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

Carboxypeptidase A was incubated at -60 degrees 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]NaCNBH3. 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 gamma-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.