The X-ray crystal structure analysis of the refined complex formed by bovine trypsin and p-amidinophenylpyruvate at 1.4 A resolution.

The X-ray crystal structure of the complex formed by bovine beta-trypsin and the potent small inhibitor p-amidinophenylpyruvate at pH 7.6, has been determined by difference Fourier methods at 1.4 A resolution and subsequently refined to a crystallographic R value of 0.191, applying diagonal matrix least-squares procedures including energy constraints. The amidino and the phenyl group of this inhibitor are bound to the specificity pocket, essentially as previously observed in benzamidine-trypsin. The reactive Ser195 O gamma of trypsin forms a covalent bond of length 1.7 A to the carbonyl carbon of the pyruvate group. The hybridization of this carbonyl carbon is just between trigonal and tetrahedral. The imidazole ring of His57 is in a correct orientation to form bonds via its N epsilon 2 hydrogen to one of the carboxylate oxygens of p-amidinophenylpyruvate and to Ser195 O gamma. The probable proton shift makes Ser195 O gamma more nucleophilic and the attacked carbonyl carbon of p-amidinophenylpyruvate more electrophilic and thus facilitates bond formation. These specific interactions offer a qualitative explanation for the unique binding properties of p-amidinophenylpyruvate and for the applicability of the quantitative structure-activity relations previously found by Markwardt and coworkers for three series of p-amidinophenylalkanone compounds with carbonyl groups in alpha-, beta- and gamma-position to the phenyl ring.