Evidence for segmental mobility in the active site of pepsin

The low hydrolytic activity (k/sub cat/ < 0.001 s-1) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a 19F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference 1H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu++ on 19F- and 1H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S3/S5 binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis