Chemically modified carboxypeptidase Y with increased amidase activity

Abstract

Treatment of carboxypeptidase Y with14C-iodoacetamide caused a drastic reduction in the peptidase activity towards FA-Phe↓Leu-OH while the esterase activity towards FA-Phe↓OMe, the amidase activity towards FA-Phe↓Nh2 and the peptidyl amino acid amide hydrolase activity towards FA-Phe↓Gly-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 P1′ position,i.e.OEt,-OBzl,-Gly-NH2,-Gly-OH, and-Leu-OH, at reduced rates while substrates with small groups in that position, i.e.,-OMe and-NH2, were hydrolysed with increased rates. These results indicate that the methionyl residue modified by phenacylbromide is located in the S1′ 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 peptid 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. © 1984 Carlsberg Laboratory.