title: Interaction with substrate sensitises caspase-3 to inactivation by hydrogen peroxide.
abstract: Caspases have an active site cysteine whose oxidation blocks catalytic activity. Caspase activity, measured in lysates of apoptotic cells, was inhibited by H2O2 with an IC50 of 7 microM. Recombinant caspase-3 was directly inhibited by H2O2, with an estimated second-order rate constant of 750 M-1 s-1. These values were determined when H2O2 was added while the caspases were cleaving a peptide substrate. There was a 40-fold decrease in sensitivity to inactivation if the substrate was absent at the time of H2O2 addition. These results rationalise conflicting reports of the sensitivity of caspase-3 to H2O2, and identify a novel mechanism for sensitising a thiol enzyme to oxidative inactivation.
authors.affiliation: Free Radical Research Group, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand. mark.hampton@chmeds.ac.nz
authors.forename: Mark B
authors.initials: MB
authors.lastname: Hampton
authors.forename: Ivan
authors.initials: I
authors.lastname: Stamenkovic
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
mesh_terms: D001665:Binding Sites; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D019169:Jurkat Cells; D009842:Oligopeptides / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D013379:Substrate Specificity
chemical_list: D061945:Caspase Inhibitors; D009842:Oligopeptides; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003545:Cysteine