Purification and characterization of two serine carboxypeptidases from Aspergillus niger and their use in C-terminal sequencing of proteins and peptide synthesis.
A procedure was developed to prepare in large amounts two carboxypeptidases, CPD-I and CPD-II, from Aspergillus niger. They were each shown to be serine proteases and single-chain monomers with molecular masses of ca. 81 kDa and containing 22% carbohydrates. Amino acid analysis, carbohydrate determination, and N-terminal sequencing (20 to 25 residues) were performed on each enzyme. CPD-I showed sequence homologies with malt carboxypeptidase II, while the N terminus of CPD-II was different from that of any known serine carboxypeptidase. Like carboxypeptidase Y from Saccharomyces cerevisiae and carboxypeptidase III from malt, CPD-II contained a free sulfhydryl group that could play a role in catalysis. Both A. niger enzymes had pH optima of about 4 and were unstable above pH 7. Their specificities for substrate positions P1 and P'1 were characterized by use of, as substrates, a series of N-blocked amino acid esters and dipeptides. Both enzymes were specific for Arg, Lys, and Phe in P1. CPD-I preferred hydrophobic residues in P'1, while CPD-II was highly specific for Arg and Lys in this position. Each displayed an original specificity when P1 and P'1 were considered together. The specificities were also studied by analyzing the time course of the release of amino acids from eight different peptides of various lengths. CPD-I and CPD-II appeared to be quite suitable for C-terminal sequence studies as well as for the synthesis of peptide bonds. The latter was studied with two peptide esters as aminolysis substrates and a series of amino acid amides as nucleophiles.(ABSTRACT TRUNCATED AT 250 WORDS)
Dal Degan
F
F
Protein Chemistry Unit, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
Ribadeau-Dumas
B
B
Breddam
K
K
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D002268:Carboxypeptidases / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013379:Substrate Specificity
D010455:Peptides; D011506:Proteins; D002268:Carboxypeptidases; C022633:serine carboxypeptidase