Elsevier

Methods

Volume 17, Issue 4, April 1999, Pages 320-328
Methods

Regular Article
Irreversible Caspase Inhibitors: Tools for Studying Apoptosis

https://doi.org/10.1006/meth.1999.0746Get rights and content

Abstract

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 constantKifor the reversible binding to a caspase and the first-order rate constantk3for the subsequentin situcovalent reaction that follows the noncovalent binding. TheKivalue provides information regarding the affinity of an inhibitor for the enzyme, whereas thek3value provides a measure of thein situreactivity 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.

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