1. Biochemistry Lecture 8

2. Why Enzymes? Higher reaction rates Greater reaction specificity Milder reaction conditions Capacity for regulation Metabolites have many potential pathways of decomposition Enzymes make the desired one most favorable

3. Enzymatic Substrate Selectivity No binding Binding but no reaction Example: Phenylalanine hydroxylase

8. H 2 O + CO 2 HOCO 2 – + H + Potential Energy Reaction EaEa EaEa '

9. How to Lower  G  ? Enzymes bind transition states best

10. How is TS Stabilization Achieved? –acid-base catalysis: give and take protons –covalent catalysis: change reaction paths –metal ion catalysis: use redox cofactors, pK a shifters –electrostatic catalysis: preferential interactions with TS End result? Rate enhancements of 10 5 to 10 17 !

11. How is TS Stabilization Achieved? –covalent catalysis: change reaction paths

12. How to Lower  G  ? Enzymes organizes reactive groups into proximity

38. Enzyme Kinetics Kinetics is the study of the rate at which compounds react Rate of enzymatic reaction is affected by –Enzyme –Substrate –Effectors –Temperature

39. How to Do Kinetic Measurements

44. What equation models this behavior? Michaelis-Menten Equation

45. Meaning of V max and K m

46. Simple Enzyme Kinetics The final form in case of a single substrate is k cat (turnover number): how many substrate molecules can one enzyme molecule convert per second K m (Michaelis constant): an approximate measure of substrate’s affinity for enzyme Microscopic meaning of K m and k cat depends on the details of the mechanism

47. Two-substrate Reactions Kinetic mechanism: the order of binding of substrates and release of products When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms –Sequential mechanism –Ping-Pong (Double Displacement) mechanism

49. Distinguishing Mechanism Ping-Pong Ternary Complex

50. Enzyme Inhibition Inhibitors are compounds that decrease enzyme’s activity Irreversible inhibitors (inactivators) react with the enzyme - one inhibitor molecule can permanently shut off one enzyme molecule - they are often powerful toxins but also may be used as drugs Reversible inhibitors bind to, and can dissociate from the enzyme - they are often structural analogs of substrates or products - they are often used as drugs to slow down a specific enzyme Reversible inhibitor can bind: –To the free enzyme and prevent the binding of the substrate –To the enzyme-substrate complex and prevent the reaction

57. Acetylcholinesterase

62. F