Principles of Biochemistry Horton • Moran • Scrimgeour • Perry • Rawn Principles of Biochemistry Fourth Edition Chapter 6 Mechanisms of Enzymes Copyright © 2006 Pearson Prentice Hall, Inc.

The Terminology of Mechanistic Chemistry Nucleophilic Substitutions Cleavage Reactions Oxidation—Reduction Reactions

Nucleophilic substitution reaction Electron-rich, or nucleophilic Electron-poor, or electrophilic unstable, high-energy state Many chemical reactions have ionic intermediates.

Cleavage Reactions *both electrons can stay with one atom carbon atom retains both electrons carbon atom loses both electrons *one electron can remain with each atom A free radical, or radical, is a molecule or atom with an unpaired electron.

Oxidation—Reduction Reactions

Energy diagram for a single-step reaction short lifetimes: 10-14 to 10-13 sec lower activation barrier more stable transition state more often reaction proceed Figure 6.1 Energy diagram for a single-step reaction. The upper arrow shows the activation energy for the forward reaction. Molecules of substrate that have more free energy than the activation energy pass over the activation barrier and become molecules of product. For reactions with a high activation barrier, energy in the form of heat must be provided in order for the reaction to proceed.

Energy diagram for a reaction with an intermediate rate-determining step can be sufficiently stable to be detected or isolated Figure 6.2 Energy diagram for a reaction with an intermediate. The intermediate occurs in the trough between the two transition states. The rate-determining step in the forward direction is formation of the first transition state, the step with the higher energy transition state. S represents the substrate, and P represents the product. substrate product

Effect of reactant binding the activation energy is lowered Figure 6.3 Enzymatic catalysis of the reaction A + B --> A - B(a) Energy diagram for an uncatalyzed reaction. (b) Effect of reactant binding. Collection of the two reactants in the EAB complex properly positions them for reaction, makes formation of the transition state more frequent, and hence lowers the activation energy. (c) Effect of transition-state stabilization. An enzyme binds the transition state more tightly than it binds substrates, further lowering the activation energy. Thus, an enzymatic reaction has a much lower activation energy than an uncatalyzed reaction. (The breaks in the reaction curves indicate that the enzymes provide multistep pathways.)

Effect of transition-state stabilization Figure 6.3 Enzymatic catalysis of the reaction A + B --> A - B(a) Energy diagram for an uncatalyzed reaction. (b) Effect of reactant binding. Collection of the two reactants in the EAB complex properly positions them for reaction, makes formation of the transition state more frequent, and hence lowers the activation energy. (c) Effect of transition-state stabilization. An enzyme binds the transition state more tightly than it binds substrates, further lowering the activation energy further lowering the activation energy. Thus, an enzymatic reaction has a much lower activation energy than an uncatalyzed reaction. (The breaks in the reaction curves indicate that the enzymes provide multistep pathways.) An enzyme binds the transition state more tightly than it binds substrates, further lowering the activation energy further lowering the activation energy.

Chemical Modes of Enzymatic Catalysis The formation of an ES complex places reactants in proximity to reactive amino acid residues in the enzyme active site. Ionizable side chains participate in two kinds of chemical catalysis: acid–base catalysis and covalent catalysis. These are the two major chemical modes of catalysis. In addition to reactive amino acid residues, there may be metal ions or coenzymes in the active site.

an acceptor or a donor The active-site cavity of an enzyme is generally lined with hydrophobic amino acid residues.

Some amino acid residues participate directly in catalyzing reactions. Enzymes usually have 2 ~ 6 key catalytic residues. Most residues contribute in an indirect way by helping to maintain the correct three-dimensional structure of a protein. .

Site-Directed Mutagenesis Modifies Enzymes In vitro mutagenesis studies of enzymes have confirmed that the key residues absolutely essential for catalysis. directly involved in the catalytic mechanism acid or base catalyst or a nucleophile indirectly to assist or enhance the role of a key residue substrate binding stabilization of the transition state interacting with essential cofactors.

