Prentice Hall c2002Chapter 61 Principles of Biochemistry Fourth Edition Chapter 6 Mechanisms of...

Prentice Hall c2002 Chapter 6 1

Principles of BiochemistryFourth Edition

Chapter 6Mechanisms of Enzymes

Copyright © 2006 Pearson Prentice Hall, Inc.

Horton • Moran • Scrimgeour • Perry • Rawn


Chapter 6 Mechanisms of Enzymes

• Mechanisms - the molecular details of catalyzed reactions

• Enzyme mechanisms deduced from:

Kinetic experiments

Protein structural studies

Studies of nonenzymatic model systems


6.1 The Terminology of Mechanistic Chemistry

• A reaction mechanism describes the details of a reaction

• Reactants, products and intermediates are identified

• Enzymatic mechanisms use the same symbolism used in organic chemistry


Nucleophilic substitution reactions

• Nucleophilic species are electron rich, and electrophilic species are electron poor

• Types of nucleophilic substitution reactions include:

(1) Formation of a tetrahedral intermediate by nucleophilic substitution

(2) Direct displacement via a transition state


Two types of nucleophilic substitution reactions

• Formation of a tetrahedral intermediate

• Direct displacement


Cleavage reactions

• Carbanion formation

• Carbocation formation

• Free radical formation


Oxidation-reduction reactions

• Electrons are transferred between two species

• Oxidizing agent gains electrons (is reduced)

• Reducing agent donates electrons (is oxidized)

• Formaldehyde is oxidized by oxygen (below)


6.2 Catalysts Stabilize Transition States

Fig 6.1 Energy diagram for a single-step reaction


Fig 6.2 Energy diagram for reaction with intermediate

• Intermediate occurs in the trough between the two transition states

• Rate determining step in the forward direction is formation of the first transition state


Enzymes lower the activation energy of a reaction

(1) Substrate binding

• Enzymes properly position substrates for reaction(makes the formation of the transition state more frequent and lowers the energy of activation)

(2) Transition state binding

• Transition states are bound more tightly than substrates (this also lowers the activation energy)


Fig 6.3 Enzymatic catalysis of the reaction A+B A-B


6.3 Chemical Modes of Enzymatic Catalysis

A. Polar Amino Acid Residues in Active Sites

• Active-site cavity of an enzyme is lined with hydrophobic amino acids

• Polar, ionizable residues at the active site participate in the mechanism

• Anions and cations of certain amino acids are commonly involved in catalysis


Table 6.1


Table 6.2 pKa Values of amino acid ionizable groups in proteins

Group pKa

Terminal -carboxyl 3-4

Side-chain carboxyl 4-5

Imidazole 6-7

Terminal -amino 7.5-9

Thiol 8-9.5

Phenol 9.5-10

-Amino ~10

Guanidine ~12

Hydroxymethyl ~16


B. Acid-Base Catalysis

• Reaction acceleration is achieved by catalytic transfer of a proton

• A general base (B:) can act as a proton acceptor to remove protons from OH, NH, CH or other XH

• This produces a stronger nucleophilic reactant (X:-)


General base catalysis reactions (continued)

• A general base (B:) can remove a proton from water and thereby generate the equivalent of

OH- in neutral solution


Proton donors can also catalyze reactions

• A general acid (BH+) can donate protons

• A covalent bond may break more easily if one of its atoms is protonated (below)


C. Covalent Catalysis

• All or part of a substrate is bound covalently to the enzyme to form a reactive intermediate

• Group X can be transferred from A-X to B in two steps via the covalent ES complex X-E

A-X + E X-E + A

X-E + B B-X + E


Sucrose phosphorylase exhibits covalent catalysis

Step one: a glucosyl residue is transferred to enzyme

*Sucrose + Enz Glucosyl-Enz + Fructose

Step two: Glucose is donated to phosphate

Glucosyl-Enz + Pi Glucose 1-phosphate + Enz

*(Sucrose is composed of a glucose and a fructose)


