Tight-bind inhibitor

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). 

Thus plots of IC50 as a function of [ TPP under conditions of Strauss and Goldstein s zone B allow one to simultaneously determine the values of Kfpp and [E T using Equations (7.13) and (7.15). Later in this chapter we will see other methods by which tight binding inhibitors can be used to provide accurate determinations of the total concentration of catalytically active enzyme in a sample. 

MORRISON S QUADRATIC EQUATION FOR FITTING CONCENTRATION-RESPONSE DATA FOR TIGHT BINDING INHIBITORS... 

A better method for analyzing concentration-response data for tight binding inhibitors was developed by Morrison and coworkers (Morrison, 1969 Williams and Morrison, 1979). This treatment is based on defining the Kt value of an inhibitor, or... 

Figure 7.4 Concentration-response plots for the data presented in Figure 7.1 fitted to Morrison s quadratic equation for tight binding inhibitors. The left panel shows the concentration-response behavior on a semilog scale, while the right panel shows the same data when the inhibitor concentration is plotted on a linear scale.

Murphy (2004) has reported an in-depth analysis of simulations for various assay conditions using Morrison s equation for tight binding inhibitors. From these studies several recommendations emerge for optimizing conditions for the determination... 

Figure 7.6 Double reciprocal plot for a tight binding competitive enzyme inhibitor, demonstrating the curvature of such plots. The dashed lines represent an attempt to fit the data at lower substrate concentrations to linear equations. This highlights how double reciprocal plots for tight binding inhibitors can be misleading, especially when data are collected only over a limited range of substrate concentrations.

TIGHT BINDING INHIBITORS OFTEN DISPLAY SLOW BINDING BEHAVIOR... 

Tight Binding Inhibitors Often Display Slow Binding Behavior 193... 

The very slow dissociation rates for tight binding inhibitors offer some potential clinical advantages for such compounds, as described in detail in Chapter 6. Experimental determination of the value of k, can be quite challenging for these inhibitors. We have detailed in Chapters 5 and 6 several kinetic methods for estimating the value of the dissociation rate constant. When the value of kofS is extremely low, however, alternative methods may be required to estimate this kinetic constant. For example, equilibrium dialysis over the course of hours, or even days, may be required to achieve sufficient inhibitor release from the El complex for measurement. A significant issue with approaches like this is that the enzyme may not remain stable over the extended time course of such experiments. In some cases of extremely slow inhibitor dissociation, the limits of enzyme stability will preclude accurate determination of koff the best that one can do in these cases is to provide an upper limit on the value of this rate constant. 

These practical approaches are by no means mutually exclusive, and attempts should be made to combine as many of these as possible to improve ones ability to experimentally measure the K-pp of tight binding inhibitors. Thus one should always work at the lowest enzyme concentration possible, and drive the substrate concentration as high as possible, when dealing with competitive inhibitors. A long preincubation step should be used before activity measurements, or the progress curves should be fitted to Equation (6.2) so that accurate determinations of the steady state velocity at each inhibitor concentration can be obtained. Finally, the concentration-response data should be fitted to Morrison s quadratic equation to obtain good estimates of the value of Arfpp. 

Potential Clinical Advantages of Tight Binding Inhibitors 207... 

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