Carboxypeptidase Y catalyzed C-terminal modifications of peptides

Abstract

It is demonstrated that carboxypeptidase Y catalyzes the exchange of C-terminal amino acid residues in peptides for various other groups. Using N-blocked dipeptides as substrates and alcohols, ammonia, glycine, glycine amide and glycine methyl ester as nucleophiles, it is shown that peptides can be converted to peptide esters, peptide amides and to peptides with altered C-terminal amino acid residues. The incorporation of the different amine nucleophiles could be studied in a wide pH range, since the products were resistant towards further degradation. However, the conversion of peptides to peptide esters by alcoholysis was limited to pH below 4, due to enzymatic hydrolysis of the reaction product. The pH profile for the incorporation of the various amine nucleophiles suggests that they bind at the same location as the C-terminal amino acid residue of the peptides. The binding of glycine amide and glycine methyl ester to the enzyme, prior to aminolysis, is dependent on the state of ionization of a residue with a pKa of 6.6-7.1. It is further demonstrated that the coupling yields for all the transacylation reactions, catalyzed by carboxypeptidase y are dependent on the hydrophobicity of the amino acid leaving the active site. A pronounced influence of the penultimate residue of the substrate is demonstrated as well. The implications are discussed. © 1981 Carlsberg Laboratory.