Gourt Home::Gourt :: Carboxypeptidase A
Description
Carboxypeptidase A (CPA) is a pancreatic exopeptidase hydrolyzing the peptide bond adjacent to the C-terminal end of a polypeptide chain.
Formula
R-CONH-CHR'COOH + H20
> R-COOH + H2NCHR'COOH
Bovine CPA
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. T
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.
Assay Method
The determination of reaction velocity is based upon the method of Folk and Schirmer (1963). The rate of hydrolysis of hippuryl-L-phenylalanine is determined by measuring the increase in absorbance at 254 nm. One unit hydrolyzes one micromole of hippuryl-L-phenylalanine per minute at pH 7.5 and 25°C under the specified conditions.
Reagents
* 0.025 M Tris⋅HCl buffer containing 0.5 M sodium chloride, pH 7.5 * 0.001 M Hippuryl-L-phenylalanine in 0.025 M Tris⋅HCl, pH 7.5 with 0.5 M sodium chloride * 10% Lithium chloride
Enzyme
Dissolve in 10% lithium chloride to a concentration of 1-3 units per ml. The enzyme crystals are not readily soluble in the diluent. Do not use solution for assay until the solution has cleared.
Read A278 in cold 10% lithium chloride.
Equation
mg/ml = A278 X 0.515
Procedure
Adjust spectrophotometer to 254 nm and 25°C.
Pipette 2.0 ml of substrate into each cuvette and incubate in spectrophotometer at 25°C for 3-4 minutes to reach temperature equilibration and establish blank rate, if any. Add 0.1 ml of diluted enzyme and record increase in A254 for 3-5 minutes. Determine ΔA254 / minute from the initial linear portion of the curve.
Calculation
Units/mg = (Delta A254/min) / 0.36* X mg enzyme/ml reaction mixture
- 0.36 = Extinction coefficient of hippuric acid formed during reaction
References
http://www.worthington-biochem.com/COA/default.html Enzymes
"Carboxypeptidase A"