Identification of a Glutamic Acid at the Active Center of Bovine Carboxypeptidase B

The glutamic acid residue at the active center of bovine carboxypeptidase B was labeled with α-N-bromoacetyl-d-[5-14C]arginine and the alkylated protein was hydrolyzed with pepsin. A 14C-labeled peptide fraction was isolated in a 52% yield by gel filtration on Sephadex G-25 followed by ion exchange chromatography on CM-cellulose. Compositional and end group analysis, in addition to enzymatic hydrolysis with carboxypeptidases A and B, aminopeptidase M, and trypsin, suggest the following amino acid sequence: Thr-Phe-Glu-Leu-Arg-Asp-Lys-Gly-Arg-Tyr-Gly-Phe. A comparison with the glutamic acid-containing sequence at the active sites of carboxypeptidases A and B revealed nearly complete homology.