Acid–Base Catalysis The acceleration of a reaction is achieved by catalytic transfer of a proton. General acid–base catalysis rely on amino acid side chains that can donate and accept protons. Catalysis by H+ or OH- is termed specific acid or specific base catalysis. B: a base, or proton acceptor BH+: conjugate acid, a proton donor

A proton acceptor can assist reactions in two ways. Cleave O-H, N-H or C-H bonds by removing a proton. Cleavage of other bonds involving carbon through removal of a proton from a molecule of water.

A covalent bond may break more easily if one of its atoms is protonated.

Covalent Catalysis 1. A substrate is bound covalently to the enzyme to form a reactive intermediate. 2. A portion of the substrate is transferred from the intermediate to a second substrate.

pH Affects Enzymatic Rates The effect of pH on the reaction rate of an enzyme can suggest which ionizable amino acid residues are in its active site. Sensitivity to pH usually reflects an alteration in the ionization state of one or more residues involved in catalysis although occasionally substrate binding is affected.

pH–rate profile for papain 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. A plot of reaction velocity versus pH most often yields a bell-shaped curve provided the enzyme is not denatured when the pH is altered.

The activity of papain depends on two ionizable residues, histidine (His-159) and cysteine (Cys-25), in the active site His-159 Cys-25 Figure 6.6 The activity of papain depends on two ionizable residues, histidine (His-159) and cysteine (Cys-25), in the active site. Three ionic forms of these residues are shown. Only the upper tautomer of the middle pair is active.

Diffusion-Controlled Reactions A reaction that occurs with every collision between reactant molecules is termed a diffusion-controlled reaction. Under physiological conditions the diffusion-controlled rate has been calculated to be about 108 to 109 M-1 S-1. The frequency of encounter can be higher if there is electrostatic attraction between the reactants.

The binding of a substrate to an enzyme is a rapid reaction. If the rest of the reaction is simple and fast, the binding step may be the rate-determining step, and the overall rate of the reaction may approach the upper limit for catalysis.

Triose phosphate isomerase catalyzes the rapid interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) in the glycolysis and gluconeogenesis pathways.

General acid–base catalysis mechanism proposed for the reaction catalyzed by triose phosphate isomerase. When dihydroxyacetone phosphate binds, the carbonyl oxygen forms a hydrogen bond with the neutral imidazole group of His-95. The carboxylate group of Glu-165 removes a proton from C-1 of the substrate to form an enediolate intermediate. His-95 forms a strong hydrogen bond to the C-2 oxygen atom of the enediolate, and protonates this oxygen atom. Figure 6.7 General acid–base catalysis mechanism proposed for the reaction catalyzed by triose phosphate isomerase.

General acid–base catalysis mechanism proposed for the reaction catalyzed by triose phosphate isomerase. Next, the imidazolate form of His-95 abstracts a proton from the hydroxyl group at C-1 and shuttles the proton between oxygen atoms, producing another unstable enediolate intermediate. Glu-165 donates a proton to C-2, producing D-glyceraldehyde 3-phosphate. Figure 6.7 General acid–base catalysis mechanism proposed for the reaction catalyzed by triose phosphate isomerase.

Structure of yeast (Saccharomyces cerevisiae) triose phosphate isomerase Glu-165 His-95 Figure 6.8 Structure of yeast (Saccharomyces cerevisiae) triose phosphate isomerase. The location of the substrate is indicated by the space-filling model of a transition state analogue. The side chains of the catalytic residues are represented by stick models: Glu-165 (red), His-95 (purple). [PDB 2YPI] transition state analogue

The imidazolate form of a histidine residue The imidazolate form of a histidine residue is unusual; the triose phosphate isomerase mechanism was the first enzymatic mechanism in which this form was implicated. The hydrogen bonds formed between histidine and the intermediates in this mechanism appear to be unusually strong.

