Chapter 15 Enzymes

Enzymes Ribbon diagram of cytochrome c oxidase, the enzyme that directly uses oxygen during respiration.

Enzyme Catalysis Enzyme: A biological catalyst. With the exception of some RNAs that catalyze their own self-cleavage, all enzymes are proteins. Enzymes can increase the rate of a reaction by a factor of 109 to 1020 over an uncatalyzed reaction. Some catalyze the reaction of only one compound. Others are stereoselective; for example, enzymes that catalyze the reactions of only L-amino acids. Others catalyze reactions of specific types of compounds or bonds; for example, trypsin catalyzes hydrolysis of peptide bonds formed by the carboxyl groups of Lys and Arg.

Enzyme Catalysis Figure 15.1 Trypsin catalyzes the hydrolysis of peptide bonds formed by the carboxyl group of lysine and arginine.

Classification of Enzymes Enzymes are commonly named after the reaction or reactions they catalyze. Example: lactate dehydrogenase, acid phosphatase. Enzymes are classified into six major groups according to the type of reaction catalyzed: Oxidoreductases: Oxidation-reduction reactions. Transferases: Group transfer reactions. Hydrolases: Hydrolysis reactions. Lyases: Addition of two groups to a double bond, or removal of two groups to create a double bond. Isomerases: Isomerization reactions. Ligases: The joining to two molecules.

Classification of Enzymes 1. Oxidoreductase: 2. Transferase:

Classification of Enzymes 3. Hydrolase: 4. Lyase:

Classification of Enzymes 5. Isomerase: 6. Ligase:

Enzyme Terminology Apoenzyme: The protein part of an enzyme. Cofactor: A nonprotein portion of an enzyme that is necessary for catalytic function; examples are metallic ions such as Zn2+ and Mg2+. Coenzyme: A nonprotein organic molecule, frequently a B vitamin, that acts as a cofactor. Substrate: The compound or compounds whose reaction an enzyme catalyzes. Active site: The specific portion of the enzyme to which a substrate binds during reaction.

Schematic of an Active Site Figure 15.2 Schematic diagram of the active site of an enzyme and the participating components.

Terms in Enzyme Chemistry Activation: Any process that initiates or increases the activity of an enzyme. Inhibition: Any process that makes an active enzyme less active or inactive. Competitive inhibitor: A substance that binds to the active site of an enzyme thereby preventing binding of substrate. Noncompetitive inhibitor: Any substance that binds to a portion of the enzyme other than the active site and thereby inhibits the activity of the enzyme.

Enzyme Activity Enzyme activity: A measure of how much a reaction rate is increased. We examine how the rate of an enzyme-catalyzed reaction is affected by: Enzyme concentration. Substrate concentration. Temperature. pH.

Enzyme Activity Figure 15.3 The effect of enzyme concentration on the rate of an enzyme-catalyzed reaction. Substrate concentration, temperature, and pH are constant.

Enzyme Activity Figure 15.4 The effect of substrate concentration on the rate of an enzyme-catalyzed reaction. Enzyme concentration, temperature, and pH are constant.

Enzyme Activity Figure 23.5 The effect of temperature on the rate of an enzyme-catalyzed reaction. Substrate and enzyme concentrations and pH are constant.

Enzyme Activity Figure 23.6 The effect of pH on the rate of an enzyme- catalyzed reaction. Substrate and enzyme concentrations and temperature are constant.

Mechanism of Action Figure 15.7 Lock-and-key model of enzyme mechanism. The enzyme is a rigid three-dimensional body. The enzyme surface contains the active site.

Mechanism of Action Figure 15.8 The Induced-fit model of an enzyme mechanism. The active site becomes modified to accommodate the substrate.

Mechanism of Action Figure 23.9 The mechanism of competitive inhibition. When a competitive inhibitor enters the active site, the substrate cannot enter.

Mechanism of Action Figure 15.10 Mechanism of noncompetitive inhibition. The inhibitor binds itself to a site other than the active site (allosterism), thereby changing the conformation of the active site. The substrate still binds but there is no catalysis.

Mechanism of Action Figure 15.11 Enzyme kinetics in the presence and the absence of inhibitors.

Mechanism of Action Both the lock-and-key model and the induced-fit model emphasize the shape of the active site. However, the chemistry of the active site is the most important. Just five amino acids participate in the active site in more than 65% of the enzymes studied to date. These five are His > Cys > Asp > Arg > Glu. Four of these amino acids have either acidic or basic side chains; the fifth has a sulfhydryl group (-SH).

