The inhibition of -chymotrypsin with 13C-enriched alanine- and phenylalanine-based α-nitrosoamides, as active-site-directed and enzyme-activated inhibitors, results in the alkylation (benzylation) of side chains and also of the amide linkages of the protein backbone (both at O and N). 13C NMR spectra of the denatured inhibited enzyme (in Gdn-HCl) indicate that alkylation has occurred at N, S, and C sites. 13C NMR spectra of the amino acid mixtures from fully hydrolyzed inhibited enzymes show that the pattern of alkylation is strikingly different for inhibitions by the alanine- and phenylalanine-based inhibitors. In the case of the phenylalanine-based inhibitor, approximately equally intense signals are observed at 52.32, 51.31, 36.78, and 32.91 ppm, while with the alanine-based inhibitor, a major signal appears at 52.35 ppm, with minor signals appearing at 36.83 and 32.9 ppm. Chromatographic and NMR evidence is presented to indicate that the 52.32-52.35-ppm signal stems from TV-benzylglycine. The chemical shift data suggest that the 51.31-ppm signal stems from N-benzylserine and the 36.78-36.83-ppm signal from S-benzylcysteine. Mechanisms are presented to account for the formation of those products.
ASJC Scopus subject areas
- Colloid and Surface Chemistry