Purification and catalytic properties of human caspase family members

Members of the caspase family of cysteine proteases are known to be key med iators of mammalian inflammation and apoptosis. To better understand the ca talytic properties of these enzymes, and to facilitate the identification o f selective inhibitors, we have systematically purified and biochemically c haracterized ten homologues of human origin (caspases 1 - 10). The method u sed for production of most of these enzymes involves folding of active enzy mes from their constituent subunits which are expressed separately in E. co li, followed by ion exchange chromatography, In cases where it was not poss ible to use this method (caspase-6 and -10), the enzymes were instead expre ssed as soluble proteins in E, coli, and partially purified by ion exchange chromatography, Based on the optimal tetrapeptide recognition motif for ea ch enzyme, substrates with the general structure Ac-XEXD-AMC were used to d evelop continuous fluorometric assays. In some cases, enzymes with virtuall y identical tetrapeptide specificities have k(cat)/k(m) values for fluoroge nic substrates that differ by more than 1000-fold. Using these assays, we h ave investigated the effects of a variety of environmental factors (e,g, pH , NaCl, Ca2+) on the activities of these enzymes. Some of these variables h ave a profound effect on the rate of catalysis, a finding that may have imp ortant biological implications.