An interactive computer graphics study of thermolysin-catalyzed peptide cleavage and inhibition by N-carboxymethyl dipeptides

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Abstract
Interactive computer graphics was used as a tool in studying the cleavage mechanism of the model substrate Z-Phe-Phe-Leu-Trp by the Zn endopeptidase thermolysin. Two Michaelis complexes and 3 binding orientations of the tetrahedral intermediate to the crystal structure of thermolysin were investigated. The results indicate that a Michaelis complex, which does not involve coordination of the scissile peptide to the Zn, is consistent with available experimental data and the most plausible of the 2 complexes. A tetrahedral intermediate complex wherein the 2 oxygens of the hydrated scissile peptide straddle the Zn in a bidentate fashion results in the most favorable interactions with the active site. The preferred tetrahedral intermediate and Michaelis complex provide a rationalization for the published substrate data. A trajectory for proceeding from the Michaelis complex to the tetrahedral intermediate is proposed. This trajectory involves a simultaneous activation of the Zn-bound water molecule concurrent with attack on the scissile peptide. A detailed ordered product release mechanism is also presented. These studies suggest some modifications and a number of extensions to the mechanism proposed earlier. The binding mode of the thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan is compared with that of the preferred tetrahedral intermediate, providing insight into this inhibitor design.