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

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

3 Enzyme Catalysis

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5 Catalyst only lower DG ‡, but not effect on the EQM positions
Stabilization the transition state Destabilizing substrate bound at the binding site Destabilizing ES complex Forming an intermediate

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

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

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

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

10 Types of Enzymatic Catalysis
6. Metal ion Catalysis (a) Metalloenzymes Contain tightly bound metal cofactors such as Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, Co2+ (b) Metal Activated Enzymes Only loosely bind the metal ions. The ions are usually Na+, K+, Mg2+, or Ca2+

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

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13 Enzyme Catalysis

14 Enzyme Catalysis 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 K1, 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 = k1/k-1

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17 Catalytic Power Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates! Urease is a good example: Catalyzed rate: 3x104/sec Uncatalyzed rate: 3x10 -10/sec Ratio is 1x1014 !

18 Catalytic Power

19 Specificity Enzymes selectively recognize proper substrates over other molecules 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

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

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

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24 What Enzymes Do.... 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 Much more of this in Chapter 16!

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26 The Michaelis-Menten Equation
You should be able to derive this! Louis Michaelis and Maude Menten's theory It assumes the formation of an enzyme-substrate complex 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

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

29 The theoretical maximal velocity
Understanding Vmax The theoretical maximal velocity Vmax is a constant Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate Vmax 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 high, the equation for rate is 0-order in S The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!

31 A measure of catalytic activity
The turnover number A measure of catalytic activity kcat, 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, k2 = kcat = Vmax/Et Values of kcat range from less than 1/sec to many millions per sec

32 The catalytic efficiency
Name for kcat/Km An estimate of "how perfect" the enzyme is kcat/Km is an apparent second-order rate constant It measures how the enzyme performs when S is low The upper limit for kcat/Km 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 Hanes-Woolf Hanes-Woolf is best - why? Smaller and more consistent errors across the plot

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

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41 Two real, one hypothetical
Classes of Inhibition Two real, one hypothetical Competitive inhibition - inhibitor (I) binds only to E, not 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

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

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53 Mechanisms of Enzyme Action

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

55 Enzyme Catalysis 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.

56 Enzyme Catalysis 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.

57 Enzyme Catalysis 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. 

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

59 Approximation Catalysis
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. Correct orientation for bond formation. 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. If DG‡ is the change in free energy between the ground state and the transition state, then DG‡=DH‡–TDS‡. In solution, the transition state would be significantly more ordered than the ground state, and DS‡ 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. 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. This is paid for by binding energy.

62 Approximation Catalysis
Bimolecular reaction (high activation energy, low rate). Unimolecular reaction, rate enhanced by factor of 105 due to increased probability of collision/reaction of the 2 groups Constraint of structure to orient groups better (elimination of freedom of rotation around bonds between reactive groups), rate enhanced by another factor of 103, for 108 total rate enhancement over bimolecular reaction

63 Approximation Catalysis
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. Expose a water charged group on the substrate for interaction with the enzyme. Also lowers the entropy of the substrate (more ordered).

64 Approximation Catalysis
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. cyclic phosphate ester Acylic phospodiester Rate: 108 1

65 Strain and Distortion

66 1. Intramolecular Catalysis
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.

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). 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 Intramolecular Catalysis
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.

69 Intramolecular Catalysis
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.

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

71 Intramolecular Catalysis
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.

72 Intramolecular Catalysis
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.

73 Intramolecular Catalysis
First consider acyl transfer with aspirin derivatives. 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.

74 Intramolecular Catalysis
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.

75 Acyl Transfer Aspirin Derivatives
Intramolecular Intermolecular

76 Intramolecular Catalysis
The first order rate constant of the intramolecular transfer of the acetyl group to the carboxyl group, k1 = 0.02 s-1. The analogous bimolecular reaction rate constant k2~ M-1s-1. 

77 Intramolecular Catalysis
Dividing k1/k2 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.  This makes the effective concentration of the carboxylate in the aspirin derivative 2 x 107 M.

78 Mechanism of Acetate with Phenylacetate

79 2. Intermolecular Catalysis
Now consider the cleavage of phenylacetate using acetate as the nucleophile. The products are acetic anhydride and phenolate. This is a bimolecular reaction (a slow one at that), with a bimolecular rate constant, k2 which I will arbitrarily set to 1 for comparison to some similar reactions.

80 Intermolecular Catalysis
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.

81 Intermolecular Catalysis

82 Intermolecular Catalysis
If you assign a second order rate constant k2 = 1 M-1s-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 105 s-1. The ratio of rate constants, k1/k2 = 105 M.

83 Intermolecular Catalysis
That is it would take 105 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 k1/k2 = 108 M.

84 Intramolecular Catalysis

85 Intermolecular Catalysis
Another example is anhydride formation between two carboxyl groups. The DGo for such a reaction is positive, suggesting an unfavorable reaction. Consider two acetic acid molecules condensing to form acetic anhydride. For this intermolecular reaction, Keq = 3x10-12 M-1.

