1. Enzyme Features Increase rate of reaction Active site binds substrate Unchanged by overall reaction

2. Enzyme Classes

3. Reaction Equilibria  G is the free energy difference  G depends upon [S] and [P]  Gº is the standard free energy change Reaction: S ↔ P  G =  Gº + RT ln ([P]/[S])  GDirection <0S → P 0equilibrium >0P → S

4. Energetics Of Catalysis GG  G ‡ )  G determines direction of reaction  G ‡ determines rate of reaction Enzymes lower  G ‡ but do not alter  G Reaction: S ↔ S T ↔ P

5. Energetics Of Coupled Reactions ammonia + glutamic acid → glutamine (unfavorable) ATP → ADP + P i (favorable) Unfavorable reaction coupled to favorable one Net reaction is favorable For example, coupled reactions catalyzed by glutamine synthase

6. Chemistry Of Catalysis How enzymes accelerate reaction rates Orient substrate(s) Stabilization of transition state Acid-base catalysis Covalent catalysis

7. Stabilization Of Transition State Enzyme binding lowers energy of reaction intermediates

8. Acid-base Catalysis Acidic residue tends to donate proton Basic residue tends to take up proton Pair with atoms in substrate and alter electron distribution

9. Covalent Catalysis Activated serine forms covalent bond with substrate Serine protease mechanism:

10. Temperature Vs. Reaction Rate Increase of temperature increases velocity Denatured at high temperature

11. pH Vs Reaction Rate Optimum pH reflects different groups being protonated or unprotonated

12. Substrate Conc Vs Reaction Rate Increase of substrate concentration increases reaction rate until V max At V max enzyme is saturated

13. Michaelis-menton Kinetics (V max [S]) (K m + [S]) v 0 = v 0 = initial reaction velocity V max = maximal velocity K m = Michaelis constant [S] = substrate concentration Reaction: E + S  ES  E + P K m reflects affinity of E for S v 0 directly proportional to [E]

14. Assumptions Of Michaelis- Menton Equation [E] << [S] Steady-state assumption: [ES] does not change with time Initial velocity is measured Reaction: E + S  ES  E + P

15. Order Of Reaction If [S]<<K m, v 0 proportional to [S] If [S]>>K m, v 0 nearly independent of [S]

16. Lineweaver-burke Plot 1 v0v0 KmKm V max [S] V max 1 = + double reciprocal plot

17. Competitive Inhibitor Binds to same site as substrate Inhibition counteracted by increasing [S]

18. Effect Of Competitive Inhibitor On K M & V MAX K m increased, V max unchanged

19. Noncompetitive Inhibitor Binds to different site than substrate Inhibition not counteracted by increasing [S]

20. Effect Of Noncompetitive Inhibitor On K M & V MAX V max decreased, K m unchanged

21. Regulation Of Enzyme Activity Allosteric effectors Phosphorylation Activation of zymogens

22. Allosteric Enzymes Allosteric effectors bind regulatory site Conformational change Positive and negative effectors

23. Positive Effectors Binding to regulatory site increases catalysis at active site

24. Negative Effectors Binding to regulatory site inhibits catalysis at active site

25. Feedback Inhibition End product often negative effector for enzyme of initial step

26. Cooperative Allosteric Effects Symmetrical assemblies of identical subunits Cooperative binding of effector Sharper response of enzyme activity

27. Regulation By Phosphorylation Reversible covalent attachment of phosphate to serine, threonine or tyrosine Can alter activity

28. Coenzymes Small organic molecules Binding site unique from substrate Provide essential chemical group Chemically changed by reaction

29. ATP Transfer of high energy phosphate Modulator of enzyme activity

30. Nicotinamide Adenine Dinucleotide Derived from nicotinic acid (niacin) Adenosine with pyrophosphate linkage to ribose and nicotinamide Oxidation-reduction reactions Niacin deficiency leads to pellagra

31. Riboflavin Coenzymes FAD = adenosine linked to riboflavin FMN = phosphate linked to riboflavin Oxidation-reduction reactions

32. Thiamine Pyrophosphate Derived from thiamine (vitamin B 1 ) Transfer of activated aldehyde unit Transketolase, pyruvate dehydrogenase,  - ketoglutarate dehydrogenase Thiamine deficiency leads to Beriberi (alcoholics)

33. Tetrahydrofolate Derived from folic acid One carbon transfers, example dTMP synthesis Folic acid deficiency leads to macrocytic (megaloblastic) anemia

34. Coenzyme B 12 (Cobalamine) Corrin ring with central cobalt Cobalt coordinated in six positions

35. Coenzyme B 12 Reactions B 12 deficiency leads to pernicious anemia Methylmalonyl CoA mutase reaction Methionine synthase reaction; THF trap

36. Coenzyme Summary CoenzymeReaction typeVitamin Consequences precursor of deficiency ATPPhospho transfer NAD + /NADP + Oxidation-reductionNicotinic acidPellagra (niacin) FAD/FMNOxidation-reductionRiboflavin (B 2 ) TPPAldehyde transferThiamine (B 1 )Beriberi TetrahydrofolateTransfer one-carbonFolic AcidAnemia units Coenzyme B 12 Transfer methyl groups, B 12 Anemia isomerization

37. Isoenzymes Different enzymes that catalyze same reaction Often have different tissue distributions

38. Isoenzyme Analysis Creatine kinase- three isoenzymes from associations of two subunits Distinguished based on charge by non-denaturing electrophoresis Diagnosis of myocardial infarction