Presentation is loading. Please wait.

Presentation is loading. Please wait.

Mechanism of Enzyme Action BCH 321 Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are not my own,

Similar presentations


Presentation on theme: "Mechanism of Enzyme Action BCH 321 Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are not my own,"— Presentation transcript:

1 Mechanism of Enzyme Action BCH 321 Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are not my own, they can be found on: The texts, tables and images contained in this course presentation are not my own, they can be found on: References supplied References supplied Atlases or Atlases or The web The web King Saud University College of Science Department of Biochemistry

2 Mechanism of Enzyme Action Enzyme Kinetic and Mechanism Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation (BCH 320) are not my own, they can be found on: The texts, tables and images contained in this course presentation (BCH 320) are not my own, they can be found on: References supplied References supplied Atlases or Atlases or The web The web King Saud University College of Science Department of Biochemistry

3 Enzyme Catalysis

4

5 Catalyst only lower  G ‡, but not effect on the EQM positions 1. Stabilization the transition state 2. Destabilizing substrate bound at the binding site 3. Destabilizing ES complex 4. Forming an intermediate

6 Types of Enzymatic Catalysis 1. Approximation (Proximity) and Orientation (Entropy Contribution) (a) Intermolecular catalysis (b) Intramolecular catalysis (c) Effective morality (concentration)

7 2.Preferential Binding of Transition Sstate (TS) (a) Oxyanion hole (b) Strain or distortion (c) Transition analongs Types of Enzymatic Catalysis

8 3. Electrostatic Catalysis 4. General Acid-base Catalysis (a) General acid (b) General base (c) Concerted acid/base Types of Enzymatic Catalysis

9 5. Nucleophilic-Electrophilic Catalysis (a) Covalent catalysis (b) Schiff base catalysis (c) Electron sink (electron flow) catalysis Types of Enzymatic Catalysis

10 6. Metal ion Catalysis (a) Metalloenzymes Contain tightly bound metal cofactors such as Fe 2+, Fe 3+, Cu 2+, Zn 2+, Mn 2+, Co 2+ (b) Metal Activated Enzymes Only loosely bind the metal ions. The ions are usually Na +, K +, Mg 2+, or Ca 2+ Types of Enzymatic Catalysis

11 Enzyme Catalysis Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality Enzymes are the agents of metabolic function Enzymes are the agents of metabolic function

12

13 Enzyme Catalysis

14 1. Enzyme works simply by lowering the energy barrier of a reaction. By doing so, the enzyme increases the fraction of molecules that have enough energy to attain the transition state, thus making the reaction go faster in both directions

15 Enzyme Catalysis 2. The position of the equilibrium (the amount of product versus reactant) is unchanged by an enzyme. 3. Even though K 1, K -1 many be greatly changed from their values in the absence of an enzyme, each one changes by the same factor and the equilibrium constant, K, is unchanged, because K = k 1 /k -1

16

17 Catalytic Power Enzymes can accelerate reactions as much as over uncatalyzed rates! Enzymes can accelerate reactions as much as over uncatalyzed rates! Urease is a good example: Urease is a good example: Catalyzed rate: 3x10 4 /sec Catalyzed rate: 3x10 4 /sec Uncatalyzed rate: 3x /sec Uncatalyzed rate: 3x /sec Ratio is 1x10 14 ! Ratio is 1x10 14 !

18 Catalytic Power

19 Specificity Enzymes selectively recognize proper substrates over other molecules Enzymes selectively recognize proper substrates over other molecules Enzymes produce products in very high yields - often much greater than 95% Enzymes produce products in very high yields - often much greater than 95% Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield

20 Enzyme Kinetics Several terms to know! rate or velocity rate or velocity rate constant rate constant rate law rate law order of a reaction order of a reaction molecularity of a reaction molecularity of a reaction

21

22 The Transition State Understand the difference between  G and  G ‡ The overall free energy change for a reaction is related to the equilibrium constant The overall free energy change for a reaction is related to the equilibrium constant The free energy of activation for a reaction is related to the rate constant The free energy of activation for a reaction is related to the rate constant It is extremely important to appreciate this distinction! It is extremely important to appreciate this distinction!

23

24 What Enzymes Do.... Enzymes accelerate reactions by lowering the free energy of activation Enzymes accelerate reactions by lowering the free energy of activation Enzymes do this by binding the transition state of the reaction better than the substrate Enzymes do this by binding the transition state of the reaction better than the substrate Much more of this in Chapter 16! Much more of this in Chapter 16!

25

26 The Michaelis-Menten Equation You should be able to derive this! Louis Michaelis and Maude Menten's theory Louis Michaelis and Maude Menten's theory It assumes the formation of an enzyme- substrate complex It assumes the formation of an enzyme- substrate complex It assumes that the ES complex is in rapid equilibrium with free enzyme It assumes that the ES complex is in rapid equilibrium with free enzyme Breakdown of ES to form products is assumed to be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S Breakdown of ES to form products is assumed to be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S

27

28 Understanding K m The "kinetic activator constant" K m is a constant K m is a constant K m is a constant derived from rate constants K m is a constant derived from rate constants K m is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S K m is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S Small K m means tight binding; high K m means weak binding Small K m means tight binding; high K m means weak binding

29 Understanding V max The theoretical maximal velocity V max is a constant V max is a constant V max is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality V max is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach V max would require that ALL enzyme molecules are tightly bound with substrate To reach V max would require that ALL enzyme molecules are tightly bound with substrate V max is asymptotically approached as substrate is increased V max is asymptotically approached as substrate is increased

30 The dual nature of the Michaelis-Menten equation Combination of 0-order and 1st-order kinetics When S is low, the equation for rate is 1st order in S When S is low, the equation for rate is 1st order in S When S is high, the equation for rate is 0- order in S When S is high, the equation for rate is 0- order in S The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S ! The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S !

31 The turnover number A measure of catalytic activity k cat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. k cat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. If the M-M model fits, k 2 = k cat = V max /E t If the M-M model fits, k 2 = k cat = V max /E t Values of k cat range from less than 1/sec to many millions per sec Values of k cat range from less than 1/sec to many millions per sec

32 The catalytic efficiency Name for k cat /K m An estimate of "how perfect" the enzyme is An estimate of "how perfect" the enzyme is k cat /K m is an apparent second-order rate constant k cat /K m is an apparent second-order rate constant It measures how the enzyme performs when S is low It measures how the enzyme performs when S is low The upper limit for k cat /K m is the diffusion limit - the rate at which E and S diffuse together The upper limit for k cat /K m is the diffusion limit - the rate at which E and S diffuse together

33 Linear Plots of the Michaelis- Menten Equation Be able to derive these equations! Lineweaver-Burk Lineweaver-Burk Hanes-Woolf Hanes-Woolf Hanes-Woolf is best - why? Hanes-Woolf is best - why? Smaller and more consistent errors across the plot Smaller and more consistent errors across the plot

34

35

36

37

38

39 Enzyme Inhibitors Reversible versus Irreversible Reversible inhibitors interact with an enzyme via noncovalent associations Reversible inhibitors interact with an enzyme via noncovalent associations Irreversible inhibitors interact with an enzyme via covalent associations Irreversible inhibitors interact with an enzyme via covalent associations

40

41 Classes of Inhibition Two real, one hypothetical Competitive inhibition - inhibitor (I) binds only to E, not to ES Competitive inhibition - inhibitor (I) binds only to E, not to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition

42

43

44

45

46

47

48 Ribozymes and Abzymes Relatively new discoveries Ribozymes - segments of RNA that display enzyme activity in the absence of protein Ribozymes - segments of RNA that display enzyme activity in the absence of protein Examples: RNase P and peptidyl transferase Examples: RNase P and peptidyl transferase Abzymes - antibodies raised to bind the transition state of a reaction of interest Abzymes - antibodies raised to bind the transition state of a reaction of interest For a great recent review, see Science, Vol. 269, pages (1995) For a great recent review, see Science, Vol. 269, pages (1995) We'll say more about transition states in Ch 16 We'll say more about transition states in Ch 16

49

50

51

52

53 Mechanisms of Enzyme Action

54 Stabilization of the Transition State Stabilization of the Transition State Enormous Rate Accelerations Enormous Rate Accelerations Binding Energy of ES Binding Energy of ES Entropy Loss and Destabilization of ES Entropy Loss and Destabilization of ES Transition States Bind Tightly Transition States Bind Tightly Types of Enzyme Catalysis Types of Enzyme Catalysis Serine Proteases Serine Proteases Aspartic Proteases Aspartic Proteases Lysozyme Lysozyme Mechanisms of Enzyme Action

55 Enzyme Catalysis Reactions in solution that are not catalyzed are slow since charge development and separation occurs in the transition state. Reactions in solution that are not catalyzed are slow since charge development and separation occurs in the transition state. When bonds are made or broken, charged intermediates are often formed which are higher in energy than the reactants. When bonds are made or broken, charged intermediates are often formed which are higher in energy than the reactants.

56 Since the intermediate is higher in energy than the reactants, the transition state would be even higher in energy, and hence more closely resemble the charged intermediate. Since the intermediate is higher in energy than the reactants, the transition state would be even higher in energy, and hence more closely resemble the charged intermediate. Enzyme Catalysis

57 Anything that can stabilize the charges on the intermediate and hence the developing charges in the transition states will lower the energy of the transition state and catalyze the reaction. Anything that can stabilize the charges on the intermediate and hence the developing charges in the transition states will lower the energy of the transition state and catalyze the reaction. In this section will investigate the mechanism underlying the catalysis by small molecules of chemical reactions. In this section will investigate the mechanism underlying the catalysis by small molecules of chemical reactions. Enzyme Catalysis

58 Presumably, biological macromolecular catalyst (like protein enzymes) will use similar mechanisms in their catalytic effects (which will be discussed in the next section). Presumably, biological macromolecular catalyst (like protein enzymes) will use similar mechanisms in their catalytic effects (which will be discussed in the next section). Enzyme Catalysis

59 Approximation Catalysis Enzyme serves as a template to bind the substrates so that they are close to each other in the reaction center. Enzyme serves as a template to bind the substrates so that they are close to each other in the reaction center. Bring substrate into contact with catalytic groups or other substrates. Bring substrate into contact with catalytic groups or other substrates. Correct orientation for bond formation. Correct orientation for bond formation. Freeze translational and rotational motion. Freeze translational and rotational motion.

60 Catalysis by Approximation The classic way that an enzyme increases the rate of a bimolecular reaction is to use binding energy to simply bring the two reactants in close proximity. The classic way that an enzyme increases the rate of a bimolecular reaction is to use binding energy to simply bring the two reactants in close proximity. If  G ‡ is the change in free energy between the ground state and the transition state, then  G ‡ =  H ‡ –T  S ‡. If  G ‡ is the change in free energy between the ground state and the transition state, then  G ‡ =  H ‡ –T  S ‡. In solution, the transition state would be significantly more ordered than the ground state, and  S ‡ would therefore be negative. In solution, the transition state would be significantly more ordered than the ground state, and  S ‡ would therefore be negative.

61 Catalysis by Approximation The formation of a transition state is accompanied by losses in translational entropy as well as rotational entropy. The formation of a transition state is accompanied by losses in translational entropy as well as rotational entropy. Enzymatic reactions take place within the confines of the enzyme active-site wherein the substrate and catalytic groups on the enzyme act as one molecule. Enzymatic reactions take place within the confines of the enzyme active-site wherein the substrate and catalytic groups on the enzyme act as one molecule. Therefore, there is no loss in translational or rotational energy in going to the transition state. Therefore, there is no loss in translational or rotational energy in going to the transition state. This is paid for by binding energy. This is paid for by binding energy.

62 a)Bimolecular reaction (high activation energy, low rate). b)Unimolecular reaction, rate enhanced by factor of 10 5 due to increased probability of collision/reaction of the 2 groups c)Constraint of structure to orient groups better (elimination of freedom of rotation around bonds between reactive groups), rate enhanced by another factor of 10 3, for 10 8 total rate enhancement over bimolecular reaction Approximation Catalysis

63 Desolvation: When substrate binds to the enzyme surrounding water in solution is replaced by the enzyme. This makes the substrate more reactive by destablizing the charge on the substrate. When substrate binds to the enzyme surrounding water in solution is replaced by the enzyme. This makes the substrate more reactive by destablizing the charge on the substrate. Expose a water charged group on the substrate for interaction with the enzyme. Expose a water charged group on the substrate for interaction with the enzyme. Also lowers the entropy of the substrate (more ordered). Also lowers the entropy of the substrate (more ordered). Approximation Catalysis

64 Strain and Distortion: Strain and Distortion:  When substrate bind to the enzyme, it may induces a conformational change in the active site to fit to a transition state.  Frequently, in the transition state, the substrate and the enzyme have slightly different structure (strain or distortion) and increase the reactivity of the substrate. Rate: cyclic phosphate esterAcylic phospodiester Approximation Catalysis

65 Strain and Distortion

66 1. Intramolecular Catalysis Consider the hydrolysis of phenylacetate. Consider the hydrolysis of phenylacetate. This reaction, a nucleophilic subsitution reaction, could be catalyzed by the addition to solution of the general base acetate, as described above. This reaction, a nucleophilic subsitution reaction, could be catalyzed by the addition to solution of the general base acetate, as described above.

67 Intramolecular Catalysis Since this reaction would double with the doubling of the solution acetate, the reaction is bimolecular (first order in reactant and catalyst). Since this reaction would double with the doubling of the solution acetate, the reaction is bimolecular (first order in reactant and catalyst). Now consider the same reaction only when the the general base part of the catalyst, the carboxyl group, is part of the reactant phenylacetate. Now consider the same reaction only when the the general base part of the catalyst, the carboxyl group, is part of the reactant phenylacetate.

68 Such a case occurs in the acetylated form of salicylic acid - i.e. aspirin. When the carboxy group is ortho compared to the acetylated phenolic OH, it is in perfect position to accept a proton from water, decreasing the charge development on the O in the transition state. Such a case occurs in the acetylated form of salicylic acid - i.e. aspirin. When the carboxy group is ortho compared to the acetylated phenolic OH, it is in perfect position to accept a proton from water, decreasing the charge development on the O in the transition state. Intramolecular Catalysis

69 The general base does not have to diffuse to the appropriate site when it is intramolecular with respect to the carbonyl C of the ester link. The general base does not have to diffuse to the appropriate site when it is intramolecular with respect to the carbonyl C of the ester link. The rate of this intramolecular base catalysis is about 100 fold greater than of an intermolecular base catalyst like acetate. The rate of this intramolecular base catalysis is about 100 fold greater than of an intermolecular base catalyst like acetate. Intramolecular Catalysis

70 It is as if the effective concentration of the intramolecular carboxyl base catalyst is much higher due to its proximity to the reaction site. It is as if the effective concentration of the intramolecular carboxyl base catalyst is much higher due to its proximity to the reaction site. Intramolecular Catalysis

71 Another type of reactions involving a carboxyl group (in addition to simple proton transfer) is when the negatively charged carboxyl O acts as a nucleophile and attacks an electrophilic carbonyl carbon. Another type of reactions involving a carboxyl group (in addition to simple proton transfer) is when the negatively charged carboxyl O acts as a nucleophile and attacks an electrophilic carbonyl carbon. When the carbonyl is part of an ester, the carboxyl group engages in a nucleophilic substitution reaction, expelling the alcohol part of the ester as a leaving group. When the carbonyl is part of an ester, the carboxyl group engages in a nucleophilic substitution reaction, expelling the alcohol part of the ester as a leaving group. Intramolecular Catalysis

