Complex of alpha-chymotrypsin and N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy

Abstract

A dipeptidyl trifluoromethyl ketone, N-acetyl-L-leucyl-L-[1-13C]phenylalanyl trifluoromethyl ketone, was synthesized. This compound inhibits chymotrypsin with Ki = 1.2 microM [Imperiali B., & Abeles, R.H. (1986) Biochemistry 25, 3760-3767]. The complex formed between this inhibitor and alpha-chymotrypsin was examined with 1H, 13C, and 19F NMR spectroscopy to establish its structure in solution. The keto group of the trifluoro ketone is present as an ionized hemiketal group as deduced from the comparison of its 13C chemical shift with those of model hemiketals. The pKa of the hemiketal hydroxyl in the complex is approximately 4.9, which is about 4.2 units lower than the pKa of model hemiketals. This observation provides direct evidence that serine proteases are able to stabilize the oxyanions of tetrahedral adducts. Evidence is also presented for the presence of an Asp-His H bond and protonation of the imidazole group of His-57 in the tetrahedral adduct. The pKa of His-57 is higher than 10. This observation directly indicates that the pKa of His-57 is elevated in a complex containing a tetrahedral adduct.