title: Characterisation of arginase from the extreme thermophile 'Bacillus caldovelox'.
abstract: A thermostable arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was purified from the extreme thermophile 'Bacillus caldovelox' (DSM 411) by a procedure including DEAE-Sepharose chromatography, and gel filtration, anion exchange and hydrophobic-interaction fast-protein liquid chromatography, with substantial retention of the metal ion cofactor. The purified enzyme is a hexamer with a subunit Mr of 31,000 +/- 2000 and contains greater than or equal to 1 Mn atom per subunit. Maximum activation on incubation with Mn2+ is 29%. Activity is optimal at pH 9 and at 60 degrees C the Km for arginine is 3.4 mM and Ki(ornithine) is 0.55 mM. Incubation in 0.1 M Mops/NaOH buffer (pH 7) causes rapid inactivation at 60 degrees C (t1/2 (half life) = 4.5 min) and individually 0.1 mM Mn2+ or 1 mg/ml BSA (bovine serum albumin) increase the t1/2 of arginase activity 4-fold, but combined they produce greater than 1000-fold increase and a t1/2 = 105 min at 95 degrees C. Aspartic acid and other species that bind Mn2+ can replace BSA, and it is suggested that arginase can be inactivated by free Mn2+. A strong chelating agent causes inactivation without subunit dissociation, but arginase dissociates rapidly at pH 2.5. Reassociation occurs at pH 9 and is unusual in that it does not require Mn2+.
authors.affiliation: Department of Biological Sciences, School of Science and Technology, University of Waikato, Hamilton, New Zealand.
authors.forename: M L
authors.initials: ML
authors.lastname: Patchett
authors.forename: R M
authors.initials: RM
authors.lastname: Daniel
authors.forename: H W
authors.initials: HW
authors.lastname: Morgan
mesh_terms: D000595:Amino Acid Sequence; D001119:Arginase / Q000096:biosynthesis / Q000302:isolation & purification*; D001407:Bacillus / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008345:Manganese; D001119:Arginase