86 Intermolecular Catalysis
Now consider the analogous intramolecular reaction of the dicarboxylic acid succinic acid. It condenses in an intramolecular reaction to form succinic anhydride with a Keq = 8x10-7 (no units). The ratio Keq-intra/Keq inter = 3 x 105 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 105 M.

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

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

89 Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction

90 Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction
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 KINTRA/KINTER and KENZ/KINTER, respectively.

91 Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction
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.

92 Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction
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.

93 Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction
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.

94 Mechanism of Binding of Ca2+ and EDTA

95 Intramolecular, Intermolecular and Enzyme-Catalyzed Reaction
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 108 M.  Another way is to look at entropy changes associated with dimer formation. 

96 Entropy and Catalysis

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98 Orientation Effects In the non-enzymatic lactonization reaction shown below, the relative rate when R = CH3 is 3.4 x1011 times that when R = H. What is the explanation?

99 Models of Approximation (1)

100 Catalysis by Approximation
In order for a reaction to take place between two molecules, the molecules must first find each other. 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. 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-1min-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-1. 5. 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 k1/k2 = 2 x 105 M Effective concentration = 2 x 107 M

104 2. Preferential Binding of TS Catalysis
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 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. 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. In order for catalysis to be effective, the energy barrier between ES and EXt must be less than S and Xt.

107 Importance of Binding Energy
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 EXt, 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. 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 Transition State Stabilization
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.  Since DGo = -RTln Keq, Keq for binding of S* to E is greater than for S binding to E.

110 Enzyme Bind the TS Tightly

111 Transition State Stabilization
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.)  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. 

112 Transition State Stabilization
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 35Cl) using 37Cl- 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. 

113 Transition State Stabilization
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.

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‡ This means that the enzyme must stabilize the EX‡ transition state more than it stabilizes ES

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117 3. Electrostatic Catalysis
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 proximity interactions perturbs the pKs of the amino acid side chains Interactions generally favor the TS

118 Electrostatic Catalysis

119 Electrostatic Catalysis

120 Electrostatic Catalysis

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

122 General Acid-base Catalysis
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] 2. Specific acid Protonation lowers the free energy of the TS Rate of reaction increases with decrease in pH

123 General Acid-base Catalysis
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

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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. 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. Specific base catalysis General base catalysis

126 Figure 6.4 pH–rate profile for papain. The left and right segments of the bell-shaped curve represent the titrations of the side chains of active-site amino acids. The inflection point at pH 4.2 reflects the pKa of Cys-25, and the inflection point at pH 8.2 reflects the pKa of His-159. The enzyme is active only when these ionic groups are present as the thiolate–imidazolium ion pair.

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 General Acid-base Catalysis
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.

131 General Acid-base Catalysis
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

132 General Acid-base Catalysis
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

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 H3O+ to N or O is diffusion-controlled: see the Table on p 31, left column, k-1  1011 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
k1 = 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. Another example: 1 2: carbon acid, k1 and k-1 relatively small 2 3: oxygen acid, k1 and k-1 relatively large (H-bond formation)

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

141 Another example: the mutarotation of glucose:
pH log kobs 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.)
kobs [H+] kobs is directly proportional to [H+]; addition of more acid (buffer) at constant pH has no effect on kobs. Proton transfer is not rate limiting, so the mechanism probably reads: x x x x x x kobs [ClCH2COOH/ ClCH2COO-] (2:1)

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 kobs [OH-] kobs is directly proportional to [OH-]. Addition of more base (in buffer) at constant pH has no effect on kobs; [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: [buffer] n {k(H3O+)[H3O+] + k(H2O)[H2O]}[III] k(buffer) The reaction is studied in a series of buffers (m-NO2-Ph-OH/m-NO2-Ph-O–): reaction rate increases with increasing buffer concentration, even if the pH remains constant n = {k(H2O)·[H2O] + k(H3O+)·[H3O+] + k(m-NO2-Ph-OH)·[m-NO2-Ph-OH]}·[III]

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 pKa Amino acid residues often have a pKa 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 group pKa N-terminus a-NH3+ a-NH C-terminus a-COOH a-COO– 3.8 aspartic acid b-COOH b-COO– 4.4 glutamic acid g-COOH g-COO– 4.6 histidine imidazolium ion imidazole 7.0 cysteine –SH –S– 8.7 tyrosine –C6H4OH –C6H4O– 9.6 lysine e-NH3+ e-NH serine b-OH b-O– 13 threonine b-OH b-O– 13 arginine –NH–(C=NH2+)NH2 –NH–(C=NH)NH2 12.5 peptide bond R–CO–NH–R’ R–CO–N––R’ 14.8 The pKa is strongly influenced by its environment: e.g., in enzymes the pKa of lysine can drop to ~7

151 5. Nucleophilic-Electrophilic (Covalent) Catalysis
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. The original nucleophile can then interact with the intermediate in a nucleophilic substitution reaction.

