Chapter 6 Enzymes
4. Examples of enzymatic reactions acid-base catalysis: give and take protons covalent catalysis: a transient covalent bond is formed between the enzyme and the substrate metal ion catalysis: metal ion participate in the catalysis
General Acid-base Catalysis Consider ester hydrolysis: O R O CH 3 O O + H-OH R OH + H + R + CH 3 OH OH O CH 3 Water is a poor nucleophile, and methanol is a poor leaving group Aqueous hydrolysis can be catalyzed either by acids or by bases Enzymes can do acid and base catalysis simultaneously
How a catalyst circumvents unfavorable charge development during cleavage of an amide. The hydrolysis of an amide bond, shown here, is the same reaction as that catalyzed by chymotrypsin and other proteases. Charge development is unfavorable and can be circumvented by donation of a proton by H 3 O + (specific acid catalysis) or HA (general acid catalysis), where HA represents any acid. Similarly, charge can be neutralized by proton abstraction by OH (specific base catalysis) or B: (general base catalysis), where B: represents any base.
Amino acids in general acid-base catalysis Many organic reactions are promoted by proton donors (general acids) or proton acceptors (general bases). The active sites of some enzymes contain amino acid functional groups, such as those shown here, that can participate in the catalytic process as proton donors or proton acceptors.
Covalent Catalysis In covalent catalysis, a transient covalent bond is formed between the enzyme and the substrate. Consider the hydrolysis of a bond between groups A and B: In the presence of a covalent catalyst (an enzyme with a nucleophilic group X:) the reaction becomes This alters the pathway of the reaction, and it results in catalysis only when the new pathway has a lower activation energy than the uncatalyzed pathway.
A representation of primary structure of chymotrypsin ( a serine protease), showing disulfide bonds and the amino acid residues crucial to catalysis.
Three-dimensional structure of chymotrypsin
A close-up of the active site of chymotrypsin with a substrate (mostly green) bound
Pre steady state kinetic evidence for an acyl-enzyme intermediate
Pre steady state kinetic evidence for an acyl-enzyme intermediate
The ph dependence of chymotrypsin-catalyzed reactions
Chymotrypsin Mechanism Step 1: Substrate Binding Hydrolytic cleavage of a peptide bond by chymotrypsin
Chymotrypsin Mechanism Step 2: Nucleophilic Attack
Chymotrypsin Mechanism Step 3: Substrate Cleavage
Chymotrypsin Mechanism Step 4: Water Comes In
Chymotrypsin Mechanism Step 5: Water Attacks
Chymotrypsin Mechanism Step 6: Break-off from the Enzyme
Chymotrypsin Mechanism Step 7: Product Dissociates General acid-base catalysis Covalent catalysis Transition-state stabilization
Peptidoglycan and Lysozyme Peptidoglycan is a polysaccharide found in many bacterial cell walls Cleavage of the cell wall leads to the lysis of bacteria Lysozyme is an antibacterial enzyme Cleavage of peptidoglycan by lysozyme: general acid-base catalysis + covalent catalysis
Ribbon diagram of lysozyme with the active-site residues Glu 35 and Asp 52 shown as blue stick structures and bound substrate shown in red
Reaction catalyzed by lysozyme
Cleavage of Peptidoglycan by Lysozyme: Two Successive Nucleophilic Displacement Reactions Asp 52 acts as a nucleophile to attack the anomeric carbon in the first S N 2 step Glu 35 acts as a general acid and protonates the leaving group in the transition state. Water hydrolyzes the covalent glycosyl-enzyme intermediate Glu 35 acts as a general base to deprotonate water in the second S N 2 step
Lysozyme reaction
Hexokinase Undergoes Induced Fit on Substrate Binding
Induced fit in hexokinase
Enolase Requires Metal Ions For Reaction
5. Regulatory Enzymes Exhibit increased or decreased catalytic activity in response to certain signals The first enzyme in most multienzyme systems Multisubunit proteins, with active site and regulatory sites on separate subunits Allosteric regulation by reversible, noncovalent binding of regulatory compounds called allosteric modulators or allosteric effectors Covalent regulation: reversible, covalent modification of one or more amino acids in enzyme Regulated steps are catalyzed by allosteric enzymes in many pathways
Factors that determine the activity of an enzyme
Factors that determine the activity of an enzyme
Subunit interactions in an allosteric enzyme, and interactions with inhibitors and activators. In many allosteric enzymes the substrate binding site and the modulator binding site(s) are on different subunits, the catalytic (C) and regulatory (R) subunits, respectively. Binding of the positive (stimulatory) modulator (M) to its specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change. This change renders the catalytic subunit active and capable of binding the substrate (S) with higher affinity. On dissociation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form.
Two views of the regulatory enzyme aspartate transcarbamoylase This allosteric regulatory enzyme has two stacked catalytic clusters, each with three catalytic polypeptide chains (in shades of blue and purple), and three regulatory clusters, each with two regulatory polypeptide chains (in red and yellow). The regulatory clusters form the points of a triangle surrounding the catalytic subunits. Binding sites for allosteric modulators are on the regulatory subunits. Modulator binding produces large changes in enzyme conformation and activity.
Feedback inhibition: The end product of a pathway inhibits the first enzyme of the pathway
Allosteric Enzymes Do Not Obey Michaelis-Menten Kinetics A homotropic enzyme: the substrate also serves as a positive (stimulatory) modulator, or activator Substrate-activity curves for representative allosteric enzymes Allosteric enzymes display a sigmoidal dependence of reaction velocity on substrate concentration.
The kinetic behavior of allosteric enzymes reflects cooperative interactions among enzyme subunits. Substrate-activity curves for representative allosteric enzymes The effects of a positive modulator (+) and a negative modulator ( ) on an allosteric enzyme in which K 0.5 is altered without a change in V max. The central curve shows the substrate-activity relationship without a modulator.
Substrate-activity curves for representative allosteric enzymes A less common type of modulation, in which V max is altered and K 0.5 is nearly constant
Some enzyme modification reactions
Protein phosphorylation and dephosphorylation
Regulation of muscle glycogen phosphorylase activity by multiple mechanisms
Multiple regulatory phosphorylations of glycogen synthase
Many proteolytic enzymes are synthesized as inactive precursors called zymogens, which are activated by cleavage of small peptide fragments. Activation of zymogens by proteolytic cleavage
Keywords catalytic capacity specificity K m V max allosteric regulation
Words of the week allosteric covalent competitive uncompetitive
Learning Goals To understand: why nature needs enzyme catalysis how enzymes can accelerate chemical reactions how to perform and analyze kinetic studies how to characterize enzyme inhibitors how chymotrypsin breaks down peptide bonds