72 The remaining examples below consider the nucleophilic (carboxyl) substitution on phenylesters, with phenolate as the leaving group. The reactions in effect transfer an acyl group to the carboxyl group to create an anhydride. The remaining examples below consider the nucleophilic (carboxyl) substitution on phenylesters, with phenolate as the leaving group. The reactions in effect transfer an acyl group to the carboxyl group to create an anhydride. Intramolecular Catalysis

73 First consider acyl transfer with aspirin derivatives. First consider acyl transfer with aspirin derivatives. Aspirin, as you know, contains a carboxyl group ortho to an ester substitutent. Aspirin, as you know, contains a carboxyl group ortho to an ester substitutent. Hence the carboxyl group can act as a nucleophile and attack the carbonyl carbon of the ester in a nucleophilic substitution reaction. Hence the carboxyl group can act as a nucleophile and attack the carbonyl carbon of the ester in a nucleophilic substitution reaction. Intramolecular Catalysis

74 The net effect is to transfer the acetyl group from the phenolic OH to the carboxyl group converting it to an anhydride. The net effect is to transfer the acetyl group from the phenolic OH to the carboxyl group converting it to an anhydride. This is an intramolecular reaction. Compare this reaction to a a comparable bimolecular reaction shown below. This is an intramolecular reaction. Compare this reaction to a a comparable bimolecular reaction shown below. Intramolecular Catalysis

75 Acyl Transfer Aspirin Derivatives Intramolecular Intermolecular

76 The first order rate constant of the intramolecular transfer of the acetyl group to the carboxyl group, k 1 = 0.02 s-1. The first order rate constant of the intramolecular transfer of the acetyl group to the carboxyl group, k 1 = 0.02 s-1. The analogous bimolecular reaction rate constant k 2 ~ M -1s-1. The analogous bimolecular reaction rate constant k 2 ~ M -1s-1. Intramolecular Catalysis

77 Dividing k 1 /k 2 gives the relative rate enhancement of the intramolecular over the intermolecular reaction. Dividing k 1 /k 2 gives the relative rate enhancement of the intramolecular over the intermolecular reaction. With units of molarity, this ratio can be interpreted as the relative effective concentration of the intramolecular nucleophile. With units of molarity, this ratio can be interpreted as the relative effective concentration of the intramolecular nucleophile. This makes the effective concentration of the carboxylate in the aspirin derivative 2 x 10 7 M. This makes the effective concentration of the carboxylate in the aspirin derivative 2 x 10 7 M. Intramolecular Catalysis

78 Mechanism of Acetate with Phenylacetate

79 Now consider the cleavage of phenylacetate using acetate as the nucleophile. Now consider the cleavage of phenylacetate using acetate as the nucleophile. The products are acetic anhydride and phenolate. The products are acetic anhydride and phenolate. This is a bimolecular reaction (a slow one at that), with a bimolecular rate constant, k 2 which I will arbitrarily set to 1 for comparison to some similar reactions. This is a bimolecular reaction (a slow one at that), with a bimolecular rate constant, k 2 which I will arbitrarily set to 1 for comparison to some similar reactions. 2. Intermolecular Catalysis

80 Now consider a monoester derivatives of succinic acid - phenyl succinate - in which the free carboxyl group of the ester attacks the carbonyl carbon of the ester derivative. Now consider a monoester derivatives of succinic acid - phenyl succinate - in which the free carboxyl group of the ester attacks the carbonyl carbon of the ester derivative. Intermolecular Catalysis

81

82 If you assign a second order rate constant k 2 = 1 M -1 s -1 to the analogous intermolecular reaction of acetate with phenylacetate (as described above), the first order rate constant for the intramolecular reaction of phenylsuccinate is 10 5 s -1. If you assign a second order rate constant k 2 = 1 M -1 s -1 to the analogous intermolecular reaction of acetate with phenylacetate (as described above), the first order rate constant for the intramolecular reaction of phenylsuccinate is 10 5 s -1. The ratio of rate constants, k 1 /k 2 = 10 5 M. The ratio of rate constants, k 1 /k 2 = 10 5 M. Intermolecular Catalysis

83 That is it would take 10 5 M concentration of acetate reacting with 1 M phenylacetate in the first bimolecular reaction to get a reaction as fast as the intramolecular reaction of phenylsuccinate. That is it would take 10 5 M concentration of acetate reacting with 1 M phenylacetate in the first bimolecular reaction to get a reaction as fast as the intramolecular reaction of phenylsuccinate. An even more sterically restricated bicyclic phenylcarboxylate shows a k 1 /k 2 = 10 8 M. An even more sterically restricated bicyclic phenylcarboxylate shows a k 1 /k 2 = 10 8 M. Intermolecular Catalysis

84 Intramolecular Catalysis

85 Another example is anhydride formation between two carboxyl groups. Another example is anhydride formation between two carboxyl groups. The  G o for such a reaction is positive, suggesting an unfavorable reaction. The  G o for such a reaction is positive, suggesting an unfavorable reaction. Consider two acetic acid molecules condensing to form acetic anhydride. Consider two acetic acid molecules condensing to form acetic anhydride. For this intermolecular reaction, K eq = 3x M -1. For this intermolecular reaction, K eq = 3x M -1. Intermolecular Catalysis

86 Now consider the analogous intramolecular reaction of the dicarboxylic acid succinic acid. Now consider the analogous intramolecular reaction of the dicarboxylic acid succinic acid. It condenses in an intramolecular reaction to form succinic anhydride with a K eq = 8x10 -7 (no units). It condenses in an intramolecular reaction to form succinic anhydride with a K eq = 8x10 -7 (no units). The ratio K eq-intra /K eq inter = 3 x 10 5 M. The ratio K eq-intra /K eq inter = 3 x 10 5 M. It is as if the effective concentration of the reacting groups. because they do not have to diffuse together to react, is 3 x 10 5 M. It is as if the effective concentration of the reacting groups. because they do not have to diffuse together to react, is 3 x 10 5 M. Intermolecular Catalysis

87 How does this apply to enzyme catalyzed reaction? How does this apply to enzyme catalyzed reaction? Enzymes bind substrates in physical steps which are typically fast. Enzymes bind substrates in physical steps which are typically fast. The slow step is chemical conversion of the bound substrate, which is effectively intramolecular. The slow step is chemical conversion of the bound substrate, which is effectively intramolecular. 3. Enzyme Catalysis

88 These three kinds of reactions, intermolecular, intramolecular, and enzyme-catalysed can be broken down into two hypothetical steps, a binding followed by catalysis. These three kinds of reactions, intermolecular, intramolecular, and enzyme-catalysed can be broken down into two hypothetical steps, a binding followed by catalysis. Enzyme Catalysis

89 Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction

90 If the rate constants for the chemical steps are all identical, the advantage of the intramolecular and enzyme- catalyzed reaction over the intermolecular reaction is K INTRA /K INTER and K ENZ /K INTER, respectively. If the rate constants for the chemical steps are all identical, the advantage of the intramolecular and enzyme- catalyzed reaction over the intermolecular reaction is K INTRA /K INTER and K ENZ /K INTER, respectively. Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction

91 The advantage of intramolecular reactions can be seen by studying the Ca-EDTA complex. The advantage of intramolecular reactions can be seen by studying the Ca-EDTA complex. Calcium in solution exists as a octahedrally coordinated complex with water occupying all the coordination sites. Calcium in solution exists as a octahedrally coordinated complex with water occupying all the coordination sites. Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction

92 EDTA, a multidentate ligand, first interacts through one of its potential six electron donors to Ca in a reaction which is entropically disfavored from the the Ca-EDTA perspective, although one water is released. EDTA, a multidentate ligand, first interacts through one of its potential six electron donors to Ca in a reaction which is entropically disfavored from the the Ca-EDTA perspective, although one water is released. Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction

93 Once this first intramolecular complex is formed, the rest of the ligands on the EDTA rapidly coordinate with the Ca and release bound water. Once this first intramolecular complex is formed, the rest of the ligands on the EDTA rapidly coordinate with the Ca and release bound water. The former is no longer entropically disfavored since it is now an intramolecular process while the later is favored through the release of the remaining five water molecules. The former is no longer entropically disfavored since it is now an intramolecular process while the later is favored through the release of the remaining five water molecules. Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction

94 Mechanism of Binding of Ca 2+ and EDTA

95 We modeled the catalytic advantage offered by intramolecular reaction in terms of a dramatic increase in the effective concentration of reactants, which sometimes reached levels of 10 8 M. We modeled the catalytic advantage offered by intramolecular reaction in terms of a dramatic increase in the effective concentration of reactants, which sometimes reached levels of 10 8 M. Another way is to look at entropy changes associated with dimer formation. Another way is to look at entropy changes associated with dimer formation. Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction

96 Entropy and Catalysis

97

98 In the non-enzymatic lactonization reaction shown below, the relative rate when R = CH 3 is 3.4 x10 11 times that when R = H. What is the explanation? Orientation Effects

99 Models of Approximation (1)

100 Catalysis by Approximation 1.In order for a reaction to take place between two molecules, the molecules must first find each other. 2.This is why the rate of a reaction is dependent upon the concentrations of the reactants, since there is a higher probability that two molecules will collide at high concentrations. 3.As an example, look at the hydrolysis of paranitrophenyl ester again catalyzed by imidazole. This reaction depends on both the concentration of imidazole and paranitrophenyl ester, therefore, it proceeds with a Second Order Rate Constant of 35 M -1 min -1.

101 Catalysis by Approximation 4. In the second reaction, the imidazole catalyst is actually part of the substrate that is being hydrolyzed. Therefore, the rate of hydrolysis is dependent only on the substrate, and therefore proceeds with a First Order Rate Constant of 839 min Rate constants of different order cannot be compared. However, the ratio of the first order rate constant to the second order rate constant gives an effective Molarity. 6. In order for the second order reaction to be as fast as the first order reaction, it would be necessary to have imidazole at a concentration of 24 M!

102 Catalysis by Approximation

103 Effective Concentration Effective concentration is k 1 /k 2 = 2 x 10 5 M Effective concentration = 2 x 10 7 M

104 2. Preferential Binding of TS Catalysis Enzymes typically bind the TS of the reactions with greater affinity than the substrates or products Enzymes typically bind the TS of the reactions with greater affinity than the substrates or products This increases the effective concentration of the TS with proportionally increases the reaction rate This increases the effective concentration of the TS with proportionally increases the reaction rate TS analogs are extremely potent enzyme inhibitors TS analogs are extremely potent enzyme inhibitors

105 Importance of Binding Energy On the left are examples of reaction coordinates of an uncatalyzed reaction, and one that is enzyme catalyzed. On the left are examples of reaction coordinates of an uncatalyzed reaction, and one that is enzyme catalyzed. The active sites of enzymes tend to be more complementary to the transition states of their respective reactions than they are to the actual substrates. The active sites of enzymes tend to be more complementary to the transition states of their respective reactions than they are to the actual substrates.

106 Importance of Binding Energy This results in lowering the energy of the enzyme–transition state complex, meaning, a lowering of the activation energy. This results in lowering the energy of the enzyme–transition state complex, meaning, a lowering of the activation energy. In order for catalysis to be effective, the energy barrier between ES and EX t must be less than S and X t. In order for catalysis to be effective, the energy barrier between ES and EX t must be less than S and X t.

107 Importance of Binding Energy Notice that the binding of substrate to enzyme lowers the free energy of the ES complex relative to substrate. Notice that the binding of substrate to enzyme lowers the free energy of the ES complex relative to substrate. If the energy is lowered too much, without a greater lowering of EX t, then catalysis would not take place. If the energy is lowered too much, without a greater lowering of EX t, then catalysis would not take place.

108 Transition State Stabilization Linus Pauling postulated long ago that the only thing that a catalyst must do is bind the transition state more tightly than the substrate. Linus Pauling postulated long ago that the only thing that a catalyst must do is bind the transition state more tightly than the substrate. That this must be the case can be seen from the diagram below, which shows how S and S* (the transition state) can react with E to form a complex which then proceeds to product, or can go to product in the absence of E. That this must be the case can be seen from the diagram below, which shows how S and S* (the transition state) can react with E to form a complex which then proceeds to product, or can go to product in the absence of E.

109 For an enzyme to be a catalyst the activation energy for the reaction in the presence of E, d, must be less than in the absence of enzyme, c. For an enzyme to be a catalyst the activation energy for the reaction in the presence of E, d, must be less than in the absence of enzyme, c. Therefore c-d = a-b > 0. Therefore c-d = a-b > 0. Since  G o = -RTln K eq, K eq for binding of S* to E is greater than for S binding to E. Since  G o = -RTln K eq, K eq for binding of S* to E is greater than for S binding to E. Transition State Stabilization

110 Enzyme Bind the TS Tightly

111 The stability of the transition state also affects the reaction kinetics (which makes sense given that the activation energy clearly affects the speed of a reaction). The stability of the transition state also affects the reaction kinetics (which makes sense given that the activation energy clearly affects the speed of a reaction). As you probably remember from organic chemistry, SN2 reactions are slow when the central atom where the substitution will occur is surrounded by bulky substitutents. (Sterics once again.) As you probably remember from organic chemistry, SN2 reactions are slow when the central atom where the substitution will occur is surrounded by bulky substitutents. (Sterics once again.) We discussed this in context to nucleophiliic substitution on a sp2 hybridized carbonyl carbon in carboxylic acid derivatives versus on a sp3 hybridized phosphorous in phosphoesters and diesters. We discussed this in context to nucleophiliic substitution on a sp2 hybridized carbonyl carbon in carboxylic acid derivatives versus on a sp3 hybridized phosphorous in phosphoesters and diesters. Transition State Stabilization

112 The explanation for this phenomena has usually been attributed to hindered access of the central atom caused by bulky substituents (intrinsic effects). Is this true? The explanation for this phenomena has usually been attributed to hindered access of the central atom caused by bulky substituents (intrinsic effects). Is this true? Recent studies on SN2 reactions of methylchloroacetonitrile and t-butylchloroacetonitrile (with the reagent labeled with 35 Cl) using 37 Cl- as the incoming nucleophile in the gas phase Recent studies on SN2 reactions of methylchloroacetonitrile and t-butylchloroacetonitrile (with the reagent labeled with 35 Cl) using 37 Cl- as the incoming nucleophile in the gas phase It shown that the more hindered t-butyl derivative's activation energy was only 1.6 kcal/mol higher than the methyl derivative, but in aqueous solution, the difference is much greater for comparable reactions. It shown that the more hindered t-butyl derivative's activation energy was only 1.6 kcal/mol higher than the methyl derivative, but in aqueous solution, the difference is much greater for comparable reactions. Transition State Stabilization

113 The explanation for this phenomena has usually been attributed to They attributed the differences to solvation effects of the transition state. The explanation for this phenomena has usually been attributed to They attributed the differences to solvation effects of the transition state. The bulkier the substituents on the central atom, the more difficult it is to solvate the transition state since water can't reorient around it as well. In effect there is steric hindrance for both reactant and solvent. The bulkier the substituents on the central atom, the more difficult it is to solvate the transition state since water can't reorient around it as well. In effect there is steric hindrance for both reactant and solvent. Transition State Stabilization

