Purification and crystallization of human carboxypeptidase A

Abstract

Human carboxypeptidase A [EC 3.4.12.2] was isolated from activated pancreatic juice by affinity chromatography employing the competitive inhibitor benzylsuccinic acid as an affinity ligand. The structural and functional features of the human and bovine enzymes are quite analogous. The molecular weights of human and bovine carboxypeptidases A are virtually identical, their amino acid compositions are similar, both contain 1 g-atom of Zn/mole, and the activities of both are restored by addition of Zn to the apoenzyme. The inhibition of human carboxypeptidase by chelating agents is reversed by either dilution or addition of a metal such as Cu2+. When other metals are substituted for the native Zn, peptidase activity of the human metallocarboxypeptidases follows the order Co > Ni > Mn > Cd, while the sequence for esterase activities is Mn > Co = Cd > Ni. The latter sequence differs from that observed for the bovine enzyme. Human carboxypeptidase A crystallizes after dialysis at low ionic strength. Hydrolysis of the dipeptide carbobenzoxyglycyl-L-phenylalanine and of the ester benzoylglycyl-L-.alpha.-hydroxy-.beta.-phenyllacetate exhibits kinetic anomalies, but that of their longer homologues does not. Chemical modification with tyrosine reagents alters esterase and peptidase activities. The affinity chromatographic method here described should greatly facilitate future studies of this enzyme from human and other sources.