A crystallographic study of the complex of phosphoramidon with thermolysin. A model for the presumed catalytic transition state and for the binding of extended substrates

The structure of the thermolysin inhibitor phosphoramidon (N-(α-l-rhamnopyranosyl-oxyhydroxyphosphinyl)-l-leucyl-l-tryptophan bound to the crystalline enzyme has been determined to a resolution of 2.3 Å by X-ray crystallography. The study shows that the complex of phosphoramidon with thermolysin resembles that of the presumed catalytic transition state inferred from the geometry of binding of dipeptide inhibitors. Also, the study reveals the mode of binding of thermolysin substrates extended on the imino side of the scissile peptide bond.The crystallographic results are consistent with a variety of other studies on the catalytic activity of thermolysin, and suggest a mechanism of action which is analogous to one of the two alternative mechanisms proposed by Lipscomb and co-workers (1968) for carboxypeptidase A. Key features of the proposed mechanism are that the substrate is initially bound to the enzyme with the carbonyl oxygen of the scissile peptide liganded to the zinc; that Glu143 promotes the nucleophilic attack of a buried water molecule on the carbonyl carbon, forming a tetrahedral intermediate; and that His231 acts as a proton donor. The observed binding of phosphoramidon to thermolysin provides further evidence supporting the mechanism in which Glu143 acts as a general base, promoting the attack of water on the carbonyl carbon, rather than the alternative mechanism in which Glu143 attacks the carbonyl carbon directly, forming an anhydride intermediate.