CHEMICALLY MODIFIED CARBOXYPEPTIDASE Y WITH INCREASED AMIDASE ACTIVITY by

Treatment of carboxypeptidase Y with ~4C-iodoacetamide caused a drastic reduction in the peptidase activity towards FA-Phe-$Leu-OH while the esterase activity towards FA-Phe 89 the amidase activity towards FA-PhelNH2 and the peptidyl amino acid amide hydrolase activity towards FA-Phe-$GIy-NH2 were much less affected. The loss of peptidase activity could be correlated with the incorporation of a single equivalent of reagent and it was demonstrated that the site of reaction was a methionyl residue, thus forming a sulfonium derivative. Analogous methionyl modifications were performed: carboxypeptidase Y modified with phenacylbromide hydrolysed substrates with bulky leaving groups in the P~ position, i.e. -OEt, -OBzl, -Gly-NH,, -Gly-OH, and -Leu-OH, at reduced rates while substrates with small groups in that position, i.e. -OMe and -NH~, were hydrolysed with increased rates. These results indicate that the methionyl residue modified by phenacylbromide is located in the S; binding site of the enzyme. Similar results were obtained with carboxypeptidase Y modified with m-nitrophenacylbromide and p-nitrophenacylbromide. The increase in amidase activity and decrease in peptidyl amino acid amide hydrolase activity of carboxypeptidase Y following modification with phenacylbromide, m-nitrophenacylbromide, and p-nitrophenacylbromide was exploited in deamidation of peptide amides. These modified enzymes deamidated peptide amides with the exception of those containing a C-terminal glycyl or seryl residue in yields of 80-100% which is significantly higher than with unmodified carboxypeptidase Y.