Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp cremoris AM2

a hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been p urified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chr omatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native M -r of 50 000-55 000 by gel filtration chromatography and by FPLC gel filtra tion on Superose 12 and to be composed of a single polypeptide chain follow ing SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amas tatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chlor omercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the purification procedures at 4 degrees C and its stability was greatly improved when 10 ml glycerol/l and 2 mM-dithiothreit ol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides. tripeptides and longer peptides p rovided that proline was not present in the penultimate position from the N -terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, t he release of N-terminal acidic residues from peptides was observed in addi tion to the release of N-terminal proline from Pro-Leu-Gly-NH2, Pro-Leu-Gly -Gly and Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr. This ability of Lys-pNA hydro lase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr-Pro-Phe-Pro-Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl amin opeptidase for metabolism of proline-containing peptides.