title: Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp. cremoris AM2.
abstract: A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography 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 filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate 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-dithiothreitol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided 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, the release of N-terminal acidic residues from peptides was observed in addition 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 hydrolase 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 aminopeptidase for metabolism of proline-containing peptides.
authors.affiliation: Biochemistry Department, National University of Ireland, Galway, Irish Republic.
authors.forename: M
authors.initials: M
authors.lastname: McDonnell
authors.forename: P
authors.initials: P
authors.lastname: Bouchier
authors.forename: R J
authors.initials: RJ
authors.lastname: Fitzgerald
authors.forename: G
authors.initials: G
authors.lastname: O'Cuinn
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006868:Hydrolysis; D013294:Lactococcus lactis / Q000201:enzymology* / Q000648:ultrastructure; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D010450:Endopeptidases; C105560:lysine-p-nitroanilide hydrolase; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase