Slow-binding enzyme inhibitors

Table 6.1 Some examples of slow binding enzyme inhibitors...

J. F. Morrison, C. T. Walsh, The Behavior and Significance of Slow-Binding Enzyme Inhibitors , Adv. InErtzy-mol. Relat. Areas Mol. Biol. 1987, 61,201-301. 

Morrison JF, Stone SR. 1985. Approaches to the study and analysis of the inhibition of enzymes by slow- and tight-binding inhibitors. Comments Mol Cell Biophys 2 347. Morrison JF, Walsh CT. 1988. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 61 201. 

Not all reversible inhibitors have an instantaneous effect on the rate of an enzymatic reaction. Some inhibitors, known as slow-binding enzyme inhibitors, can take a considerable time to establish the equilibrium between the free enzyme and inhibitor, and the enzyme-inhibitor complex. This time period may be on the scale of seconds, minutes, or even longer. The enzyme-inhibitor complexes have slow off (dissociation) rates, but the on (association) rates may be either slow or fast. Hence, the term slow binding does not necessarily indicate a slow binding of inhibitor to enzyme but rather the fact that reaching equilibrium is a... 

A case similar to the slow, practically irreversible inhibition of jack bean a-D-mannosidase by swainsonine is represented by the interaction of castanospermine with isomaltase and rat-intestinal sucrase. Whereas the association constants for the formation of the enzyme-inhibitor complex were similar to those of other slow-binding glycosidase inhibitors (6.5 10 and 0.3 10 M s for sucrase and isomaltase, respectively), the dissociation constant of the enzyme-inhibitor complex was extremely low (3.6 10 s for sucrase) or could not be measured at all (isomaltase), resulting in a virtually irreversible inhibition. Danzin and Ehrhard discussed the strong binding of castanospermine in terms of the similarity of the protonated inhibitor to a D-glucosyl oxocarbenium ion transition-state, but were unable to give an explanation for the extremely slow dissociation of the enzyme-inhibitor complex. 

Unlike dehydrogenases, enzymes cleaving the glycosyl bond are extremely sensitive to modifications of the sugar moiety. The arabino analogs, arabino-NAD+ and 2 -fluoro-araW/K>-NAD (12) are both potent, slow-binding competitive inhibitors of calf spleen NADase with ATj values of 170 nAf and 2 fiM,... 

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). 

If the inhibition is found to be rapidly reversible, we must next determine if the approach to equilibrium for the enzyme-inhibitor complex is also rapid. As described in Chapter 4, some inhibitors bind slowly to their target enzymes, on a time scale that is long in comparision to the time scale of the reaction velocity measurement. The effect of such slow binding inhibition is to convert the linear progress curve seen in the absence of inhibitor to a curvilinear function (Figure 5.10). When nonlinear progress curves are observed in the presence of inhibitor, the analysis of... 

Figure 5.10 Progress curves for an enzyme in the absence (open circles) and presence (closed circles) of an slow-binding inhibitor. See Chapter 6 for more details on this form of inhibition.

The hallmark of slow binding inhibition is that the degree of inhibition at a fixed concentration of compound will vary over time, as equilibrium is slowly established between the free and enzyme-bound forms of the compound. Often the establishment of enzyme-inhibitor equilibrium is manifested over the time course of the enzyme activity assay, and this leads to a curvature of the reaction progress curve over a time scale where the uninhibited reaction progress curve is linear. We saw... 

Figure 6.1 Typical progress curve for an enzyme reaction in the presence of a slow binding inhibitor. The initial (v,) and steady state (vs) velocities are defined by the slope values in the early and late stages of the progress curve, respectively, as indicated by the dashed lines.

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 ... 

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.

Often high-affinity, or tight binding, interactions with enzymes is the result of a very slow dissociation rate of the enzyme-inhibitor binary complex. 

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