Enzyme Catalysis

1. Enzyme Catalysis • Please turn in your take-home part for midterm 1 BEFORE class • I will hand back in-class exams at the end of class today • NO class on THURSDAY 10/30/14, Jim will be back NEXT week and he will continue with Chapter 12 (Enzyme Kinetics) 28 October 2014 Katja Dove PhD Candidate, Department of Biochemistry, University of Washington Email: Katja.Dove@seattlecolleges.edu

2. How long would it take you to digest a fried breakfast without the help of digestive enzymes? 1 day 1 week 1 month 1 year 50 years Never

3. How long would it take you to digest a fried breakfast without the help of digestive enzymes? 1 day 1 week 1 month 1 year 50 years Never

4. How long would it take you to digest a fried breakfast without the help of digestive enzymes? 1 day 1 week 1 month 1 year 50 years Never What is a “spontaneous” reaction?

5. How long would it take you to digest a fried breakfast without the help of digestive enzymes? 1 day 1 week 1 month 1 year 50 years Never What is a “spontaneous” reaction? Free Energy (G) Reaction coordinate

6. How long would it take you to digest a fried breakfast without the help of digestive enzymes? 1 day 1 week 1 month 1 year 50 years Never What is a “spontaneous” reaction? Free Energy (G) ΔG < 0, for spontaneous reactions Reaction coordinate

7. How long would it take you to digest a fried breakfast without the help of digestive enzymes? 1 day 1 week 1 month 1 year 50 years Never What is a “spontaneous” reaction? ΔGǂactivation energy Free Energy (G) ΔG < 0, for spontaneous reactions Reaction coordinate

8. What are Enzymes? Proteins that perform biochemical reactions (really fast) R P Uncatalyzed (non-enzymatic) reaction: Catalyzed (enzymatic) reaction: S + E ES EP P + E Free Energy (G) Reaction coordinate

9. What are Enzymes? Proteins that perform biochemical reactions (really fast) R P Uncatalyzed (non-enzymatic) reaction: Catalyzed (enzymatic) reaction: S + E ES EP P + E • Terminology • S – substrates ( reactants (R) for enzymes) • ES – Enzyme-substrate complex • EP – Enzyme-product complex • P – products • Active site = substrate binding and transformation (aka business end)  specify rearrangement of amino acids Free Energy (G) Reaction coordinate

10. What are Enzymes? Proteins that perform biochemical reactions (really fast) R P Uncatalyzed (non-enzymatic) reaction: Catalyzed (enzymatic) reaction: S + E ES EP P + E • Terminology • S – substrates ( reactants (R) for enzymes) • ES – Enzyme-substrate complex • EP – Enzyme-product complex • P – products • Active site = substrate binding and transformation (aka business end)  specify rearrangement of amino acids Free Energy (G) • Enzymes = biological catalysts • speed up reactions by lowering activation Energy • DO NOT make reactions spontaneous (e.i. no influence on thermodynamics, but “only” kinetics) Reaction coordinate

11. Examples of Speediness Rate enhancement = rate catalyzed/rate uncatalyzed

12. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

13. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms Two-Step Reaction: Which one is the rate-limiting step? • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

14. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

15. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Temp below 100°C, neutral pH, atmospheric pressure • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

16. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Temp below 100°C, neutral pH, atmospheric pressure • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

17. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Temp below 100°C, neutral pH, atmospheric pressure • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

18. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Temp below 100°C, neutral pH, atmospheric pressure • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

19. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Temp below 100°C, neutral pH, atmospheric pressure • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

20. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Temp below 100°C, neutral pH, atmospheric pressure • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

21. General Properties of Enzymes Naming convention: -ases • Increase reaction rates • lowering the activation energy • High specificity • substrate binding (“Key & Lock”) • Stereospecific • Mild reaction conditions • Temp below 100°C, neutral pH, atmospheric pressure • Regenerated (not used up) • Cofactors (e.g. metals) • Capacity for regulation (e.g. glycolysis) • Drug targets (e.g. NSAIDs, antibiotics) Catalytic mechanisms • Covalent • Acid/Base • Metal ions • Proximity and orientation of reactants

22. Covalent Catalysis • Covalent mechanisms often need a nucleophile • uncatalyzed: A B A + B • catalyzed: A B + :X A X + B A + B + :X (:X)

23. Covalent Catalysis • Covalent mechanisms often need a nucleophile • uncatalyzed: A B A + B • catalyzed: A B + :X A X + B A + B + :X (:X)

24. Covalent Catalysis • Covalent mechanisms often need a nucleophile • uncatalyzed: A B A + B • catalyzed: A B + :X A X + B A + B + :X (:X)

25. Covalent Catalysis • Covalent mechanisms often need a nucleophile • uncatalyzed: A B A + B • catalyzed: A B + :X A X + B A + B + :X (:X)

26. Acid-Base catalysis • Enzymes use amino acid residues as proton acceptors/donor to aid in catalysis • Covalent catalysis: A B + :X A X + B A + B + :X • Acid-Base catalysis: A B + X H + :Y A X + B +Y HA + B + X H :Y +H+ Product #1 Products base substrate Regenerated active site of enzyme Conjugated base Nucleophile/acid Enzyme-intermediate complex

27. Acid-Base catalysis • Enzymes use amino acid residues as proton acceptors/donor to aid in catalysis • Covalent catalysis: A B + :X A X + B A + B + :X • Acid-Base catalysis: A B + X H + :Y A X + B +Y HA + B + X H :Y +H+ Product #1 Products base substrate Regenerated active site of enzyme Conjugated base Nucleophile/acid Enzyme-intermediate complex

