Biochemistry. Lecture 8 Enzyme Kinetics

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1 Biochemistry Lecture 8 Enzyme Kinetics

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

4 Enzymes Affect Reaction Rates Catalyst Protein (globular) or RNA Classified based on the reaction catalyzed 4

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7 2 + C 2 C C Potential Energy E a 2 + C E ' a C œ + + Reaction

8 ow to Lower ΔG? Enzymes bind transition states best

9 ow Do Enzymes Stabilize the Transition State and Increase Reaction Rate? Conserved active site amino acid residues in the enzyme can help with orientation of the substrate and stabilizing transition state 9

10 Serine Protease Mechanism 10

11 ow Do Enzymes Stabilize the Transition State and Increase Reaction Rate? Conserved active site amino acid residues, metal ions and organic molecules in the enzyme can help with orientation of the substrate and stabilizing transition state 11

12 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!

13 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

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

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16 (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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19 (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

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

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

39 (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

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

41 ow to Do Kinetic Measurements

42 Steady-State Assumption 42

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45 What equation models this behavior? Michaelis-Menten Equation

46 Michaelis-Menten Kinetics V 0 = V max [S] K m +[S] k 1 E + S ß à ES à E + P k -1 k 2 Derived using a few assumptions: steady state assumption: formation of ES = breakdown of ES (until a significant amount of S has been consumed). consider initial velocity at early time-points, [P] = 0: rate of reaction depends exclusively on the breakdown of ES (k -2 can be ignored). free ligand assumption: [S] is in such excess that its decrease in concentration when forming ES is negligible (total [S] = free [S] + [ES]). 46

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48 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]

49 Calculating V max and K m : The Double V 0 = V max[s] K m +[S] 1 = K m +[S] V 0 V max [S] Reciprocal Plot K m V max [S] + 1 V max 49

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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52 Reversible Inhibition - Competitive No change in V max ; apparent increase in K m Lineweaver- Burk: lines intersect at the y- axis 52

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54 Reversible Inhibition - Uncompetitive Decrease in V max ; apparent decrease in K m No change in K m /V max Lineweaver- Burk: lines are parallel 54

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56 Reversible Inhibition Mixed Inhibition Decrease in V max ; apparent change in K m Lineweaver- Burk: lines intersect le@ from the y- axis NoncompeBBve inhibitors are mixed inhibitors such that there is no change in K m 56

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