Carboxypeptidase Y catalyzed C-terminal modification in the B-chain of porcine insulin

Abstract

It is demonstrated that carboxypeptidase Y can be used to exchange the C-terminal alanyl residue of porcine insulin with a threonyl residue, thus forming human insulin. Using threonine amide as nucleophile, the reaction proceeds as a transpeptidation via human insulin amide, des(Ala)B30(Thr-NH2)B30insulin. However, since the amidase activity of carboxypeptidase Y towards this particular insulin derivative is significant in comparison with its peptidase activity towards porcine insulin, various secondary products are subsequently formed. One of these products is human insulin, formed by deamidation of the initial coupling product. Unfortunately, this product cancer be separated chromatographically from the hydrolysis product des(Ala)B30insulin and residual porcine insulin. Another secondary product is des(Ala)B30(Thr-Thr-NH2)B30-31insulin, formed by coupling of an additional threonine amide residue via the amidase activity of carboxypeptidase Y. This product, together with the primary coupling product, des(Ala)B30(Thr-NH2)B30insulin, can be isolated from the reaction mixture by ion exchange chromatography, and both of these insulin derivatives can subsequently be converted to human insulin through the peptidyl amino acid amide hydrolase activity and amidase activity of carboxypeptidase Y. The semisynthetic human insulin formed in this fashion is essentially pure. It is further demonstrated that carboxypeptidase Y can be used to replace the C-terminal residue in another insulin derivative without significant side-reactions. Des(Ala)B30insulin is a much better substrate of CPD-Y than porcine insulin, resulting in a coupling product exhibiting a much higher stability towards degradation. Thus, the conversion of des(Ala)B30insulin to des(Lys-Ala)B29-30(Thr-OH)B29insulin is completely free of side reactions.