114 Transition State Analogs

115 Stabilizing the Transition State Rate acceleration by an enzyme means that the energy barrier between ES and EX ‡ must be smaller than the barrier between S and X ‡ Rate acceleration by an enzyme means that the energy barrier between ES and EX ‡ must be smaller than the barrier between S and X ‡ This means that the enzyme must stabilize the EX ‡ transition state more than it stabilizes ES This means that the enzyme must stabilize the EX ‡ transition state more than it stabilizes ES

116

117 3. Electrostatic Catalysis Water is generally excluded from an enzyme active site Water is generally excluded from an enzyme active site The chemical environment of an enzyme active site is more like that of found in organic solvents The chemical environment of an enzyme active site is more like that of found in organic solvents The proximity interactions perturbs the pKs of the amino acid side chains The proximity interactions perturbs the pKs of the amino acid side chains Interactions generally favor the TS Interactions generally favor the TS

118 Electrostatic Catalysis

119

120

121 1. General Acid Catalysis It donates a proton to substrate Enzyme active site residue must be protonated 2. General Base Catalysis It accepts a proton from the substrate Enzyme active site residue must be deprotonated 3. Concerted Acid-base Catalysis It is acid and base both participate in the reaction 4. General Acid-base Catalysis

122 1. General acid (a) Partial transfer for a proton from a Bronsted acid lowers the free energy of TS (b) Rate of reaction increases with decrease in pH and increase in [Bronsted acid] (b) Rate of reaction increases with decrease in pH and increase in [Bronsted acid] 2. Specific acid Protonation lowers the free energy of the TS Rate of reaction increases with decrease in pH General Acid-base Catalysis

123 3. General base Partial abstraction of a proton by a Bronsted base lowers the free energy of TS Rate of reaction increases with increase in Bronsted base 4. Specific base Abstraction of a proton (or nucleophilic attack) by OH- lowers the free energy of TS Rate of reaction increases with increase in pH General Acid-base Catalysis

124

125 General Acid-Base Catalysis General acid-base catalysis is involved in a majority of enzymatic reactions. General acid–base catalysis needs to be distinguished from specific acid–base catalysis. General acid-base catalysis is involved in a majority of enzymatic reactions. General acid–base catalysis needs to be distinguished from specific acid–base catalysis. Specific acid–base catalysis means specifically, – OH or H + accelerates the reaction. The reaction rate is dependent on pH only, and not on buffer concentration. Specific acid–base catalysis means specifically, – OH or H + accelerates the reaction. The reaction rate is dependent on pH only, and not on buffer concentration. In General acid–base catalysis, the buffer aids in stabilizing the transition state via donation or removal of a proton. Therefore, the rate of the reaction is dependent on the buffer concentration, as well as the appropriate protonation state. In General acid–base catalysis, the buffer aids in stabilizing the transition state via donation or removal of a proton. Therefore, the rate of the reaction is dependent on the buffer concentration, as well as the appropriate protonation state. Specific base catalysis General base catalysis

126

127 General Acid-Base Catalysis

128 Conventions for Describing General Acid/Base Catalysis The dehydration reaction below is catalyzed by an enzyme at pH 7 and 25°C. This reaction does not occur nonenzymatically under these conditions. Sketch a mechanism to show how an enzyme can easily catalyze this reaction.

129 Models for General Acid-Base Catalysis

130 Charge development in the TS can be decreased by either donation of a proton from general acids (like acetic acid or a protonated indole ring) to an atom such as a carbonyl O which develops a partial negative charge in the TS when it is attached by a nucleophile. Charge development in the TS can be decreased by either donation of a proton from general acids (like acetic acid or a protonated indole ring) to an atom such as a carbonyl O which develops a partial negative charge in the TS when it is attached by a nucleophile. General Acid-base Catalysis

131 Proton donation decreases the developing negative in the TS. Proton donation decreases the developing negative in the TS. Alternatively, a nucleophile such as water which develops a partial positive charge in the TS as it begins to form a bond to an electrophilic C in a carbonyl Alternatively, a nucleophile such as water which develops a partial positive charge in the TS as it begins to form a bond to an electrophilic C in a carbonyl General Acid-base Catalysis

132 Can be stabilized by the presence of a general base (such as acetate or the deprotonated indole ring). Can be stabilized by the presence of a general base (such as acetate or the deprotonated indole ring). Proton abstraction decreases the developing positive charge Proton abstraction decreases the developing positive charge General Acid-base Catalysis

133 Charge Development in TS for Ester Hydrolysis

134 Mechanism of General Acid Catalysis

135 Mechanism of General Base Catalysis

136 Acid-bases Catalysis The rate of proton transfer Mechanism: 1) The diffusion-controlled formation of a hydrogen bond between the base B – and the acid HA; 2) The transfer of a proton, leading to the formation of a new hydrogen bonded complex; 3) The diffusion-controlled dissociation of the product.

137 - proton transfer to N, O, S is fast; - proton transfer to carbon (C) is slow - proton transfer from H 3 O + to N or O is diffusion-controlled: see the Table on p 31, left column, k -1  l.mol -1.s -1 Formation of the hydrogen bond between the proton donor and the proton acceptor is the rate determining step: this step is slower than the actual proton transfer! Typical for cases where a proton is both bound to and transferred to oxygen or nitrogen atoms, where the negative charge is localised on one atom.

138 Nevertheless, diffusion of H + in water is much faster than diffusion of other ions:

139 Proton transfer from/to carbon acids/bases k 1 = 4x10 -8 l.mol -1.s -1, slow process due to: - lack of hydrogen bond formation before proton transfer - low acidity of the hydrogen atoms. k -1 = 16 l.mol -1.s -1, also much slower than diffusion rate, because: - upon protonation a considerable redistribution of charge has to take place, including the concomitant change of solvation. 12: carbon acid, k 1 and k -1 relatively small 23: oxygen acid, k 1 and k -1 relatively large (H-bond formation) Another example:

140 Influence of pH on reaction rate The hydrolysis of esters is catalysed by both acid and base: pH log k obs 7.0

141 Another example: the mutarotation of glucose: pH log k obs 7.0

142 Two mechanisms for acid catalysis Specific acid catalysis: - A proton is transferred to the substrate in a rapid preequilibrium; subsequently, the protonated substrate reacts further to the product(s) in the rate determining step: General acid catalysis: - Proton transfer occurs in a slow, rate determining step; subsequently, the protonated substrate rapidly reacts to give the product(s):

143 Specific acid/base catalysis Usually found for electronegative elements (O, N), where proton transfer is fast: The second step is rate determining and can be mono- or bimolecular: Reaction rate: since we can now write: So the rate is only dependent on the pH, not on [HA] !!

144 Example of specific acid catalysis: hydrolysis of acetals (A1 mech.) x x x x x x k obs [H + ] k obs is directly proportional to [H + ]; addition of more acid (buffer) at constant pH has no effect on k obs. Proton transfer is not rate limiting, so the mechanism probably reads: k obs [ClCH 2 COOH/ ClCH 2 COO - ] (2:1) x x x x xx

145 Example of a reaction that is specific acid catalysed according to the A2 mechanism: the hydrolysis of ethyl acetate:

146 Specific base catalysis Example: the retro-aldol reaction of I: x x x x x x k obs [OH - ] k obs is directly proportional to [OH - ]. Addition of more base (in buffer) at constant pH has no effect on k obs ; [OH - ] is the only base that occurs in the rate equation.

