title: Purification and characterization of a dipeptidyl carboxypeptidase from Pseudomonas sp. WO24.
abstract: A dipeptidyl carboxypeptidase (DCP) activity was detected in cell-free extracts of Pseudomonas sp. WO24. After purification and characterization the enzyme was found to be homogeneous by SDS-PAGE, and had a molecular mass of 74,000 Da by SDS-PAGE and 72,000 Da by gel filtration, indicating that it is monomeric. The isoelectric point was 5.2 and optimum pH was 6.5-7.0. It showed a specific activity of 780 mumol/min/mg, which is the highest of the values shown by known enzymes. The enzyme hydrolyzed angiotensin I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from the C-terminus bradykinin. The DCP could not cleave imido-bonds, Gly-Gly bonds, or tripeptides. The enzymatic activity was completely inhibited by 0.001 mM EDTA and 0.1 mM O-phenanthroline, but it was not affected by general serine and cysteine protease inhibitors. Addition of Zn2+ completely restored the original activity of the inactivated DCP treated with EDTA. These results suggest that this enzyme is a zinc metalloprotease. The characteristics of the purified enzyme are slightly different from those of the DCPs from Escherichia coli, Pseudomonas maltophilia, and Corynebacterium equi, and considerably from those of the DCP from Bacillus pumilus.
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
authors.forename: W
authors.initials: W
authors.lastname: Ogasawara
authors.forename: N
authors.initials: N
authors.lastname: Abe
authors.forename: T
authors.initials: T
authors.lastname: Hagio
authors.forename: H
authors.initials: H
authors.lastname: Okada
authors.forename: Y
authors.initials: Y
authors.lastname: Morikawa
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D013045:Species Specificity
chemical_list: D010450:Endopeptidases; C020071:dipeptidyl carboxypeptidase