Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin

Abstract

The X-ray crystal structures of native penicillopepsin and of its complex with a synthetic analog of the inhibitor pepstatin were refined recently at 1.8-.ANG. resolution. These highly refined structures permit a detailed examination of peptide hydrolysis in the aspartic proteinases. Complexes of penicillopepsin with substrate and catalytic intermediates were modeled, by using computer graphics, with minimal perturbation of the observed inhibitor complex. A thallium ion binding experiment shows that the position of solvent molecule O39, between Asp-33(32) and Asp-213(215) in the native structure, is favorable for cations, a fact that places constraints on possible mechanisms. A mechanism for hydrolysis is proposed in which Asp-213(215) acts as an electrophile by protonating the carbonyl oxygen of the substrate, thereby polarizing the C.sbd.O bond, a water molecule bound to Asp-33(32) (O284 in the native structure) attacks the carbonyl carbon as the nucleophile in a general-base mechanism, the newly pyramidal peptide N is protonated, either from the solvent after N inversion or by an internal proton transfer via Asp-213(215) from a hydroxyl of the tetrahedral carbon and the tetrahedral intermediate breaks down in a manner consistent with the stereoelectronic hypothesis. The models permit the rationalization of observed subsite preferences for substrates and may be useful in predicting subsite preferences of other aspartic proteinases.