Identification of the reactive histidine of cucumisin, a plant serine protease: modification with peptidyl chloromethyl ketone derivative of peptide substrate.

A radioactive peptidyl chloromethyl ketone derived from substrates of cucumisin, 3H-labeled Z-Ala-Ala-Pro-Phe-chloromethyl ketone (3H-ZAAPFCK), was synthesized. When cucumisin was incubated with a 100-fold molar excess of 3H-ZAAPFCK for 16 h, 98% of the cucumisin activity was inhibited and about 0.93 mol of 3H-ZAAPFCK was incorporated in 1 mol of cucumisin. The 3H-ZAAPFCK-modified cucumisin was reduced and pyridylethylated, and then digested by trypsin. The radioactive peptide fragment was isolated and its amino acid sequence was determined. The radioactive fragment contained 32 amino acid residues and the sequence around the labeled residue was found to be -Asp-Thr-Asn-Gly-(His)-Gly-Thr-His-Thr-Ala-. This sequence is analogous to that around the reactive site histidine residue of the subtilisin family.