Carboxypeptidase A

I.U.B.: 3.4.17.1
Peptidyl-L-amino-acid hydrolase

Carboxypeptidase A (CPA) is a pancreatic exopeptidase hydrolyzing the peptide bond adjacent to the C-terminal end of a polypeptide chain.

Bovine CPA is derived from a trimeric zymogen by trypsin activation. See Puigserver and Desnuelle (1975) on bovine 6S procarboxypeptidase A. It is indicated that only Subunit I is activated to CPA. See also Behnke et al. (1970); Freishein et al. (1967); Brown et al. (1963). Kokkonen et al. (1986) report on a CPA in rat serosal mast cells. Burgess et al. (1975) report on the enhanced thermal stability of immobilized CPA. The enzyme has been reviewed by

Characteristics of Carboxypeptidase A from Bovine Pancreas:

Molecular weight: 35,268 (307 residues; Bradshaw et al. 1969b).

Composition: CPA contains 1 atom of zinc per molecule probably at the active site. Rosenberg et al. (1975) have reported on the substitution of zinc by other metals (Cu²⁺ CPA shows no peptidase nor esterase activities). The amino acid sequence has been reported (Bradshaw 1969; Bradshaw et al. 1969; and Nomoto et al. 1969). Structural studies have been reviewed by Lipscomb (1970). Depending upon the method of isolation and activation there are differences in amino acid composition: CPA α, -β, -γ have 307, 305, and 300 amino acid residues respectively. (See Table I in Hartsuck and Lipscomb 1971). In solution the activities are the same but activities of the crystals differ. Lipscomb 1973; Quiocho et al. 1972). Johansen and Vallee (1975) report on differences in conformation of the crystals as compared with the molecules in solution. Pétra and Neurath (1969) have reported that under certain conditions five components can be obtained from Anson CPA (CPAγ). See also Pétra et al. (1969).

Studies on the active site have been reported by Turk and Marshall (1975). Auld and Holmquist (1974) indicate there to be different binding sites for esters and peptides. King et al. (1987) discuss the interaction of carboxypeptidase A with carbamate and carbonate esters. Suh and Kaiser (1975) report on the hydrolysis of different enantiomers of a substrate.

Optimum pH: 7 - 9, depending upon substrate.

Extinction coefficient: = 19.4.

Inhibitors: CPA is inhibited by cysteine, sulfides, and cyanide but not by diisopropylphosphofluoridate (DFP) or phenylmethanesulfonyl flouride (PMSF). It is strongly inhibited by the chelating agent 1,10-phenanthroline. The Anson enzyme is insoluble in H₂O and freezing and lyophilization inactivate the crystalline preparation. Ochratoxin A is a competitive inhibitor of the enzyme (Pitout and Nel 1969).

Activator: The zinc component is essential for activity; if lost on dialysis it may be replaced.

Specificity: C-terminal L-amino acids that have aromatic or branched sidechains are preferentially cleaved off the peptide chain. See Libscomb (1970). Auld and Holmquist (1974) report on peptide and ester hydrolytic mechanisms.