Crystal Structure of the Covalent Complex Formed by a Peptidyl α,α-Difluoro-β-keto Amide with Porcine Pancreatic Elastase at 1.78-Å Resolution

Abstract

The crystal structure analysis of the covalent enzyme-inhibitor complex of porcine pancreatic elastase (PPE) with a peptidyl α,α-difluoro-β-keto amide has shown that the tightly bound inhibitor forms an hemiketal complex with the Oϒatom of the catalytic Ser-195 and is stabilized by five intermolecular H bonds and optimal van der Waals' surface interactions. The inhibitor is bound to the enzyme in an antiparallel β-pleated sheet arrangement. The carbonyl oxygen atom of the inhibitor is situated in the “oxyanion hole”, hydrogen bonded to the amido nitrogen atoms of Ser-195 and Gly-193. A strong hydrogen bond between His-57 and a fluorine atom also aids in stabilizing the complex. The H-bonding catalytic tetrad of elastase is structurally intact. Covalent attachment of ligand plus active site Ser-195 is based upon contiguous electron density found in the initial, unbiased difference Fourier electron density map. The resulting hemiketal linkage has chemical and structural similarities with the putative tetrahedral intermediate of a productive enzyme-peptide ligand complex. This analysis provides structural evidence for the preferred binding of a novel class of inhibitors of the serine proteinases. Two refinement programs, Eref and Tnt, were used to refine the enzyme + inhibitor model. © 1989, American Chemical Society. Alle Rechte vorbehalten.