Purification and Characterization of a Dipeptidyl Carboxypeptidase from Pseudomonas sp . W 024

A dipeptidy} cftrbexypeptidase (DCP) activity was detected in cell-free extracts of Pseudomonas sp. W024. After purification and characterization the enzyme was found to be homogeneous by SDS-PAGE, and had a molecu)ar mass of 74,eOODa by SDS-PAGE and 72,OOODa by gel filtration, i dicating that it is monomeric. The isoeleenic point was 5.2 and optimum pH was 6.5-7.0. It showed a specific activity of 780"molfmintmg, -'hich is the highest of the yalues shown by known enzymes. The enzyme hydrolyzed angiotensin I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from the C-terminus bradykinim. The DCP ceuld not c]eaye imido-bonds, Gly-Gly bonds, or tripeptides. The enzymati ¢ actiyity was compgetely inhibited by O.OOgmM EDTA and O.1mM o-phenanthroline, but it was llot affected by general serine and cysteimae protease inhibiters. Addition ef Zn2" completely restored the original activity of the ina¢ tivated DCP treated with EDTA. These results suggest that this enzyme is a zinc metalloprotease.