IMPROVEMENT OF THE APPLICABILITY OF CARBOXYPEPTIDASE-Y IN PEPTIDE-SYNTHESIS BY PROTEIN ENGINEERING

Asn51 and Glu145 of(serine) carboxy-peptidase Y function as binding sites for dc 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 ann on the maximum yield of aminolysis. Replacement of Asn51 and Glu145 with Ala or G!v 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 tire 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 wild-type enzyme when amino acid asters are wed as nucleophiles.