Distinguishing Features of Mechanism-Based Inactivation

Despite the mechanistic differences in the definitions of kimcl and A) between quiescent affinity labels and mechanism-based inactivators, the dependence of kohs on [/] is the same for both mechanism. Hence we cannot determine whether or not a compound is acting as a mechanism-based inhibitor, based merely on this two-step kinetic behavior. However, there is a set of distinguishing features of mechanism-based inactivation that are experimentally testable. Compounds that display all of these features can be safely defined as mechanism-based inactivators. 

Several authors have discussed specific criteria for designating a compound as a mechanism-based inactivator. Abeles and Maycock (1976) and Walsh (1978) were among the first to set out specific experimental tests for mechanism-based inactivation. More recently Silverman (1988, 1992, 1995) has described a comprehensive set of seven distinguishing features that mechanism-based inactivators must display. These are described here. 

As described above, mechanism-based inactivation conforms to a two-step reaction and should therefore display saturation behavior. The value of should be a rectangular hyperbolic function of [/]. This was described in detail above in Section 8.1. 

Note that in some cases one may follow the time course of covalent E-A formation by equilibrium binding methods (e.g., LC/MS, HPLC, NMR, radioligand incorporation, or spectroscopic methods) rather than by activity measurements. In these cases substrate should also be able to protect the enzyme from inactivation according to Equation (8.7). Likewise a reversible competitive inhibitor should protect the enzyme from covalent modification by a mechanism-based inactivator. In this case the terms. S and Ku in Equation (8.7) would be replaced by [7r] and K respectively, where these terms refer to the concentration and dissociation constant for the reversible inhibitor. 

Mechanism-based inactivation results in formation of a covalent adduct between the active inhibitor and the enzyme, or between the active inhibitor and a substrate or cofactor molecule. If the mechanism involves covalent modification of the enzyme, then one should not be able to demonstrate a recovery of enzymatic activity after dialysis, gel filtration, ultrafiltration, or large dilution, as described in Chapters 5 to 7. Additionally, if the inactivation is covalent, denaturation of the enzyme should fail to release the inhibitory molecule into solution. If a radiolabeled version of the inactivator is available, one should be able to demonstrate irreversible association of radioactivity with the enzyme molecule even after denaturation and separation by gel filtration, and so on. In favorable cases one should likewise be able to demonstrate covalent association of the inhibitor with the enzyme by a combination of tryptic digestion and LC/MS methods. 

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