A NOVEL DIPEPTIDYL AMINOPEPTIDASE FROM PSEUDOMONAS SP STRAIN WO24

An activity similar to that of dipeptidyl aminopeptidase I (DAP I) whi ch releases dipeptide from Gly-Arg-p-nitroanilide (Gly-Arg-pNA) was de tected in a Pseudomonas sp, An enzyme was isolated and purified about 400-fold by a series of column chromatographies. The enzyme, named DAP BI (DAP from bacteria, type I), was revealed to be homogeneous by sod ium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing, The molecular mass was estimated to be 82 kDa by SDS-PAGE and 65 kDa by gel filtration, suggesting that the enzyme may be a monomer, The enzyme had an isoelectric point of 4.7. It is optim ally active at pH 9.0. The K-m and V-max of the enzyme for Gly-Arg-pNA were 0.25 mM and 195 mu mol/min/mg, respectively, The purified enzyme did not hydrolyze Gly-Phe-pNA, which was also a substrate for DAP I, whereas it hydrolyzed Arg-Arg-4-methoxy-beta-naphthylamide (Arg-Arg-MN A), a model substrate for DAP III. The K-m and V-max for Arg-Arg-MNA w ere 0.019 mM and 145 mu mol/min/mg, respectively. This purified enzyme can also catalyze the removal of Asp-Arg from the N termini of angiot ensins I and II. The enzyme activity was completely inhibited by Zn(II ) (0.5 mM), tosyl-L-lys-chloromethyl ketone (0.1 mM), and leupeptin (0 .1 mM) and partially inhibited by Co(II) (0.5 mM) and chymostatin (0.1 mM), whereas the enzyme was not affected by general serine protease i nhibitors (phenylmethylsulfonyl fluoride and diisopropylfluorophosphat e) and thiol protease inhibitors, The substrate specificity, classific ation of catalytic site, and other enzymatic properties demonstrate th at this enzyme is distinct from the previously described mammalian DAP s I and III and Saccharomyces cerevisiae DAP III. These results indica te that DAP BI may be a new type of the DAP family.