α-Chymotrypsin was irreversibly inhibited with an enzyme-activated N-nitrosoamide inhibitor, N-nitroso-N-(1-naphthylmethyl)-N′-isobutyrylalanine; alkylation of the active-site residues by the naphthylmethyl cation produced in the enzymatic reaction occurred. The inhibited enzyme was reduced and aminoethylated and then subjected to tryptic and chymotryptic digestion. Separation of the digest by reversed-phase HPLC revealed one major new peak relative to that of a control run from the native enzyme. Subsequent amino acid analysis and sequencing along with fast atom bombardment-mass spectrometry measurements indicated that this new peak stemmed from an active-site peptide, Met-192-Leu-199, and that the naphthylmethyl label was attached to the side-chain oxygen of Ser-195. The general approach employed can be applied to labeling active sites of a variety of hydrolytic enzymes.
ASJC Scopus subject areas
- Molecular Biology