147 General acid/base catalysis Proton transfer is the rate determining step. Example: the hydrolysis of ortho esters: The reaction is studied in a series of buffers (m-NO 2 -Ph-OH/m-NO 2 -Ph-O – ): reaction rate increases with increasing buffer concentration, even if the pH remains constant = {k(H 2 O)·[H 2 O] + k(H 3 O + )·[H 3 O + ] + k(m-NO 2 -Ph-OH)·[m-NO 2 -Ph-OH]}·[III] [buffer] {k(H 3 O + )[H 3 O + ] + k(H 2 O)[H 2 O]}[III] k(buffer)

148 The relation between general and specific catalysis Why is there sometimes general and sometimes specific acid/base catalysis? pH: [H + ] and [OH - ] are very low in neutral solution, whereas [HA] or [B - ] can be high  beneficial for general catalysis rate of proton transfer: H transfer to and from C atoms is slower than transfer to N, O, etc.  beneficial for general catalysis stability of reaction intermediates plays an important role. Example: compare the hydrolysis of ortho esters and acetals

149 General acid/base catalysis by enzymes Enzymes often use general acid or base catalysis: They work at neutral pH, so low [H + ] and [OH - ] High effective concentration of general acid/base Correct orientation of the acidic/basic group around the substrate Optimum catalysis at pH around pK a Amino acid residues often have a pK a that is close to neutral pH and are therefore able to act as a general acid or base catalyst:

150 Prototropic groups of enzymes Amino acid Acidic group Basic grouppK a N-terminus  -NH 3 +  -NH C-terminus  -COOH  -COO – 3.8 aspartic acid  -COOH  -COO – 4.4 glutamic acid  -COOH  -COO – 4.6 histidine imidazolium ionimidazole7.0 cysteine–SH–S – 8.7 tyrosine–C 6 H 4 OH–C 6 H 4 O – 9.6 lysine  -NH 3 +  -NH serine  -OH  -O – 13 threonine  -OH  -O – 13 arginine –NH–(C=NH 2 + )NH 2 –NH–(C=NH)NH peptide bond R–CO–NH–R’R–CO–N – –R’14.8 The pK a is strongly influenced by its environment: e.g., in enzymes the pK a of lysine can drop to ~7

151 One way to change the activation energy of the reaction is to change the reaction mechanism in ways which introduces new steps with lower activation energy. One way to change the activation energy of the reaction is to change the reaction mechanism in ways which introduces new steps with lower activation energy. A typical way is to add a nucleophilic catalyst which forms a covalent intermediate with the reactant. A typical way is to add a nucleophilic catalyst which forms a covalent intermediate with the reactant. The original nucleophile can then interact with the intermediate in a nucleophilic substitution reaction. The original nucleophile can then interact with the intermediate in a nucleophilic substitution reaction. 5. Nucleophilic-Electrophilic (Covalent) Catalysis

152

153

154

155

156 Nucleophilic power There is no simple correlation between chemical structure and nucleophilic power. Nucleophilicity, among others, depends on: 1.The solvation energy of the nucleophile (which is influenced by the solvent); 2.The strength of the chemical bond to the electrophile (the C-Nu bond); 3.The size (steric hindrance); 4.The electronegativity and the polarisability of the nucleophilic atom in the nucleophile The effects of the latter factors have been quantified by Edwards:

157 Edwards equation: - k 0 is the rate constant of the reaction with a standard nucleophile (H 2 O) - P = polarisability, related to the refractive index: (R Nu = refractive index of the nucleophile) - H = basicity, related to the pK a : H = pK a  and  are dependent on the reaction (usually  >>  )  and  can be determined by performing a reaction of a substrate with a set of nucleophiles, like: Nucleophilic power =

158 What kind of groups in enzymes are good nucleophiles: Aspartate caboxylates Glutamates caboxylates Cystinethiol- Cystinethiol- Serinehydroxyl- Tyrosinehydroxyl- Lysineamino- Lysineamino- Histadineimidazolyl-

159 Nucleophile Groups

160 Electrophilie Groups

161 Covalent Catalysis If the nucleophilic catalyst is a better nucleophile than the original nucleophile (usually water) then the reaction is catalyzed. If the nucleophilic catalyst is a better nucleophile than the original nucleophile (usually water) then the reaction is catalyzed. The nucleophilic catalyst and the original nucleophile usually interact with a carbonyl C in a substitution reaction, initially forming the tetrahedral oxyanion intermediate. The nucleophilic catalyst and the original nucleophile usually interact with a carbonyl C in a substitution reaction, initially forming the tetrahedral oxyanion intermediate.

162 Covalent Catalysis

163 If an amine is used as the nucleophilic catalyst, then the initial addition product (a carbinolamine) can become dehydrated, If an amine is used as the nucleophilic catalyst, then the initial addition product (a carbinolamine) can become dehydrated, Since the free pair of electrons on the N are more likely to be shared with the carbon to form a double bond than electrons from the original carbonyl O, which is more electronegative than the N). Since the free pair of electrons on the N are more likely to be shared with the carbon to form a double bond than electrons from the original carbonyl O, which is more electronegative than the N). An imine or Schiff Base forms, with a pKa of about 7. An imine or Schiff Base forms, with a pKa of about 7. Covalent Catalysis

164 Mechanism of Schiff Base Formation

165 This is easily protonated to form a positively charged N at the former carbonyl O center. This is easily protonated to form a positively charged N at the former carbonyl O center. This serves as an excellent electron sink for decarboxylation reactions of beta-keto acids and illustrates an important point. This serves as an excellent electron sink for decarboxylation reactions of beta-keto acids and illustrates an important point. Electrons in chemical reactions can be viewed as flowing from a source (such as a carboxyl group) to a sink (such as an nucleophilic carbonyl O or a positively charged N in a Schiff base). Electrons in chemical reactions can be viewed as flowing from a source (such as a carboxyl group) to a sink (such as an nucleophilic carbonyl O or a positively charged N in a Schiff base).

166 Electron Flow (Electron Sink) In a subsequent section, we will discuss how protein enzymes use these same catalytic strategies. In a subsequent section, we will discuss how protein enzymes use these same catalytic strategies. An intriguing question arises: how much of the structure of a large protein is really needed for catalysis? Much work has been directed to the development of small molecule catalysis mimetics of large protein enzymes. An intriguing question arises: how much of the structure of a large protein is really needed for catalysis? Much work has been directed to the development of small molecule catalysis mimetics of large protein enzymes. Just how small can you go in reducing the size of a protein and still get catalysis. Just how small can you go in reducing the size of a protein and still get catalysis.

167 Electron Flow (Electron Sink) One important feature of enzyme catalysis is that they catalyze reactions in which only one enantiomer is produced. That is, the synthesis is assymertric. One important feature of enzyme catalysis is that they catalyze reactions in which only one enantiomer is produced. That is, the synthesis is assymertric. This is typically a consequence of the asymmetric enzyme (itself chiral) binding only one enantiomer as a reactant and/or the imposition of steric restrictions on the possible reactions of the bound substrate. This is typically a consequence of the asymmetric enzyme (itself chiral) binding only one enantiomer as a reactant and/or the imposition of steric restrictions on the possible reactions of the bound substrate. Recently, it has been show that L-Pro alone can act as such an assymetric catalyst in an aldol condensation reaction. Recently, it has been show that L-Pro alone can act as such an assymetric catalyst in an aldol condensation reaction.

168 Mechanism of Electron Sink

169 L-Proline Catalysis of Aldol Condensation Mechanism

170 How to distinguish between nucleophilic catalysis and general base catalysis? 1. “Common ion effect” Add anions that are identical to the leaving group in the reaction (assuming that the pK b of the leaving group is such that the group effectively acts as a base) and determine the reaction rate: - faster: general base catalysis, since addition of the leaving group increases the concentration of base in solution and =  [B i ][S]. - slower: nucleophilic catalysis, addition of extra leaving group drives the reaction equilibria back from product to the covalent intermediate.