Catalytic Power Figure 23.12 Enzymes provide an alternative pathway for reaction. (a) The activation energy profile for a typical reaction. (b) A comparison of the activation energy profiles for a catalyzed and uncatalyzed reactions.

Enzyme Regulation Feedback control: An enzyme-regulation process where the product of a series of enzyme-catalyzed reactions inhibits an earlier reaction in the sequence. The inhibition may be competitive or noncompetitive.

Enzyme Regulation Proenzyme (zymogen): An inactive form of an enzyme that must have part of its polypeptide chain hydrolyzed and removed before it becomes active. An example is trypsin, a digestive enzyme. It is synthesized and stored as trypsinogen, which has no enzyme activity. It becomes active only after a six-amino acid fragment is hydrolyzed and removed from the N-terminal end of its chain. Removal of this small fragment changes not only the primary structure but also the tertiary structure, allowing the molecule to achieve its active form.

Enzyme Regulation Allosterism: Enzyme regulation based on an event occurring at a place other than the active site but that creates a change in the active site. An enzyme regulated by this mechanism is called an allosteric enzyme. Allosteric enzymes often have multiple polypeptide chains. Negative modulation: Inhibition of an allosteric enzyme. Positive modulation: Stimulation of an allosteric enzyme. Regulator: A substance that binds to an allosteric enzyme.

Enzyme Regulation Figure 15.14 The allosteric effect. Binding of the regulator to a site other than the active site changes the shape of the active site.

Enzyme Regulation Figure 15.15 Effects of binding activators and inhibitors to allosteric enzymes. The enzyme has an equilibrium between the T form and the R form.

Enzyme Regulation Protein modification: The process of affecting enzyme activity by covalently modifying it. The best known examples of protein modification involve phosphorylation/dephosphorylation. Example: Pyruvate kinase (PK) is the active form of the enzyme; it is inactivated by phosphorylation to pyruvate kinase phosphate (PKP).

Enzyme Regulation Isoenzyme (Isozymes): An enzyme that occurs in multiple forms; each catalyzes the same reaction. Example: lactate dehydrogenase (LDH) catalyzes the oxidation of lactate to pyruvate. The enzyme is a tetramer of H and M chains. H4 is present predominately in heart muscle. M4 is present predominantly in the liver and in skeletal muscle. H3M, H2M2, and HM3 also exist. H4 is allosterically inhibited by high levels of pyruvate while M4 is not. H4 in serum correlates with the severity of heart attack.

Enzyme Regulation Figure 15.16 The isozymes of lactate dehydrogenase (LDH). The electrophoresis gel depicts the relative isozyme types found in different tissues.

Glycogen phosphorylase activity is subject to allosteric control and covalent modification via phosphorylation. The phosphorylated form is more active. The enzyme that puts a phosphate group on phosphorylase is called phosphorylase kinase. p649

Enzymes Used in Medicine Table 15.2 – Enzyme Assays useful in Medical Diagnosis Insert Table 23.2, page 648

Transition-State Analogs Transition state analog: A molecule whose shape mimics the transition state of a substrate. Figure 15.17 The proline racemase reaction. Pyrrole- 2-carboxylate mimics the planar transition state of the reaction (next screen).

Transition-State Analog

Transition-State Analogs Abzyme: An antibody that has catalytic activity because it was created using a transition state analog as an immunogen. (a) The molecule below is a transition analog for the reaction of an amino acid with pyridoxal-5’-phosphate. (b) The abzyme is then used to catalyze the reaction on the next screen.

Transition-State Analogs

Catalytic Antibodies Against Cocaine The mechanism of action of cocaine. (a) Dopamine acts as a neurotransmitter. It is released from the presynaptic neuron, travels across the synapse, and bonds to dopamine receptors on the postsynaptic neuron. It is later released and taken up into vesicles in the presynaptic neuron. (b) Cocaine increases the amount of time that dopamine is available to the dopamine receptors by blocking its uptake. (Adapted from Immunotherapy for Cocaine Addiction, by D. W. Landry, Scientific American, February 1997, pp 42–45.) p652

Catalytic Antibodies Against Cocaine Degradation of cocaine by esterases or catalytic antibodies. Cocaine (a) passes through a transition state (b) on its way to being hydrolyzed to benzoic acid and ecgonine methyl ester (c). Transition-state analogs are used to generate catalytic antibodies for this reaction. (Adapted from Immunotherapy for Cocaine Addiction, by D. W. Landry, Scientific American, February 1997, pp 42–45.) p652