fits into which classification? • Group transfer • Isomerization • Carbon-carbon bond breaking • Carbon-carbon bond formation • Oxidation-reduction • 5. The type of enzyme that catalyzes • this reaction is which of the following? • Kinase • Dehydrogenase • Glycosyltransferase • Transaminase • isomerase