171 Example: General base catalysis would involve an intermediate like: Mechanism of nucleophilic catalysis: A rate enhancement was found upon addition of F -  general base catalysis

172 2. Detection of a covalent intermediate is a proof for nucleophilic catalysis. The existence of the intermediate can be proven by: - isolation - spectroscopic detection:

173 - trapping, i.e. the in situ modification of the intermediate by a “trapping agent” that is deliberately added to the reaction mixture. With caution, the failure to detect an intermediate can be used as a proof for the occurrence of general base catalysis, e.g.:

174 3. Nonlinearity of the Brønsted plot: In general base catalysis there is a good correlation, data points (x) are on a straight line; in nucleophilic catalysis there are sometimes strong deviations (o). pK a (cat.) log k x x x x o o o o Reasons for deviations in the Brønsted plot: a) A difference in polarisability at the same pK a. Substrate k im/phosphate k OH - /im type of catalysis (~same pK a ) (~same nucleophilicity) ethyl acetate general base catalysis ethyl dichloroacetate general base catalysis p-nitrophenyl acetate nucleophilic catalysis acetic anhydride nucleophilic catalysis im = imidazole

175 b) Steric hindrance Not important for base catalysis (H-transfer), but very important in nucleophilic catalysis, e.g.: This reaction is not catalysed by sterically hindered bases like: c) The  -effect.

176 Substrate k H/D type of catalysis ethyl dichloroacetate3general base catalysis p-nitrophenyl acetate1nucleophilic catalysis 4. Determine the solvent isotope effect (H 2 O vs. D 2 O): The rate determining step in general base catalysis = cleavage of a O-H (O-D) bond, which is not the case in nucleophilic catalysis. E.g.: N.B.: the isotope effect can be obscured by solvation effects!

177 A metal such as Cu 2+ or Zn 2+ can also stabilize the TS. A metal such as Cu 2+ or Zn 2+ can also stabilize the TS. The metal must be able to be bound to the charged intermediate and hence the TS. The metal must be able to be bound to the charged intermediate and hence the TS. 6. Metal Ion Catalysis

178

179 The tetrahedral oxyanion intermediate of the reaction of an electrophilic carbonyl C can interact with a metal if there is an O on an adjacent atom which can help coordinate the metal ion. T The tetrahedral oxyanion intermediate of the reaction of an electrophilic carbonyl C can interact with a metal if there is an O on an adjacent atom which can help coordinate the metal ion. T His charge stabilization of the developing negative in the TS and the full negative in the intermediate is often called electrostatic catalysis. His charge stabilization of the developing negative in the TS and the full negative in the intermediate is often called electrostatic catalysis. 6. Metal Ion Catalysis

180 This method is likely to be found in many enzymes since nearly 1/3 of all enzymes require metal ions. This method is likely to be found in many enzymes since nearly 1/3 of all enzymes require metal ions. A classic example of an enzyme using metal ion catalysis is carboxypeptidase A. A classic example of an enzyme using metal ion catalysis is carboxypeptidase A. 6. Metal Ion Catalysis

181 Metal ion catalysis Roles of metals in catalysis: 1. As “super acid”: comparable to H + but stronger 2. As template: metal ions are able to coordinate to more than 2 ligands and can thereby bring molecules together 3. As redox catalyst: many metal ions can accept or donate electrons by changing their redox state Super acid catalysis Features: Introduces positive charge into the substrate, making it more susceptible toward nucleophilic attack. Exchange of metal ions is fast ( s -1 ), but slower than exchange of H + (10 11 s -1 )

182 Metal ion catalysis in C-C bond cleavage Decarboxylation of oxalosuccinate by isocitrate dehydrogenase: Mn 2+ is very well able to accept the developing negative charge (“electron sink”); M 3+ like Al 3+ are also good, M + like Na +, K + (and H + !) are much less effective. Other acceptable substrates: - both COO - and C=O are needed for correct binding of Mn 2+ - cleaving COO - group on  -position

183 Metal ion catalysis in additions to C=O(N) bonds Cu 2+ ions are very effective catalysts for the hydrolysis of  - amino acid esters: They are less effective in the hydrolysis of amides, because of a tighter bond between the metal and the substrate (= ground state stabilisation): E reaction co-ordinate amide ester uncatalysed catalysed

184 Metal ion catalysis in the hydrolysis of phosphate esters and anhydrides Hydrolysis of phosphate esters (e.g. acetyl phosphate) or anhydrides (e.g. ATP) is always catalysed by metal ions, usually Mg 2+ : The role of the metal ion is twofold: - neutralisation of the negative charge in the substrate, to enable the the approach of the nucleophile; - stabilisation of the leaving group (neutralisation of charge)

185 Metal Ion Catalysis (Stabilization of TS)

186 Binding Energy of ES Competing effects determine the position of ES on the energy scale Try to mentally decompose the binding effects at the active site into favorable and unfavorable Try to mentally decompose the binding effects at the active site into favorable and unfavorable The binding of S to E must be favorable The binding of S to E must be favorable But not too favorable! But not too favorable! K m cannot be "too tight" - goal is to make the energy barrier between ES and EX ‡ small K m cannot be "too tight" - goal is to make the energy barrier between ES and EX ‡ small

187

188

189 Entropy Loss and Destabilization of ES Raising the energy of ES raises the rate For a given energy of EX ‡, raising the energy of ES will increase the catalyzed rate For a given energy of EX ‡, raising the energy of ES will increase the catalyzed rate This is accomplished by This is accomplished by (a) loss of entropy due to formation of ES (a) loss of entropy due to formation of ES (b) destabilization of ES by (b) destabilization of ES by strain strain distortion distortion desolvation desolvation

190

191

192

193 Transition State Analogs Very tight binding to the active site! The affinity of the enzyme for the transition state may be M! The affinity of the enzyme for the transition state may be M! Can we see anything like that with stable molecules? Can we see anything like that with stable molecules? Transition state analogs (TSAs) do pretty well! Transition state analogs (TSAs) do pretty well! Proline racemase was the first case Proline racemase was the first case

194

195 Mechanism of Ribonuclease A 2,3’-cyclic phosphate Divalent TS stabilized by Lys-41

196 Mechanism of Acetoacetate Decarboxylase

197 Mechanism of Enolase

198 Mechanism of Carboxypeptidase A

199 Zn 2+ is acting as a Lewis acid Zn 2+ is acting as a Lewis acid It coordinates to the non-bonding electrons of carbonyl group It coordinates to the non-bonding electrons of carbonyl group Including charge separation and making the carbon more electrophilic or Including charge separation and making the carbon more electrophilic or More susceptible to nucleophilic attack More susceptible to nucleophilic attack

200 Mechanism of Carbonic Anhydrase

201 Zn 2+ function to make potential nucleophiles (such as water) more nucleophilic group. Zn 2+ function to make potential nucleophiles (such as water) more nucleophilic group. For example, the pKa of water drop from 15.7 to 6-7 when it is coordinate to Zn 2+ For example, the pKa of water drop from 15.7 to 6-7 when it is coordinate to Zn 2+ OH - is 4 orders of magnitude more nucleophilic than is water OH - is 4 orders of magnitude more nucleophilic than is water

202 Mult-Substrate Enzyme Mechanism In reality, many enzymes have more than one substrate (A, B) and more than one product (P, Q). In reality, many enzymes have more than one substrate (A, B) and more than one product (P, Q). For example, the enzyme alcohol dehydrogenase catalyzes the oxidation of ethanol with NAD (a biological oxidizing agent) to form acetaldehyde and NADH. For example, the enzyme alcohol dehydrogenase catalyzes the oxidation of ethanol with NAD (a biological oxidizing agent) to form acetaldehyde and NADH. How do you do enzymes kinetics on these more complicated systems? How do you do enzymes kinetics on these more complicated systems?