D. pH Affects Enzymatic Rates

• Catalytic mechanisms are dependent upon the state of ionization of catalytic groups

• Plots of reaction velocity versus pH can indicate important ionizable catalytic groups


Fig 6.4 pH-rate profile for papain

• The two inflection points approximate the pKa values of the two ionizable residues


Fig 6.5

• Papain’s activity depends upon ionizable residues:His-159 and Cys-25

(a) Ribbon model(b) Active site residues

(N blue, S yellow)


Fig. 6.6

Three ionic forms of papain. Only the upper tautomer of the middle pair is active


6.4 Diffusion-Controlled Reactions

• Enzyme rates can approach the physical limit of the rate of diffusion of two molecules in solution

• Under physiological conditions the encounter frequency is about 108 to 109 M-1s-1

• A few enzymes have rate-determining steps that are roughly as fast as the binding of substrates to the enzymes


Table 6.4


A. Triose Phosphate Isomerase (TPI)

• TPI catalyzes a rapid aldehyde-ketone interconversion


Role of active site residues in TPI catalysis

• TPI has two ionizable active-site residues

• Glu acts as a general acid-base catalyst

• His shuttles protons between oxygens of an enzyme-bound intermediate


Fig 6.7 Proposed mechanism for TPI

• General acid-base catalysis mechanism (4 slides)


Fig 6.7 TPI mechanism (continued)


Fig 6.9 Energy diagram for the TPI reaction


B. Superoxide Dismutase

• Superoxide dismutase is a very fast catalyst because: (1) The negatively charged substrate is

attracted by a positively charged electric field near the active site

(2) The reaction itself is very fast


Two step reaction of superoxide dismutase

• An atom of copper bound to the enzyme is reduced and then oxidized


6.5 Binding Modes of Enzymatic Catalysis

• Proper binding of reactants in enzyme active sites provides substrate specificity and catalytic power

• Two catalytic modes based on binding properties can each increase reaction rates over 10,000-fold :

(1) Proximity effect - collecting and positioning substrate molecules in the active site

(2) Transition-state (TS) stabilization - transition states bind more tightly than substrates


Binding forces utilized for catalysis

1. Charge-charge interactions

2. Hydrogen bonds

3. Hydrophobic interactions

4. Van der Waals forces


A. The Proximity Effect

• Correct positioning of two reacting groups (in model reactions or at enzyme active sites):

(1) Reduces their degrees of freedom

(2) Results in a large loss of entropy

(3) The relative enhanced concentration of substrates (“effective molarity”) predicts the rate acceleration expected due to this effect


Effective molarity

k1(s-1)

Effective molarity =

k2(M-1s-1)


Fig 6.11 Reactions of carboxylates with phenyl esters

• Increased rates are seen when the reactants are held more rigidly in proximity (continued next slide)


Fig. 6.11 (continued)


B. Weak Binding of Substrates to Enzymes

• Energy is required to reach the transition state from the ES complex

• Excessive ES stabilization would create a “thermodynamic pit” and mean little or no catalysis

• Most Km values (substrate dissociation constants) indicate weak binding to enzymes


Fig 6.12 Energy of substrate binding

• If an enzyme binds the substrate too tightly (dashed profile), the activation barrier (2) could be similar to that of the uncatalyzed reaction (1)


C. Induced Fit

• Induced fit activates an enzyme by substrate-initiated conformation effect

• Induced fit is a substrate specificity effect, not a catalytic mode

• Hexokinase mechanism requires sugar-induced closure of the active site

Glucose + ATP Glucose 6-phosphate + ADP


Fig 6.13 Stereo views of yeast hexokinase

• Yeast hexokinase contains 2 domains connected by a hinge region. Domains close on glucose binding.