The reaction coordinate of triose phosphate isomerase In the mid-1970s, Jeremy Knowles and his coworkers determined the rate constants of all four kinetically measurable enzymatic steps in both directions.

Energy diagram for the reaction catalyzed by triose phosphate isomerase. Glu165->Asp165 (1000X slower) Figure 6.9 Energy diagram for the reaction catalyzed by triose phosphate isomerase. The solid line is the profile for the wild-type (naturally occurring) enzyme. The dotted line is the profile for a mutant enzyme in which the active-site glutamate residue has been replaced by an aspartate residue. In the mutant enzyme, the activation energies for the proton-transfer reactions (Steps 2 and 3 in Reaction 6.17) are significantly higher. [Adapted from Raines, R. T., Sutton, E. L., Strauss, D. R., Gilbert, W., and Knowles, J. R. (1986). Reaction energetics of a mutant triose phosphate isomerase in which the active-site glutamate has been changed to aspartate. Biochemistry 25:7142–7154.] wild-type

Superoxide Dismutase conversion of superoxide to molecular oxygen and hydrogen peroxide The reaction catalyzed by superoxide dismutase proceeds in two steps

Surface charge on human superoxide dismutase kcat /Km = 2*109 M-1 s-1 (faster than the substrate association with the enzyme based on typical diffusion rates) An electric field around the superoxide dismutase active site enhances the rate of formation of the ES complex about 30-fold. Figure 6.10 Surface charge on human superoxide dismutase. The structure of the enzyme is shown as a model that emphasizes the surface of the protein. Positively charged regions are colored blue and negatively charged regions are colored red. The copper atom at the active site is green. Note that the channel leading to the binding site is lined with hydrophilic residues. [PDB 1HL5] Blue: positively charged Red: negatively charged

The Proximity Effect For entropy traps: decreasing their entropy and increasing the probability of their interaction. The Proximity Effect: William Jencks and his colleagues two molecules at the active site work as an intramolecular (unimolecular) The acceleration is expressed in terms of the enhanced relative concentration, called the effective molarity, of the reacting groups in the unimolecular reaction.

Reactions of carboxylates with substituted phenyl esters Figure 6.11 Reactions of a series of carboxylates with substituted phenyl esters. The proximity effect is illustrated by the increase in rate observed when the reactants are held more rigidly in proximity. Reaction 4 is 50 million times faster than Reaction 1, the bimolecular reaction. The proximity effect is illustrated by the increase in rate observed when the reactants are held more rigidly in proximity.

Energy of substrate binding Enzyme-substrate weak binding Figure 6.12 Energy of substrate binding. In this hypothetical reaction, the enzyme accelerates the rate of the reaction by stabilizing the transition state. In addition, the activation barrier for formation of the transition state ES‡ from ES must be relatively low. If the enzyme bound the substrate too tightly (dashed profile), the activation barrier (2) would be comparable to the activation barrier of the nonenzymatic reaction (1). Enzyme-substrate tight binding

Yeast hexokinase contains two structural domains connected by a hinge region (induced fit) Open conformation Closed conformation glucose Figure 6.13 Yeast hexokinase. Yeast hexokinase contains two structural domains connected by a hinge region. On binding of glucose, these domains close, shielding the active site from water. (a) Open conformation. (b) Closed conformation. [PDB 2YHX and 1HKG].

2-Phosphoglycolate a transition-state analog for the enzyme triose phosphate isomerase Figure 6.14 2-Phosphoglycolate, a transition-state analog for the enzyme triose phosphate isomerase. 2-Phosphoglycolate is presumed to be an analog of C-2 and C-3 of the transition state (center) between dihydroxyacetone phosphate (right) and the initial enediolate intermediate in the reaction. 100 times tight binding

Inhibition of adenosine deaminase by a transition-state analog the transition state—through interaction with the hydroxyl group at C-6 Figure 6.15 Inhibition of adenosine deaminase by a transition-state analog. (a) In the deamination of adenosine, a proton is added to N-1 and a hydroxide ion is added to C-6 to form an unstable covalent hydrate, which decomposes to produce inosine and ammonia. (b) The inhibitor purine ribonucleoside also rapidly forms a covalent hydrate, 6-hydroxy-1,6-dihydropurine ribonucleoside. This covalent hydrate is a transition-state analog that binds more than a million times more avidly than another competitive inhibitor, 1,6-dihydropurine ribonucleoside (c), which differs from the transition-state analog only by the absence of the 6-hydroxyl group.