28. Acid-Base catalysis • Enzymes use amino acid residues as proton acceptors/donor to aid in catalysis • Covalent catalysis: A B + :X A X + B A + B + :X • Acid-Base catalysis: A B + X H + :Y A X + B +Y HA + B + X H :Y +H+ Product #1 Products base substrate Regenerated active site of enzyme Conjugated base Nucleophile/acid Enzyme-intermediate complex

29. Acid-Base catalysis • Enzymes use amino acid residues as proton acceptors/donor to aid in catalysis • Covalent catalysis: A B + :X A X + B A + B + :X • Acid-Base catalysis: A B + X H + :Y A X + B +Y HA + B + X H :Y +H+ Product #1 Products base substrate Regenerated active site of enzyme Conjugated base Nucleophile/acid Enzyme-intermediate complex

30. Acid-Base catalysis • Enzymes use amino acid residues as proton acceptors/donor to aid in catalysis • Covalent catalysis: A B + :X A X + B A + B + :X • Acid-Base catalysis: A B + X H + :Y A X + B +Y HA + B + X H :Y +H+ Product #1 Products base substrate Regenerated active site of enzyme Conjugated acid Nucleophile/acid Enzyme-intermediate complex

31. Acid-Base catalysis • Enzymes use amino acid residues as proton acceptors/donor to aid in catalysis • Covalent catalysis: A B + :X A X + B A + B + :X • Acid-Base catalysis: A B + X H + :Y A X + B +Y HA + B + X H :Y +H+ Product #1 Products base substrate Regenerated active site of enzyme Conjugated acid Nucleophile/acid Enzyme-intermediate complex

32. Acid/Base reaction with Covalent Intermediate: Serine proteases • Proteases • Also called peptidase or proteinase • Perform proteolysis = breakdown of peptides by hydrolysis of peptides bonds • Serine proteases – Serine acts as nucleophile • Catalytic triad • Stabilize transition state (oxyanion hole)

33. 2 1

34. 2 1 Catalytic triad

35. 2 1 Catalytic triad

36. 2 1 Catalytic triad Oxyanion hole stabilizes intermediate

37. 2 1 3

38. 2 1 4 3

39. 4 5

40. 4 6 5

41. Flash-Back to 1st step 4 Enzymes are always regenerated! 6 5

42. Metal Ions Cofactors assist in Catalysis: Carbonic anhydrase: H2O+CO2 H+ + HCO3- • Metal Ions can: • Bind and orient substrates • Stabilize charged intermediate • Perform oxidation/reduction chemistry

43. Enzyme Inhibition • Many therapeutic drugs are enzyme inhibitors • Enzyme kinetics important to drug design (effectiveness) • Natural toxins are also enzyme inhibitors • Often Enzyme inhibitors either prevent/interfere with substrate binding OR lower catalytic activity of enzyme OR both

44. Classes of enzyme inhibitors Reversible Irreversible “inactivators” “suicide” inhibitors Covalent modification  enzyme-inhibitor complex Aspirin • Competitive • Inhibitor looks like substrate • HIV protease inhibitors • Mixed • Non-competitive

45. Irreversible Enzyme Inhibition – mechanism based (suicide inhibitors) “bad” - toxic “good” - antibiotics Diisopropylfluorophosphate (DIFP) penicillin Enzyme Enzyme Inactivation through covalent bond Inactivation through covalent bond Enzyme Enzyme Enzyme = acetylcholinesterase (hydrolase that hydrolysis acetylcholine) Enzyme = transpeptidase (catalysis cross-linkages in peptidoglycan cell walls)

46. Irreversible Enzyme Inhibition – mechanism based (suicide inhibitors) “bad” - toxic “good” - antibiotics Diisopropylfluorophosphate (DIFP) penicillin Enzyme Enzyme Inactivation through covalent bond Inactivation through covalent bond Enzyme Enzyme Enzyme = acetylcholinesterase (hydrolase that hydrolysis acetylcholine) Enzyme = transpeptidase (catalysis cross-linkages in peptidoglycan cell walls)

47. Irreversible Enzyme Inhibition – mechanism based (suicide inhibitors) “bad” - toxic “good” - antibiotics Diisopropylfluorophosphate (DIFP) penicillin Enzyme Enzyme Inactivation through covalent bond Inactivation through covalent bond Enzyme Enzyme Enzyme = acetylcholinesterase (hydrolase that hydrolysis acetylcholine) Enzyme = transpeptidase (catalysis cross-linkages in peptidoglycan cell walls)

48. Irreversible Enzyme Inhibition – mechanism based (suicide inhibitors) “bad” - toxic “good” - antibiotics Diisopropylfluorophosphate (DIFP) penicillin Enzyme Enzyme Inactivation through covalent bond Inactivation through covalent bond Enzyme Enzyme Enzyme = acetylcholinesterase (hydrolase that hydrolysis acetylcholine) Enzyme = transpeptidase (catalysis cross-linkages in peptidoglycan cell walls)

49. How NSAIDs work by inhibiting COX-2 naproxen aspirin adds 2 oxygen molecules to arachidonic acid to make prostaglandin pain and inflammation pulls one molecule of arachidonic acid out of membrane How can NSAIDs prevent pain and inflammation?

50. Aspirin- Mechanism of action Cox-2 HO-CH2-