(a) Open conformation

(b) Closed conformation


D. Transition-State (TS) Stabilization

• An increased interaction of the enzyme and substrate occurs in the transition-state (ES‡)

• The enzyme distorts the substrate, forcing it toward the transition state

• An enzyme must be complementary to the transition-state in shape and chemical character

• Enzymes may bind their transition states 1010 to 1015 times more tightly than their substrates


Transition-state (TS) analogs

• Transition-state analogs are stable compounds whose structures resemble unstable transition states

• Fig. 6.14 2-Phosphoglycolate, a TS analog for the enzyme triose phosphate isomerase


Fig 6.15 Inhibition of adenosine deaminase by a TS analog


Catalytic antibodies

• Fig 6.16 Antibody-catalyzed amide hydrolysis

(a) TS analog antigen

(b) Reaction catalyzed by the catalytic antibody


6.6 Lysozyme Binds an Ionic Intermediate Tightly

• Lysozyme binds polysaccharide substrates (the sugar in subsite D of lysozyme is distorted into a half-chair conformation)

• Binding energy from the sugars in the other subsites provides the energy necessary to distort sugar D

• Lysozyme binds the distorted transition-state type structure strongly


Fig 6.17 Bacterial cell-wall polysaccharide

• Lysozyme cleaves bacterial cell wall polysaccharides (a four residue portion of a bacterial cell wall with lysozyme cleavage point is shown below)


Fig 6.18 Lysozyme from chicken with a triaccharide molecule (pink)


Fig 6.19 Conformations of N-acetylmuramic acid

(a) Chair conformation

(b) D-Site sugar residue is distorted into a higher energy half-chair conformation


Lysozyme reaction mechanisms

1. Proximity effects

2. Acid-base catalysis

3. TS stabilization (or substrate distortion toward the transition state)


Fig 6.20 Mechanism of lysozyme (2 slides)


6.7 Properties of Serine Proteases

A. Zymogens Are Inactive Enzyme Precursors

• Digestive serine proteases including trypsin, chymotrypsin, and elastase are synthesized and stored in the pancreas as zymogens

• Storage of hydrolytic enzymes as zymogens prevents damage to cell proteins

• Zymogens are activated by selective proteolysis


Fig 6.21 Activation of some pancreatic zymogens


Fig 6.22 Stereo view of the backbones of chymotrypsinogen and -chymotrypsin

Chymotrypsinogen (blue), -chymotrypsin (green)Catalytic Asp, His, Ser (red), Ile-16, Asp-194 (yellow)


B. Substrate Specificity of Serine Proteases

• Many digestive proteases share similarities in 1o,2o and 3o structure

• Chymotrypsin, trypsin and elastase have a similar backbone structure

• Active site substrate specificities differ due to relatively small differences in specificity pockets


Fig 6.23 Comparison of the backbones of (a) chymotrypsin (b) trypsin and (c) elastase

• Backbone conformations and active-site residues (red) are similar in these three enzymes


Fig 6.24 Binding sites of chymotrypsin, trypsin, and elastase

• Substrate specificities are due to relatively small structural differences in active-site binding cavities


C. Serine Proteases Use Both the Chemical and Binding Modes of Catalysis

• -Chymotrypsin active site groups include:

Ser-195 - identified by covalent DFP labeling

His-57 - identified by affinity labeling with TosPheCH2Cl

Asp-102 - identified by X-ray crystallography


Fig 6.25 Stereo view of the catalytic site of chymotrypsin

• Active-site Asp-102, His-57, Ser-195 are arrayed in a hydrogen-bonded network (O red, N blue)


Fig 6.26 Catalytic triad of chymotrypsin

• Imidazole ring (His-57) removes H from Ser-195 hydroxyl to make it a strong nucleophile (-CH2O-)

• Buried carboxylate (Asp-102) stabilizes the positively-charged His-57 to facilitate serine ionization


Fig 6.27 -Chymotrypsin mechanism (8 slides)

Step (1): E + S


Fig 6.27 (E-S)


Fig 6.27 (E-TI1)


Fig 6.27 (Acyl E + P1)


Fig 6.27 (Acyl E + H2O)

(4)


Fig 6.27 (E-TI2)

(5)


Fig 6.27 (E-P2)

(6)


Fig 6.27 (E + P2)

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