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Horton • Moran • Scrimgeour • Perry • Rawn Principles of Biochemistry Fourth Edition Chapter 6 Mechanisms of Enzymes Prentice Hall c2002 Chapter 6 Copyright © 2006 Pearson Prentice 1 Hall, Inc. 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 Prentice Hall c2002 Chapter 6 2 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 Prentice Hall c2002 Chapter 6 3 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 Prentice Hall c2002 Chapter 6 4 Two types of nucleophilic substitution reactions • Formation of a tetrahedral intermediate • Direct displacement Prentice Hall c2002 Chapter 6 5 Cleavage reactions • Carbanion formation • Carbocation formation • Free radical formation Prentice Hall c2002 Chapter 6 6 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) Prentice Hall c2002 Chapter 6 7 6.2 Catalysts Stabilize Transition States Fig 6.1 Energy diagram for a singlestep reaction Prentice Hall c2002 Chapter 6 8 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 Prentice Hall c2002 Chapter 6 9 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) Prentice Hall c2002 Chapter 6 10 Fig 6.3 Enzymatic catalysis of the reaction A+B A-B Prentice Hall c2002 Chapter 6 11 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 Prentice Hall c2002 Chapter 6 12 Table 6.1 Prentice Hall c2002 Chapter 6 13 Table 6.2 pKa Values of amino acid ionizable groups in proteins Group Terminal a-carboxyl Side-chain carboxyl Imidazole Terminal a-amino Thiol Phenol pKa 3-4 4-5 6-7 7.5-9 8-9.5 9.5-10 e-Amino ~10 Guanidine ~12 Hydroxymethyl ~16 Prentice Hall c2002 Chapter 6 14 Prentice Hall c2002 Chapter 6 15 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:-) Prentice Hall c2002 Chapter 6 16 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 Prentice Hall c2002 Chapter 6 17 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) Prentice Hall c2002 Chapter 6 18 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 Prentice Hall c2002 Chapter 6 19 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) Prentice Hall c2002 Chapter 6 20 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 Prentice Hall c2002 Chapter 6 21 Fig 6.4 pH-rate profile for papain • The two inflection points approximate the pKa values of the two ionizable residues Prentice Hall c2002 Chapter 6 22 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) Prentice Hall c2002 Chapter 6 23 Fig. 6.6 Three ionic forms of papain. Only the upper tautomer of the middle pair is active Prentice Hall c2002 Chapter 6 24 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 Prentice Hall c2002 Chapter 6 25 Table 6.4 Prentice Hall c2002 Chapter 6 26 A. Triose Phosphate Isomerase (TPI) • TPI catalyzes a rapid aldehyde-ketone interconversion Prentice Hall c2002 Chapter 6 27 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 Prentice Hall c2002 Chapter 6 28 Fig 6.7 Proposed mechanism for TPI • General acid-base catalysis mechanism (4 slides) Prentice Hall c2002 Chapter 6 29 Fig 6.7 TPI mechanism (continued) Prentice Hall c2002 Chapter 6 30 Fig 6.7 TPI mechanism (continued) Prentice Hall c2002 Chapter 6 31 Fig 6.7 TPI mechanism (continued) Prentice Hall c2002 Chapter 6 32 Prentice Hall c2002 Chapter 6 33 Fig 6.9 Energy diagram for the TPI reaction Prentice Hall c2002 Chapter 6 34 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 Prentice Hall c2002 Chapter 6 35 Two step reaction of superoxide dismutase • An atom of copper bound to the enzyme is reduced and then oxidized Prentice Hall c2002 Chapter 6 36 Prentice Hall c2002 Chapter 6 37 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 Prentice Hall c2002 Chapter 6 38 Binding forces utilized for catalysis 1. Charge-charge interactions 2. Hydrogen