203 The answer is fairly straightforward. You keep one of the substrates (B) fixed, and vary the other substrate (A) and obtain a series of hyperbolic plots of v vs A at different fixed B concentrations. The answer is fairly straightforward. You keep one of the substrates (B) fixed, and vary the other substrate (A) and obtain a series of hyperbolic plots of v vs A at different fixed B concentrations. This would give a series of linear 1/v vs 1/A double-reciprocal plots (Lineweaver-Burk plots) as well. The pattern of Lineweaver-Burk plots depends on how the reactants and products interact with the enzyme. This would give a series of linear 1/v vs 1/A double-reciprocal plots (Lineweaver-Burk plots) as well. The pattern of Lineweaver-Burk plots depends on how the reactants and products interact with the enzyme. Mult-Substrate Enzyme Mechanism

204 1. Sequential Mechanism: 1. Sequential Mechanism: In this mechanism, both substrates must bind to the enzyme before any products are made and released. In this mechanism, both substrates must bind to the enzyme before any products are made and released. The substrates might bind to the enzyme in a random fashion (A first then B or vice- versa) or in an ordered fashion (A first followed by B). The substrates might bind to the enzyme in a random fashion (A first then B or vice- versa) or in an ordered fashion (A first followed by B).

205 Sequential Mechanism: Sequential Mechanism: An abbreviated notation scheme is shown below for the sequential random and sequential ordered mechanisms. An abbreviated notation scheme is shown below for the sequential random and sequential ordered mechanisms. For both mechanisms, Lineweaver-Burk plots at varying A and different fixed values of B give a series of intersecting lines. For both mechanisms, Lineweaver-Burk plots at varying A and different fixed values of B give a series of intersecting lines.

206 Sequential Mechanism: Sequential Mechanism:

207 Bi-substrate Enzyme Kinetics Sequential 1. ordered 2. random Ping-pong

208 Equations for Bi-substrate Kinetics V max [A][B] K a [B] + K b [A] + [A][B] v = [A][B] + K a [B] + K b [A] + K a K b V max [A][B] v = 1/[A] 1/v [B] 1/[A] 1/v Ping Pong Mechanism Sequential Mechanism

209 Secondary plot (Replot) 1/[B] Ping Pong Mechanism Sequential Mechanism Intercept Kb/V 1/V -1/Kb Slope 1/[B] Ka/V Intercept Slope 1/V Kb/V -1/Kb 1/[B] Ka/V KiaKb/V

210 Sequential Kinetics Sequential kinetics can be distinguished from ping-pong kinetics by initial rate studies. In practice, measure initial rates as a function of the concentration of one substrate while holding the concentration of the second constant. Next, vary the concentration of the second substrate and repeat.

211 Sequential Kinetics Lineweaver-Burk (double- reciprocal) analysis should yield a family of lines that intersect at the left of the y- axis of the graph. Within the realm of sequential reactions lies ordered sequential and random sequential at the extreme ends. The equations for the two are identical; therefore, simple initial rate studies cannot differentiate between the two.

212 Sequential Kinetics In ordered sequential reactions, one substrate is obligated to bind to the enzyme before a second substrate. In random sequential mechanisms there is no preference. In practice, there is usually some degree of order in binding.

213 Adenylate Kinase Kinetic Pathway Adenylate kinase displays a random ordered kinetic mechanism. In this case, the two substrates are bound randomly, and are in equilibrium with the “ternary complex” (EMgATPAMP). As in our derivation, this necessitates that the off rate for each of the substrates is less than the forward rate constant for the chemical step. This allows us to replace K m with K s. However, it would not be incorrect to use K m values. Below is typical shorthand notation for kinetic schemes.

214 Random E ES1S2 E + P1 + P2 ES1 ES2 S2 S1 e.g. hexokinase (E) catalyzed phosphorylation of glucose (S1) by ATP (S2)

215 Ordered E ES1S2 E + P1 + P2ES1 S2 S1 e.g. oxidation reactions involving nicotinamide adenine dinucleotide coenzyme

216 2. Ping Pong Mechanism In this mechanism, one substrate bind first to the enzyme followed by product P release. Typically, product P is a fragment of the original substrate A. The rest of the substrate is covalently attached to the enzyme E, which we now designate as E'. In this mechanism, one substrate bind first to the enzyme followed by product P release. Typically, product P is a fragment of the original substrate A. The rest of the substrate is covalently attached to the enzyme E, which we now designate as E'. Now the second reactant, B, binds and reacts with the enzyme to form a covalent adduct with the covalent fragment of A still attached to the enzyme to form product Q. Now the second reactant, B, binds and reacts with the enzyme to form a covalent adduct with the covalent fragment of A still attached to the enzyme to form product Q.

217 Ping Pong Mechanism This is now released and the enzyme is restored to its initial form, E. This mechanism is term ping-pong. This is now released and the enzyme is restored to its initial form, E. This mechanism is term ping-pong. An abbreviated notation scheme is shown below for the ping-pong mechanisms. For this mechanisms, Lineweaver-Burk plots at varying A and different fixed values of B give a series of parallel lines. An abbreviated notation scheme is shown below for the ping-pong mechanisms. For this mechanisms, Lineweaver-Burk plots at varying A and different fixed values of B give a series of parallel lines.

218 Ping Pong Mechanism Water (B) then comes in and covalently attacks the enzyme, forming an adduct with the phosphate which is covalently bound to the enzyme, releasing it as inorganic phosphate. Water (B) then comes in and covalently attacks the enzyme, forming an adduct with the phosphate which is covalently bound to the enzyme, releasing it as inorganic phosphate. In this particular example, however, you can't vary the water concentration and it would be impossible to generate the parallel Lineweaver- Burk plots characteristic of ping-pong kinetics. In this particular example, however, you can't vary the water concentration and it would be impossible to generate the parallel Lineweaver- Burk plots characteristic of ping-pong kinetics.

219 Ping Pong Mechanism

220 E E* E S2 EES1 S2 S1 P1 P2 H2OH2O OH + H e.g. Cleavage of polypeptide chain by serine protease

221 Ping-Pong Reaction

222 Galactose-1-P Uridylytransferase

223 Lysozyme Lysozyme hydrolyzes polysaccharide chains and ruptures certain bacterial cells by breaking down the cell wall Lysozyme hydrolyzes polysaccharide chains and ruptures certain bacterial cells by breaking down the cell wall Hen egg white enzyme has 129 residues with four disulfide bonds Hen egg white enzyme has 129 residues with four disulfide bonds The first enzyme whose structure was solved by X-ray crystallography (by David Phillips in 1965) The first enzyme whose structure was solved by X-ray crystallography (by David Phillips in 1965)

224

225

226 Substrate Analog Studies Natural substrates are not stable in the active site for structural studies Natural substrates are not stable in the active site for structural studies But analogs can be used - like (NAG) 3 But analogs can be used - like (NAG) 3 Fitting a NAG into the D site requires a distortion of the sugar Fitting a NAG into the D site requires a distortion of the sugar This argues for stabilization of a transition state via destabilization (distortion and strain) of the substrate This argues for stabilization of a transition state via destabilization (distortion and strain) of the substrate

227

228

229

230 The Lysozyme Mechanism Studies with 18 O-enriched water show that the C 1 -O bond is cleaved on the substrate between the D and E sites Studies with 18 O-enriched water show that the C 1 -O bond is cleaved on the substrate between the D and E sites This incorporates 18 O into C 1 This incorporates 18 O into C 1 Glu 35 acts as a general acid Glu 35 acts as a general acid Asp 52 stabilizes a carbonium ion intermediate Asp 52 stabilizes a carbonium ion intermediate

231

232

233 Mechanism of Lysozyme Asp-52 acting to stabilize positively charged intermediate at TS


Download ppt "Mechanism of Enzyme Action BCH 321 Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are not my own,"

Similar presentations


Ads by Google