Comparison of the structures of carboxypeptidase A and thermolysin.

Abstract

Chemistry and Physics, University of Oregon, Eugene, Oregon 97403 Using the known three-dimensional structures of the zinc metallopeptidases carboxypeptidase A and thermolysin, the two enzymes are compared by superimposing one active site upon the other and identifying corresponding structural and functional elements. The overall folding of the respec- tive enzymes is quite different, but the active sites have several features in common, and the modes of binding of dipeptide inhibitors are very similar. The known differences in specificity of the two enzymes are readily explained in terms of the observed differences between the respective substrate binding sites. Also, it is clear that the similarities between carboxypeptidase A and thermolysin are due to convergent rather than divergent evolution. The close struc- tural correspondence in the immediate vicinity of the zinc suggests that these two neutral proteases from mammalian and bacterial sources may have similar mechanisms for peptide hydrolysis. For both thermolysin and carboxypepti- dase A, peptide substrates bind to the enzyme with the carbonyl oxygen of the scissile peptide displacing a bound water molecule and becoming the fourth zinc ligand. Evi- dence is presented that for both enzymes, hydrolysis pro- ceeds by a general base mechanism with Glu 143 of thermol- ysin, or Glu 270 of carboxypeptidase A, promoting the attack of a water molecule on the carbonyl carbon of the substrate. There is no tyrosine in the thermolysin active site which is analogous to Tyr 248 of carboxypeptidase A. In contrast, the imidazole of His 231 is the presumptive proton donor. The comparison of the active sites of thermol- ysin and carboxypeptidase A does not obviously confirm that in carboxypeptidase A Tyr 248 acts as a proton donor in peptide hydrolysis, but neither does it rule out this possibility.