bonds 3. Hydrophobic interactions 4. Van der Waals forces Prentice Hall c2002 Chapter 6 39 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 Prentice Hall c2002 Chapter 6 40 Effective molarity k1(s-1) Effective molarity = k2(M-1s-1) Prentice Hall c2002 Chapter 6 41 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) Prentice Hall c2002 Chapter 6 42 Fig. 6.11 (continued) Prentice Hall c2002 Chapter 6 43 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 Prentice Hall c2002 Chapter 6 44 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) Prentice Hall c2002 Chapter 6 45 C. Induced Fit • Induced fit activates an enzyme by substrateinitiated 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 Prentice Hall c2002 Glucose 6-phosphate + ADP Chapter 6 46 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 Prentice Hall c2002 Chapter 6 47 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 Prentice Hall c2002 Chapter 6 48 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 Prentice Hall c2002 Chapter 6 49 Fig 6.15 Inhibition of adenosine deaminase by a TS analog Prentice Hall c2002 Chapter 6 50 Catalytic antibodies • Fig 6.16 Antibodycatalyzed amide hydrolysis (a) TS analog antigen (b) Reaction catalyzed by the catalytic antibody Prentice Hall c2002 Chapter 6 51 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 Prentice Hall c2002 Chapter 6 52 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) Prentice Hall c2002 Chapter 6 53 Fig 6.18 Lysozyme from chicken with a triaccharide molecule (pink) Prentice Hall c2002 Chapter 6 54 Fig 6.19 Conformations of N-acetylmuramic acid (a) Chair conformation (b) D-Site sugar residue is distorted into a higher energy halfchair conformation Prentice Hall c2002 Chapter 6 55 Lysozyme reaction mechanisms 1. Proximity effects 2. Acid-base catalysis 3. TS stabilization (or substrate distortion toward the transition state) Prentice Hall c2002 Chapter 6 56 Fig 6.20 Mechanism of lysozyme (2 slides) Prentice Hall c2002 Chapter 6 57 Prentice Hall c2002 Chapter 6 58 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 Prentice Hall c2002 Chapter 6 59 Fig 6.21 Activation of some pancreatic zymogens Prentice Hall c2002 Chapter 6 60 Fig 6.22 Stereo view of the backbones of chymotrypsinogen and a-chymotrypsin Chymotrypsinogen (blue), a-chymotrypsin (green) Catalytic Asp, His, Ser (red), Ile-16, Asp-194 (yellow) Prentice Hall c2002 Chapter 6 61 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 Prentice Hall c2002 Chapter 6 62 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 Prentice Hall c2002 Chapter 6 63 Fig 6.24 Binding sites of chymotrypsin, trypsin, and elastase • Substrate specificities are due to relatively small structural differences in active-site binding cavities Prentice Hall c2002 Chapter 6 64 C. Serine Proteases Use Both the Chemical and Binding Modes of Catalysis • a-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 Prentice Hall c2002 Chapter 6 65 Fig 6.25 Stereo view of the catalytic site of chymotrypsin • Active-site Asp102, His-57, Ser195 are arrayed in a hydrogenbonded network (O red, N blue) Prentice Hall c2002 Chapter 6 66 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 positivelycharged His-57 to facilitate serine ionization Prentice Hall c2002 Chapter 6 67 Fig 6.27 a-Chymotrypsin mechanism (8 slides) Step (1): E + S Prentice Hall c2002 Chapter 6 68 Fig 6.27 (E-S) Prentice Hall c2002 Chapter 6 69 Fig 6.27 (E-TI1) Prentice Hall c2002 Chapter 6 70 Fig 6.27 (Acyl E + P1) Prentice Hall c2002 Chapter 6 71 Fig 6.27 (Acyl E + H2O) (4) Prentice Hall c2002 Chapter 6 72 Fig 6.27 (E-TI2) (5) Prentice Hall c2002 Chapter 6 73 Fig 6.27 (E-P2) (6) Prentice Hall c2002 Chapter 6 74 Fig 6.27 (E + P2) Prentice Hall c2002 Chapter 6 75