The X-Ray Crystal Structure Analysis of the Refined Complex formed by Bovine Trypsin andp-Amidinophenylpyruvate at 1.4 Å Resolution

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Abstract
The X-ray crystal structure of the complex formed by bovine .beta.-trypsin and the complex formed by bovine .beta.-trypsin and the potent small inhibitor p-amidinophenylpyruvate at pH 7.6, was determined by difference Fourier methods at 1.4 .ANG. 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 .ANG. to the carbonyl C of the pyruvate group. The hybridization of this carybonyl C 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 O of p-amidinophenylpyruvate and to Ser195 O.gamma.. The probable proton shift makes Ser195 O.gamma. more nucleophilic and the attacked carbonyl C 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 3 series of p-amidinophenylalkanone compounds with carbonyl groups in .alpha.-, .beta.- and .gamma.-position to the phenyl ring.