Nonexclusive inhibitors

If an enzyme is able to accommodate a molecule of substrate and a molecule of only one inhibitor at a time, but not the molecule of the other inhibitor, we have the case of two exclusive inhibitors thus, the two inhibitors are mutually exclusive. If the enzyme can bind a substrate and both inhibitors simultaneously, we have the case of two nonexclusive inhibitors. We assume that the presence of either inhibitor prevents the catalytic reaction. 

Let us now examine the three cases of inhibition with a mixture of two nonexclusive inhibitors, derived again from the general scheme (5.34) Table 4 summarizes these cases. Note that, in contrast to the preceding case, in aU the rate equations with two nonexclusive inhibitors, the cross term IX) is always present. 

Table 4. Inhibition by a mixture of two nonexclusive inhibitors Case 4. Ordered bindii of two nonexclusive inhibitors...

Consider the pure noncompetitive inhibition by two nonexclusive inhibitors, depicted by Eq. (5.35). If there are two dead-end inhibitors I and X, the kinetic question is whether an EIX complex forms, and if so, whether the dissociation constant of I from EIX is the same as from El (and similarly for X from EIX and EX). To answer this, the substrate concentration is held constant, the concentration of the two inhibitors is varied, and initial velocities are determined (Yonetani Theorell, 1965 Yonetani, 1982). 

Let us, now, depart from monosubstrate reactions and turn our attention to a much more realistic case of a hyperbolic inhibition in bisubstrate reactions (Segel, 1975 Dixon Webb, 1979 f rich Allison, 2000). In the rapid equilibrium reaction (6.14), A and B are the substrates while I is a nonexclusive inhibitor ... 

Figure 3.9 Apparent value of the dissociation constant (K,) for a labeled inhibitor, I, as a function of the concentration of a second inhibitor, J when measured by equilibrium binding methods. The solid circles represent the behavior expected when compounds I and J bind in a mutually exclusive fashion with one another. The other symbols represent the behavior expected when compounds I and J bind in a nonexclusive, but antagonistic (i.e., noncompetitive, a > 1) fashion, to separate binding sites. The data for mutually exclusive binding were fit to the equation (apparent)K, = A, 1 + ([f ] A",) I and that for nonexclusive binding were fit to the equation (apparent)Kt = ( [J] + Kj / Kj + f[I]/y) ) for y values of 5 (closed triangles), 10 (open squares), 20 (closed squares), and 50 (open circles).

Figure 3.10 Concentration of labeled compound I bound to an enzyme as a function of the concentration of a second inhibitor J. (A) Response of bound I to concentration of / when I and / bind in a mutually exclusive fashion. Note that here the concentration of the bound I is driven to zero at high concentrations of J. (B) Response of bound I to concentration of J when the two compounds bind in a nonexclusive, antagonistic manner to the target enzyme. Note that at high concentrations of J one does not drive the concentration of bound I to zero. Rather, the concentration of bound I at high concentrations of /reflects the concentration of ternary E I J complex. Condition of simulations I IK, = 1 (closed circles), 3 (open circles), and 5 (closed squares). For panel B, y = 5.

This analysis predicts that the addition of a negative effector should not inhibit the oscillations in the case of an exclusive binding of the substrate to the R state of the enzyme. As indicated in fig. 2.26, indeed, the curve H vs L loses its bell shape when coefficient c 0 the degree of cooperativity remains maximal at large values of the allosteric constant so that the steady state remains unstable regardless of the amount of inhibitor added to the system. The fact that an inhibitor of PFK, such as citrate, suppresses glycolytic oscillations would therefore suggest that the substrate binds in a nonexclusive manner to the two conformations of the enzyme. 

The terms exclusive and nonexclusive binding, will be applied throughout this book not only to inhibitors, but also to ligands other then substrates. 

In monosubstrate and bisubstrate reactions with a mixture of two inhibitors, the two inhibitors may bind nonexclusively to the enzyme or may be mutually... 

Nonexclusive substrate and effector binding may be even more complex thus, the binding of substrate. A, can take place in the presence of an inhibitor, I, and an activator, X. In this case, the rate equation becomes even more complex ... 

Figure 14, Nonexclusive binding of effectors. Velocity curves were drawn according to Eqs. (13.68)-Ci3.70), assuming that the substrate, A, binds preferentially to the T state (c = 0.01), the inhibitor, 1, binds almost exclusively to the T state (f = looo), and the activator, X, binds exclusively to the R state (e == o).

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