The enzyme from Sigma has been used as a comparison to study the specificity of Metarhizium anisopliae carboxypeptidase A (MeCPA). MeCPA had been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. It has also been used to de-tyrosinate α-tubulin, in vitro, in order to induce high affinity to ethyl-N-phenylcarbamate (EPC) sepharose.
An enzyme that catalyzes the cleavage of amino acids
from a protein at the C-terminus of a polypeptide
chain.
Carboxypeptidase A-agarose product is prepared by the immobilization of carboxypeptidase A, originally isolated from the bovine pancreas, to activated 4% crosslinked beaded agarose.
Carboxypeptidase A(CPA) is an enzyme of the digestive system that is known to cleave amino acids favouring the C-terminal end as well as certain esters. This enzymatic activity depends on the metal at the catalytic site. Zn2+ and some Co2+-containing CPAs exhibit peptidase function, whilst esterase function has been seen by CPAs containing a variety of divalent d-block metals. CPA has a size similar to CA, consisting of about 300 amino acids and a molecular mass of 34 kDa.
Carboxypeptidase as isolated from bovine pancreas glands is a metalloenzyme that contains 1 g atom of zinc per mole of protein. It catalyzes the hydrolysis of the carboxyl-terminal peptide bond in peptides and proteins. It is primarily specific to aromatic and hydrophobic side chains such as phenylalanine, tryptophan or leucine. The enzyme also exhibits esterase activity. It is inhibited by beta-phenylpropionate and indole acetate.
Carboxypeptidase A is purified by DEAE-cellulose chromatography, activation with trypsin and dialysed against 0.1M NaCl, yielding crystals. It is recrystallised by dissolving in 20 mL of M NaCl and dialysed for 24hours each against the following salts present in 500mL of 0.02M sodium veronal pH 8.0, 0.5M NaCl, 0.2M NaCl and 0.15M NaCl. The last dialysate usually induces crystallisation. If it does not crystallise, then dialyse the last solution against 0.02M sodium veronal containing 0.10M NaCl. Only 2 or 3 recrystallisations are required to attain maximum activity. [Cox et al. Biochemistry 3 44 1964.] Enzyme activity is measured by hydrolysing hippuryl-L-phenylalanine (or phenylacetic acid) and observing the rate of change of optical density at 254nm (reaction extinction coefficient is ~0.592 cm2/Wmole at pH 7.5) [Bergmyer Methods in Enzymatic Analysis (Academic Press) 1 436 1974].
