Direct chemical evidence for the mixed anhydride intermediate of carboxypeptidase A in ester and peptide hydrolysis

Abstract

Carboxypeptidase A was incubated at −60°C with an excess of O -( trans - p -chlorocinnamoyl)-L-phenyllactate, O -(hippuryl)-glycolate or N -(hippuryl)-L-phenylalanine. After rapid denaturation with trichloracetic acid the precipitated protein was reduced with [ 3H]NaCNBH 3. 3H Labeled enzyme was isolated by gel chromatography on Sephadex G-25. After complete acid hydrolysis the specific label within the protein was identified by high voltage paper electrophoresis and paper chromatography as [ 3H]2-amino-5-hydroxyvaleric acid, the reduction product of a γ-acylated glutamic acid. These results give strong evidence that a mixed anhydride intermediate is formed, which for the first time was identified during the hydrolysis of classical ester as well as peptide substrates by direct chemical means.