Purification and catalytic properties of human caspase family members.
Members of the caspase family of cysteine proteases are known to be key mediators of mammalian inflammation and apoptosis. To better understand the catalytic properties of these enzymes, and to facilitate the identification of selective inhibitors, we have systematically purified and biochemically characterized ten homologues of human origin (caspases 1 - 10). The method used for production of most of these enzymes involves folding of active enzymes from their constituent subunits which are expressed separately in E. coli, followed by ion exchange chromatography. In cases where it was not possible to use this method (caspase-6 and -10), the enzymes were instead expressed as soluble proteins in E. coli, and partially purified by ion exchange chromatography. Based on the optimal tetrapeptide recognition motif for each enzyme, substrates with the general structure Ac-XEXD-AMC were used to develop continuous fluorometric assays. In some cases, enzymes with virtually identical tetrapeptide specificities have kcat/Km values for fluorogenic substrates that differ by more than 1000-fold. Using these assays, we have investigated the effects of a variety of environmental factors (e.g. pH, NaCl, Ca2+) on the activities of these enzymes. Some of these variables have a profound effect on the rate of catalysis, a finding that may have important biological implications.
Garcia-Calvo
M
M
Department of Enzymology, Merck Research Laboratories, R80W-250, P.O. Box 2000, Rahway, New Jersey 07065, USA. margarita_garcia-calvo@merck.com
Peterson
E P
EP
Rasper
D M
DM
Vaillancourt
J P
JP
Zamboni
R
R
Nicholson
D W
DW
Thornberry
N A
NA
D017209:Apoptosis / Q000276:immunology*; D002118:Calcium / Q000494:pharmacology; D020170:Caspase 1 / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020134:Catalytic Domain; D003374:Coumarins / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D005470:Fluorometry; D015971:Gene Expression Regulation, Enzymologic / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007249:Inflammation; D007371:Interferon-gamma / Q000378:metabolism; D007375:Interleukin-1 / Q000378:metabolism; D020382:Interleukin-18 / Q000378:metabolism; D007700:Kinetics; D005810:Multigene Family / Q000502:physiology; D009842:Oligopeptides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012492:Salts / Q000494:pharmacology
C112287:Ac-aspartyl-glutamyl-valyl-aspartyl-aminomethylcoumarin; D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D007375:Interleukin-1; D020382:Interleukin-18; D009842:Oligopeptides; D011994:Recombinant Proteins; D012492:Salts; C096429:L 709049; D007371:Interferon-gamma; D010450:Endopeptidases; D020169:Caspases; D020170:Caspase 1; D002118:Calcium