Crystallographic Study of the Binding of Dipeptide Inhibitors to Thermolysin: Implications for the Mechanism of Catalysis

Abstract

The mode of binding to thermolysin of a number of dipeptide inhibitors including β-phenylpropionyl-L-phenylalanine and carbobenzoxy-L-phenylalanine has been determined by x-ray crystallography to a nominal resolution of 2.3 Å. These results indicate the probable mode of binding of extended substrates to thermolysin and also suggest a mechanism of action for this neutral metalloendopeptidase which is similar in a number of respects to one of the two alternate mechanisms for peptide hydrolysis by carboxypeptidase A proposed by Lipscomb and co-workers (Lipscomb, W. N., et al. (1968), Brookhaven Symp. Biol. 21, 24). The crystallographic results suggest that substrates bind to thermolysin with the carbonyl oxygen of the scissile peptide bond displacing a water molecule and becoming the fourth zinc ligand. Also, in the case of β-phenylpropionyl-L-phenylalanine, the side chain of Glu-143 and a neighboring water molecule are seen to be close to the carbonyl carbon of the scissile peptide, as is the imidazole of His-231 to the peptide nitrogen, suggesting that these groups may participate in catalysis. It is proposed that following substrate binding, Glu-143, acting as a general base, promotes the attack of a water molecule on the carbonyl carbon of the scissile peptide bond. Concurrent with, or following this step, His-231 donates a proton to the peptide nitrogen, forming a tetrahedral intermediate which is stabilized by both hydrogen bonds and hydrophobic interactions. Finally, the carbon-nitrogen bond of the intermediate breaks to yield the products. Thus, Glu-143 in thermolysin appears to be a counterpart of Glu-270 in carboxypeptidase A, while the role of His-231 as a proton donor is analogous to that of Tyr-248. The proposed mechanism for thermolysin is similar to that suggested by Lipscomb for carboxypeptidase A in which Glu-270 acts as a general base. In contrast, however, in the case of thermolysin, stereochemical restrictions appear to exclude the possibility of direct nucleophilic attack of the carbonyl carbon by the acid, as occurs in the alternative mechanism of action for carboxypeptidase A proposed by Lipscomb and co-workers. © 1977, American Chemical Society. All rights reserved.