Slow binding inhibitors mechanisms

Slow-binding inhibitors operate by one of two mechanisms. Either the inhibitor binds slowly in an initial step, or the initial binding step occurs quickly, followed by a slow rearrangement of the E-I complex. 

Figure 6.2 Effect of preincubation time with inhibitor on the steady state velocity of an enzymatic reaction for a very slow binding inhibitor. (A) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the single-step binding mechanism of scheme B of Figure 6.3. (B) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the two-step binding mechanism of scheme C of Figure 6.3. Note that in panel B both the initial velocity (y-intercept values) and steady state velocity are affected by the presence of inhibitor in a concentration-dependent fashion.

In the mechanism illustrated by scheme B, significant inhibition is only realized after equilibrium is achieved. Hence the value of vs (in Equations 6.1 and 6.2) would not be expected to vary with inhibitor concentration, and should in fact be similar to the initial velocity value in the absence of inhibitor (i.e., v, = v0, where v0 is the steady state velocity in the absence of inhibitor). This invariance of v, with inhibitor concentration is a distinguishing feature of the mechanism summarized in scheme B (Morrison, 1982). The value of vs, on the other hand, should vary with inhibitor concentration according to a standard isotherm equation (Figure 6.5). Thus the IC50 (which is equivalent to Kfv) of a slow binding inhibitor that conforms to the mechanism of scheme B can be determined from a plot of vjv0 as a function of [/]. 

Figure 6.7 Concentratiom-esponse plots for the initial (A) and final (B) inhibited states of an enzyme reaction inhibited by a slow binding inhibitor that conforms to die mechanism of scheme C of Figure 6.3. The values of Vj and vs at each inhibitor concentration were obtained by fitting the data in Figure 6.6Ato Equation (6.1). These were then used to calculate the fractional velocity (Vj/v0 in panel A and vs/v0 in panel B), and die data in panels A and B were fit to Equations (6.8) and (6.9) to obtain estimates of Kf99 and Kf 9, respectively.

Figure 6.8 Replot of kobs as a function of inhibitor concentration for a slow binding inhibitor that conforms to the mechanism of scheme C of Figure 6.3 when the value of k6 is too small to estimate from the y-intercept of the data fit.

Some inhibitors interact very slowly with the enzyme protein, and onset of inhibition thus exhibits time-dependence. These inhibitors are generally referred to as slow-binding inhibitors, and as slow tight-binding inhihitors if the potency of inhibition is extremely high. Analysis of these inhibitory mechanisms is complex because binding and dissociation rate constants may be determined in addition to values. Indeed, a complete analysis may require extensive use of specialized computer software, and the complexities of such analyses preclude their discussion in this chapter. However, the reader is directed to several publications from Morrison s laboratory if a slow-binding mechanism is suspected for an inhibitor of interest (Morrison, 1982 Morrison and Stone, 1985 Sculley and Morrison, 1986 Morrison and Walsh, 1988). 

Transition-state inhibitors, especially those with peptidyl or peptidomimetic extensions, are slow-binding inhibitors, and the protease-inhibitor binding mechanism includes one or more weakly bound intermediates before the formation of the tightly bound E I complex. This slow-binding inhibition is a hallmark of inhibitors that bind in the active site in a substrate-like manner. In this way, transition-state analogs mimic the association... 

Slow-Binding Inhibitors. Two different mechanisms have been suggested to rationalize the slow-binding behavior of competitive inhibitors (71, 78, 80). In the one-step mechanism A, the direct binding process of the inhibitor to the enzyme is slow (Equation 17.26) that is, the magnitude of 3[I] is small relative to i[S] and k, the rate constants for the conversionof substrate to product. 

The first mechanism-based inhibitor (11) of 3-deoxy-D-arahino heptulosonate 7-phosphate (DAH7P synthase) has been synthesized in 12 steps from D-arabinose and has been found to be a very slow binding inhibitor against E. coli DAH7P synthase/ ... 

Slow, tight-binding inhibition occurs when slow-binding inhibition takes place at inhibitor concentrations comparable to that of the enzyme, in which case the previous two mechanisms can still apply. Comprehensive review articles on the subject of tight, slow, and slow, tight-binding inhibitors ate available in the literature (12,14). 

To distinguish between simple, reversible slow binding (scheme B) and an enzyme isomerization mechanism (scheme C), one can examine the dependence of kobs on inhibitor concentration. If the slow onset of inhibition merely reflects inherently slow binding and/or dissociation, then the term kobs in Equations (6.1) and (6.2) will depend only on the association and dissociation rate constants k3 and k4 as follows ... 

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