Reversible Inhibitors

The rate and extent of inhibition of enzymes by reversible inhibitors also depend on the factors listed above. However, the rate of inhibition is very rapid (msec) as a noncovalent complex is formed. Also, the inhibition can be reversed by removing the inhibitor (by dialysis or gel filtration). Our attention in this chapter is focused on the reversible enzyme inhibitors. Defined kinetically, a reversible inhibitor is one that reacts with an enzyme in a reversible manner as shown in Eq. (32), where Ki is an equilibrium (dissociation) constant. Ki = [E][/]/E • I.

The reaction occurs within a few msec and the extent of inhibition is controlled by the concentration of E and I and Ki. Generally, Ki for the reversible inhibitors will be in the range of 10—2-10—7 M. If the Ki is < 10—8 M, the dissociation rate is so slow that there is not a true equilibrium of the process when perturbed by addition of substrate.

Based on their different effects on the rates of enzyme-catalyzed reactions, the reversible inhibitors can be divided into three types, as shown in Figure 8. The three types are the competitive inhibitors (C), noncompetitive inhibitors (N), and the uncompetitive inhibitors (U). The competitive inhibitors (C) compete with the substrate for binding to the active site of the enzyme. Therefore, the equilibrium involves the species E, EA, and EC and the free A and C (Fig. 8). The noncompetitive inhibitors (N) affect the rate of the enzyme-catalyzed reaction by binding outside the active site in a way that permits substrate to bind into the active site but no catalysis of substrate to product occurs. As shown in Figure 8, the species E, A, EA, EA', EN, EAN, EA'N, and N can be present. The uncompetitive inhibitors (U) bind to the enzyme only after the substrate has bound. The uncompetitive inhibitors can also bind to other intermediate enzyme-product complexes. As shown in Figure 8, at equilibrium the species A, E, U, EAU, and EP'U can exist. Actually, the three types of inhibitors are distinguishable only experimentally where different effects on the kinetics are shown. The experiments must be performed in the absence and presence of the reversible inhibitor.

Figure 9 shows the kinetic effect of a competitive inhibitor on the rate of an enzyme-catalyzed reaction, when the data are plotted by the Lineweaver-Burk method. As noted, there is no effect of inhibitor on the y-axis intercept. Therefore V (= Vmax) is the same in the presence and absence of the competitive inhibitor. But the slope of the line is larger in the presence of the competitive inhibitor, reflecting the effect of the inhibitor on K (= Km) for the reaction as shown also by the difference in the —x intercept. The numerical difference in slopes and —x intercepts in the presence and absence of inhibitors is 1 + [I]o/Ki. Knowing the [I]o added to the reaction, Ki can be calculated. These data were determined for a linear competitive inhibitor.

Figure 10 shows the kinetic effect of a noncompe-titive inhibitor when added to an enzyme-catalyzed reaction. Again, data must be obtained for a control with no inhibitor and one with a fixed concentration of the inhibitor. Figure 10 shows that both the slope and the y-intercept are changed by the term 1 + [I]o/Ki. There is no effect on the —x intercept. This is the definition of a simple linear noncompeti-tive inhibitor.

Figure 11 shows the effect of an uncompetitive inhibitor when added to an enzyme-catalyzed reaction. Again, a control without inhibitor must be included. There is no effect of the uncompetitive inhibitor on the slope of the lines, while there is a marked difference in the y-intercept (and the —x intercept). The difference in y-intercept is given by 1 + [I]o/Ki. The example chosen is for a linear uncompetitive inhibitor.

The examples given in Figures 9-11 are linear-behaving inhibitors, as defined in Figure 12. This is determined from a plot of slope (or intercept) vs. [I]o where experiments at several different [I]o are done. In many cases, one finds that the type of inhibition is linear. Generally, they will be linear for most competitive inhibitors. But for noncompetitive and uncom-petitive inhibitors there are more opportunities for observing hyperbolic or parabolic type behavior.

Figure 8 Schematic representation of inhibition of enzyme activity by various types of reversible inhibitors. The model for E shows only the active site with the binding locus (inner void) and the transforming locus, with catalytic groups A and B. The symbols for the species involved (in parentheses) are: E, free enzyme; EA, enzyme-substrate complex; EA', acylenzyme intermediate; C, competitive inhibitor; N, noncompetitive inhibitor; U, uncompetitive inhibitor; P1 and P2 are products formed from the substrate, A, and EN, EAN, EA'N, EC, and EA'U are complexes with respective enzyme species and inhibitors. All complexes formed involve noncovalent bonds except EA', where a covalent bond is formed with catalytic group A. (From Ref. 1.)

Figure 8 Schematic representation of inhibition of enzyme activity by various types of reversible inhibitors. The model for E shows only the active site with the binding locus (inner void) and the transforming locus, with catalytic groups A and B. The symbols for the species involved (in parentheses) are: E, free enzyme; EA, enzyme-substrate complex; EA', acylenzyme intermediate; C, competitive inhibitor; N, noncompetitive inhibitor; U, uncompetitive inhibitor; P1 and P2 are products formed from the substrate, A, and EN, EAN, EA'N, EC, and EA'U are complexes with respective enzyme species and inhibitors. All complexes formed involve noncovalent bonds except EA', where a covalent bond is formed with catalytic group A. (From Ref. 1.)

Figure 9 Linear competitive inhibition, plotted by the Lineweaver-Burk method. V = Vmax in absence of inhibitor. V' = Vmax(1 + (Io)/Ki) in presence of inhibitor, K = Km in absence of inhibitor. K' = (1 + (Io)/Ki) in presence of inhibitor. (Ref. 1.)
Figure 10 Simple linear noncompetitive inhibition, plotted by the Lineweaver-Burk method. See Figure 9 for additional explanation. (From Ref. 1.)

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