Raaschou-Nielsen_1994_Pept.Res_7_132

Asn51 and Glu145 of (serine) carboxypeptidase Y function as binding sites for the C-terminal carboxylate group of peptide substrates, and Glu65 is involved in orienting these two amino acid residues. A series of mutants of carboxypeptidase Y where these three amino acid residues have been replaced were investigated for their applicability in transacylation reactions with amino acid esters as acceptors. With H-Val-OMethyl as the nucleophile, the fraction of aminolysis is significantly higher than with the corresponding amino acid, suggesting a beneficial effect of blocking the alpha-carboxylate group. Increasing the size of the alcohol moiety, i.e., -OEthyl, -OPropyl or OButyl, has an adverse effect on the binding of the nucleophile and on the maximum yield of aminolysis. Replacement of Asn51 and Glu145 with Ala or Gly has a pronounced beneficial effect both on binding and the maximum fraction of aminolysis. However, the results do not establish a specific type of interaction between the enzyme and these valine esters. It is probable that the rotational freedom around the ester bond allows multiple binding modes, depending on both the leaving group and type of structural change within the binding site. From a synthetic point of view, some of the mutant enzymes are much better than the wildtype enzyme when amino acid esters are used as nucleophiles.