Biochemistry. Lecture 8

Transcription

1 Biochemistry Lecture 8

2 Why Enzymes? igher reaction rates Greater reaction specificity Milder reaction conditions Capacity for regulation C - - C N 2 - C N 2 - C - C Chorismate mutase - C - C - C Metabolites have many potential pathways of decomposition Enzymes make the desired one most favorable

3 Enzymatic Substrate Selectivity - C + N 3 - C + N C N 3 No binding N C 3 Binding but no reaction Example: Phenylalanine hydroxylase

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8 2 + C 2 C C Potential Energy E a 2 + C E ' a C œ + + Reaction

9 ow to Lower ΔG? Enzymes bind transition states best

10 ow 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 ow is TS Stabilization Achieved? covalent catalysis: change reaction paths C 3 C 3 N.. C 3 C 3 2 slow C C + 2 fast N C 3 + C

12 ow to Lower ΔG? Enzymes organizes reactive groups into proximity

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14 (C 3 ) 3 N C 2 C 2 C ac et ylc holine (ACh) C AChE (C 3 ) 3 N C 2 C 2 + C C 3 choline (Ch) ac et ic ac id

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17 (C 3 ) 3 N C 2 C 2 C ac et ylc holine (ACh) C AChE (C 3 ) 3 N C 2 C 2 + C C 3 choline (Ch) ac et ic ac id

18 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

19 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

20 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

21 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

22 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

23 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

24 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

25 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

26 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

27 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

28 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

29 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

30 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

31 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

32 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

33 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

34 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

35 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

36 Ser Glu C 2 C Asp C N is N (C 3 ) 3 N C 2 C 2 C C 3

37 (C 3 ) 3 N C 2 C 2 C ac et ylc holine (ACh) C AChE (C 3 ) 3 N C 2 C 2 + C C 3 choline (Ch) ac et ic ac id

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 ow to Do Kinetic Measurements

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44 What equation models this behavior? Michaelis-Menten Equation

45 Meaning of Vmax and Km

46 Simple Enzyme Kinetics The final form in case of a single substrate is v = k cat K [ E m ][ S] 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 tot + [ S]

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

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49 Distinguishing Mechanism Ternary Complex Ping-Pong

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

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57 Acetylcholinesterase Ser Glu C 2 C Asp C N is N

58 Ser Glu C 2 C Asp C F N is N C 3 C P C 3 C 3

59 Ser Glu C 2 C Asp C F N is N C 3 C P C 3 C 3

60 Ser Glu C 2 C Asp C F N is N C 3 C P C 3 C 3

61 Ser Glu C 2 C Asp C F N is N C 3 C P C 3 C 3

62 Ser Glu C 2 C Asp C F N is N C 3 C P C 3 C 3

63 I N C N C 3 Pyridine aldoxime me thiodide (PAM) Br Br (C 3 ) 3 NœC 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 œn(c 3 ) 3 decamethonium bromide (C 3 ) 3 N C 2 C 2 CC 2 C 2 CC 2 C 2 N(C 3 ) 3 succ inylcholine

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