Interaction with substrate sensitises caspase-3 to inactivation by hydrogen peroxide.

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.