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156 Nucleophilic power There is no simple correlation between chemical structure and nucleophilic power. Nucleophilicity, among others, depends on: The solvation energy of the nucleophile (which is influenced by the solvent); The strength of the chemical bond to the electrophile (the C-Nu bond); The size (steric hindrance); 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: Nucleophilic power =
- k0 is the rate constant of the reaction with a standard nucleophile (H2O) - P = polarisability, related to the refractive index: (RNu = refractive index of the nucleophile) - H = basicity, related to the pKa: H = pKa - a and b are dependent on the reaction (usually a >> b) a and b can be determined by performing a reaction of a substrate with a set of nucleophiles, like:

158 What kind of groups in enzymes are good nucleophiles:
Aspartate caboxylates Glutamates caboxylates Cystine thiol- Serine hydroxyl- Tyrosine hydroxyl- Lysine amino- Histadine imidazolyl-

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. 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 Covalent Catalysis 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).  An  imine or Schiff Base forms, with a pKa of about 7.

164 Mechanism of Schiff Base Formation

165 Mechanism of Schiff Base Formation
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. 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.  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. 

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.   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. 

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 pKb 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 n = S[Bi][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:
pKa (cat.) log k x o 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). Reasons for deviations in the Brønsted plot: a) A difference in polarisability at the same pKa. Substrate kim/phosphate kOH-/im type of catalysis (~same pKa) (~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 a-effect.

176 4. Determine the solvent isotope effect (H2O vs. D2O):
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.: Substrate kH/D type of catalysis ethyl dichloroacetate 3 general base catalysis p-nitrophenyl acetate 1 nucleophilic catalysis N.B.: the isotope effect can be obscured by solvation effects!

177 6. Metal Ion Catalysis A metal such as Cu2+ or Zn2+ can also stabilize the TS. The metal must be able to be bound to the charged intermediate and hence the TS.

178

179 6. Metal Ion Catalysis 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.

180 6. Metal Ion Catalysis 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.

181 Metal ion catalysis Roles of metals in catalysis: Super acid catalysis
As “super acid”: comparable to H+ but stronger As template: metal ions are able to coordinate to more than 2 ligands and can thereby bring molecules together 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+ (1011 s-1)

182 Metal ion catalysis in C-C bond cleavage
Decarboxylation of oxalosuccinate by isocitrate dehydrogenase: Mn2+ is very well able to accept the developing negative charge (“electron sink”); M3+ like Al3+ 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 Mn2+ - cleaving COO- group on b-position

183 Metal ion catalysis in additions to C=O(N) bonds
Cu2+ ions are very effective catalysts for the hydrolysis of a-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 Mg2+: 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 Competing effects determine the position of ES on the energy scale
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 The binding of S to E must be favorable But not too favorable! Km cannot be "too tight" - goal is to make the energy barrier between ES and EX‡ small

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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 This is accomplished by (a) loss of entropy due to formation of ES (b) destabilization of ES by strain distortion desolvation

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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! Can we see anything like that with stable molecules? Transition state analogs (TSAs) do pretty well! Proline racemase was the first case

194

195 Mechanism of Ribonuclease A
Divalent TS stabilized by Lys-41 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 Mechanism of Carboxypeptidase A
Zn2+ is acting as a Lewis acid It coordinates to the non-bonding electrons of carbonyl group Including charge separation and making the carbon more electrophilic or More susceptible to nucleophilic attack

200 Mechanism of Carbonic Anhydrase

201 Mechanism of Carbonic Anhydrase
Zn2+ 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 Zn2+ 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).  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? 

203 Mult-Substrate Enzyme Mechanism
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.

204 1. Sequential Mechanism:
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). 

205 Sequential Mechanism:
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.

206 Sequential Mechanism:

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

208 Equations for Bi-substrate Kinetics
Ping Pong Mechanism [B] 1/v Vmax[A][B] v = Ka[B] + Kb[A] + [A][B] 1/[A] Sequential Mechanism [B] Vmax[A][B] 1/v v = [A][B] + Ka[B] + Kb[A] + KaKb 1/[A]

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

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” (E•MgATP•AMP). 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 Km with Ks. However, it would not be incorrect to use Km values. Below is typical shorthand notation for kinetic schemes.

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

215 Ordered E E•S1•S2 E + P1 + P2 E•S1 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'.   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.    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.  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 Ping Pong Mechanism E E* E •S2 E E•S1 S2 S1 P1 P2
e.g. Cleavage of polypeptide chain by serine protease H2O OH + H

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 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)

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225

226 Substrate Analog Studies
Natural substrates are not stable in the active site for structural studies But analogs can be used - like (NAG)3 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

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229

230 The Lysozyme Mechanism
Studies with 18O-enriched water show that the C1-O bond is cleaved on the substrate between the D and E sites This incorporates 18O into C1 Glu35 acts as a general acid Asp52 stabilizes a carbonium ion intermediate

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232

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


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