Amide hydrolysis catalyzed by an antibody Catalytic antibodies, with catalytic activity, can be induced by using transition-state analogs bound to carrier proteins as antigens. The catalytic antibody exhibits fairly narrow substrate specificity. The antibody uses covalent catalysis, possibly through acylation of a histidine residue. The antigen used to induce the antibody was a phosphonamidate coupled to a carrier protein. Figure 6.16 Amide hydrolysis catalyzed by an antibody. (a) The antigen used to induce the antibody was a phosphonamidate coupled to a carrier protein. (b) The substrate of the reaction catalyzed by the antibody is a p-nitroanilide.

Amide hydrolysis catalyzed by an antibody Figure 6.16 Amide hydrolysis catalyzed by an antibody. (a) The antigen used to induce the antibody was a phosphonamidate coupled to a carrier protein. (b) The substrate of the reaction catalyzed by the antibody is a p-nitroanilide. An catalytic antibody was isolated and found to catalyze the hydrolysis of the synthetic amide, with the greatest rates at pH values of 9 or above. At 37°C, the catalytic activity is about 1/25 of the chymotrypsin-catalyzed hydrolysis of an amide at 25°C and pH 7, which shows that the antibody is a potent catalyst.

Lysozyme Lysozyme catalyzes the hydrolysis of some polysaccharides, especially those that make up the cell walls of bacteria. Lysozyme causes lysis, or disruption, of bacterial cells. Many secretions such as tears, saliva, and nasal mucus contain lysozyme activity to help prevent bacterial infection. Lysozyme specifically catalyzes hydrolysis of the glycosidic bond between C-1 of a MurNAc residue and the oxygen atom at C-4 of a GlcNAc residue.

Structure of a four-residue portion of a bacterial cell-wall polysaccharide Figure 6.17 Structure of a four-residue portion of a bacterial cell-wall polysaccharide. Lysozyme catalyzes hydrolytic cleavage of the glycosidic bond between C-1 of MurNAc and the oxygen atom involved in the glycosidic bond.

Lysozyme from chicken with a trisaccharide molecule Figure 6.18 Lysozyme from chicken with a trisaccharide molecule (pink). The ligand is bound in sites A, B, and C. Three more monosaccharide residues can fit into a model of this active site, but the sugar residue in site D must be distorted. [PDB 1HEW]. trisaccharide molecule

Conformations of N-acetylmuramic acid Figure 6.19 Conformations of N-acetylmuramic acid. (a) Chair conformation. (b) Half-chair conformation proposed for the sugar bound in site D of lysozyme. R represents the lactyl group of MurNAc. proposed for the sugar bound in site D of lysozyme

Mechanism of lysozyme Figure 6.20 Mechanism of lysozyme. R1 represents the lactyl group, and R2 represents the N-acetyl group of MurNAc. The substrate-binding cleft of lysozyme accommodates six saccharide residues at six sites (designated A through F).

Mechanism of lysozyme Figure 6.20 Mechanism of lysozyme. R1 represents the lactyl group, and R2 represents the N-acetyl group of MurNAc.

Mechanism of lysozyme Figure 6.20 Mechanism of lysozyme. R1 represents the lactyl group, and R2 represents the N-acetyl group of MurNAc.

