Irreversible caspase inhibitors: tools for studying apoptosis.

Irreversible inhibitors of caspase proteases are often used in studies of apoptosis. However, vigorous interpretation of data generated with irreversible inhibitors requires quantitative analysis of their effects on enzyme kinetics. A simple method for the quantitative analysis of affinity irreversible inhibitors is introduced. The method allows simultaneous measurement of the dissociation constant Ki for the reversible binding to a caspase and the first-order rate constant k3 for the subsequent in situ covalent reaction that follows the noncovalent binding. The Ki value provides information regarding the affinity of an inhibitor for the enzyme, whereas the k3 value provides a measure of the in situ reactivity between the reactive functional groups of the bound inhibitor and the nearby nucleophilic side chain at the protease active site. This two-step kinetic analysis offers a more complete description of the characteristics of an irreversible inhibitor than does the commonly used second-order rate constant. The method has been applied to a library of irreversible caspase inhibitors. We demonstrate how the resulting quantitative inhibitory constants can be used to identify key caspase activities responsible for apoptosis in specific cellular models. Copyright 1999 Academic Press.