CHARACTERIZATION OF X-PROLYL DIPEPTIDYL AMINOPEPTIDASE FROM LACTOCOCCUS-LACTIS SUBSP LACTIS

A dipeptidyl aminopeptidase catalysing hydrolysis of X-prolyl amidomethylcoumarin (AMC) substrates has been purified from Lactococcus lactis subsp. lactis H1. The active enzyme has a molecular mass of approximately 150 kDa, a subunit molecular mass of 82 to 83 kDa and is inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. The K(m) and k(cat) values for five different dipeptidyl AMC substrates (Gly-Pro; Leu-Pro; Lys-Pro; Phe-Pro- and Glu-Pro-AMC) are similar except for the K(m) value for Glu-Pro-AMC, which is about threefold higher than that for the other substrates. The enzyme also catalyses hydrolysis of X-Ala-AMC substrates but with much lower k(cat) and higher K(m) values than the corresponding X-Pro-AMC substrates. The beta-casein-derived heptapeptides Lys-Ala-Val-Pro-Tyr-Pro-Gln and Tyr-Pro-Phe-Pro-Gly-Pro-Ile were hydrolysed, but bradykinins with N-terminal sequences Arg-Pro-Pro- and Lys-Pro-Pro- were not. Dipeptidyl aminopeptidase specific activity is the same in a plasmid-free strain of L. lactis subsp. lactis H1 and in the wild-type, indicating that the enzyme is chromosomally encoded.