Proposed Transition State for a Bimolecular Reaction catalyzed by arginine kinase Proposed Transition State for a Bimolecular Reaction

Properties of Serine Proteases Cleave the peptide bond of proteins Serine residue in their active sites Ex: trypsin, chymotrypsin, and elastase

Activation of some pancreatic zymogens cleavage of the Lys-6—Ile-7 bond of trypsinogen Figure 6.21 Activation of some pancreatic zymogens. Initially, enteropeptidase catalyzes the activation of trypsinogen to trypsin. Trypsin then activates chymotrypsinogen, proelastase, and additional trypsinogen molecules.

The catalytic-site residues Asp-102, His-57, and Ser-195 Polypeptide chains of chymotrypsinogen (blue) and -chymotrypsin (green) Figure 6.22 Polypeptide chains of chymotrypsinogen (blue) [PDB 2CGA] and -chymotrypsin (green) [PDB 5CHA]. Ile-16 and Asp-194 in both zymogen and the active enzyme are shown in yellow. The catalytic-site residues (Asp-102, His-57, and Ser-195) are shown in red. The catalytic-site residues Asp-102, His-57, and Ser-195

Comparison of the polypeptide backbones chymotrypsin trypsin elastase Figure 6.23 Comparison of the polypeptide backbones of (a) chymotrypsin [PDB 5CHA] (b) trypsin [PDB 1TLD] and (c) elastase [PDB 3EST]. Residues at the catalytic center are shown in red. Residues at the catalytic center are shown in red

Substrate Specificity of Serine Proteases Different binding sites of chymotrypsin, trypsin, and elastase shallow binding pocket For glycine and alanine Figure 6.24 Binding sites of chymotrypsin, trypsin, and elastase. The differing binding sites of these three serine proteases are primary determinants of their substrate specificities. (a) Chymotrypsin has a hydrophobic pocket that binds the side chains of aromatic or bulky hydrophobic amino acid residues. (b) A negatively charged aspartate residue at the bottom of the binding pocket of trypsin allows trypsin to bind the positively charged side chains of lysine and arginine residues. (c) In elastase, the side chains of a valine and a threonine residue at the binding site create a shallow binding pocket. Elastase binds only amino acid residues with small side chains, especially glycine and alanine residues. hydrophobic pocket lysine and arginine only

The catalytic site of chymotrypsin The imidazole ring of His-57 removes the proton from the hydroxymethyl side chain of Ser-195, thereby making Ser-195 a powerful nucleophile. Figure 6.25 The catalytic site of chymotrypsin. The active-site residues Asp-102, His-57, and Ser-195 are arrayed in a hydrogen-bonded network. The conformation of these three residues is stabilized by a hydrogen bond between the carbonyl oxygen of the carboxylate side chain of Asp-102 and the peptide-bond nitrogen of His-57. Oxygen atoms of the active-site residues are red, and nitrogen atoms are dark blue. [PDB 5CHA].

Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond The noncovalent enzyme-substrate complex is formed. R1 group binding in the specificity pocket (shaded). The carbonyl carbon of the scissile peptide bond next to the oxygen of Ser-195. Figure 6.27 Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond.

Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond The raised pKa of His-57 enables the imidazole ring to remove a proton from the hydroxyl group of Ser-195. The nucleophilic oxygen of Ser-195 attacks the carbonyl carbon of the peptide bond to form a tetrahedral intermediate (ETI1). Figure 6.27 Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond.

Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond The imidazolium ring of His-57 acts as an acid catalyst, donating a proton to the nitrogen of the scissile peptide bond, thus facilitating its cleavage. Figure 6.27 Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond.

Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond Figure 6.27 Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond.

Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond His-57, once again an imidazolium ion, donates a proton, leading to the collapse of the second tetrahedral intermediate. Figure 6.27 Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond. A second tetrahedral intermediate (E-TI2) is formed and stablized by the oxyanion hole.

Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond The carboxylate product is released from the active site, and free chymotrypsin is regenerated. Figure 6.27 Mechanism of chymotrypsin-catalyzed cleavage of a peptide bond.