Inhibition of Enzyme Activity

INHIBITION OF ENZYME ACTIVITY

Any substance that can decrease the velocity of an enzyme-catalyzed reaction is called an inhibitor. Inhibitors can be reversible or irreversible. Irreversible inhibitors bind to enzymes through covalent bonds. Lead, for example, forms covalent bonds with the sulfhydryl side chain of cysteine in proteins. Ferrochelatase, an enzyme involved in heme synthesis, is irreversibly inhibited by lead. [Note: An important group of irreversible inhibitors are the mechanism-based inhibitors that are converted by the enzyme itself to a form that covalently links to the enzyme, thereby inhibiting it. They also are referred to as “suicide” inhibitors.] Reversible inhibitors bind to enzymes through noncovalent bonds and, thus, dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor and recovery of enzyme activity. The two most commonly encountered types of reversible inhibition are competitive and noncompetitive.

A. Competitive inhibition

This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for that site.

1. Effect on V_max: The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the V_max observed in the absence of inhibitor (Figure 5.12).

2. Effect on K_m: A competitive inhibitor increases the apparent K_m for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve 1⁄2V_max.

3. Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y axis at 1/Vmax (Vmax is unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because - 1/Km moves closer to zero from a negative value (see Figure 5.12). [Note: An important group of competitive inhibitors are the transition state analogs, stable molecules that approximate the structure of the transition state and, therefore, bind the enzyme with a higher affinity than the substrate.]

Figure 5.12 A. Effect of a competitive inhibitor on the reaction velocity versus substrate ([S]) plot. B. Lineweaver-Burk plot of competitive inhibition of an enzyme.

4. Statin drugs as examples of competitive inhibitors: This group of antihyperlipidemic agents competitively inhibits the rate-limiting (slowest) step in cholesterol biosynthesis. This reaction is catalyzed by hydroxymethylglutaryl–CoA reductase (HMG-CoA reductase). Statins, such as atorvastatin (Lipitor) and pravastatin (Pravachol), are structural analogs of the natural substrate for this enzyme and compete effectively to inhibit HMG-CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels (Figure 5.13).

Figure 5.13 Pravastatin competes with HMGCoA for the active site of HMGCoA reductase. HMG-CoA = hydroxymethylglutaryl-coenzyme A.

B. Noncompetitive inhibition

This type of inhibition is recognized by its characteristic effect on Vmax (Figure 5.14). Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The noncompetitive inhibitor can bind either free enzyme or the enzyme-substrate complex, thereby preventing the reaction from occurring (Figure 5.15).

Figure 5.14 A. Effect of a noncompetitive inhibitor on the reaction velocity versus substrate ([S]) plot. B. Lineweaver-Burk plot of noncompetitive inhibition of an enzyme.

Figure 5.15 A noncompetitive inhibitor binding to both free enzyme and enzyme-substrate (ES) complex.

1. Effect on V_max: Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Therefore, noncompetitive inhibitors decrease the apparent V_max of the reaction.

2. Effect on K_m: Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Therefore, the enzyme shows the same K_m in the presence or absence of the noncompetitive inhibitor.

3. Effect on Lineweaver-Burk plot: Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/v_o versus 1/[S] and noting that the apparent V_max decreases in the presence of a noncompetitive inhibitor, whereas K_m is unchanged (see Figure 5.14). [Note: Oxypurinol, a metabolite of the drug allopurinol, is a noncompetitive inhibitor of xanthine oxidase, an enzyme of purine degradation.]

C. Enzyme inhibitors as drugs

At least half of the ten most commonly prescribed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed β-lactam antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may also act by inhibiting extracellular reactions. This is illustrated by angiotensin-converting enzyme (ACE) inhibitors. They lower blood pressure by blocking the enzyme that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II.  These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and, therefore, a reduction in blood pressure. Aspirin, a nonprescription drug, irreversibly inhibits prostaglandin and thromboxane synthesis.