title: Purification and characterization of inorganic pyrophosphatase from Methanobacterium thermoautotrophicum (strain delta H).
abstract: Inorganic pyrophosphatase (EC 3.6.1.1.) has been isolated from the archaebacterium Methanobacterium thermoautotrophicum (strain delta H). The enzyme was purified 850-fold in three steps to electrophoretic homogeneity. The soluble pyrophosphatase consists of four identical subunits: the molecular mass of the native enzyme estimated by gel filtration was approx. 100 kDa and denaturing polyacrylamide gel electrophoresis gave a single band of 25 kDa. The enzyme also may occur as an active dimer formed by dissociation of the tetramer. The pyrophosphate showed an optimal activity at 70 degrees C and a pH of 7.7 (at 60 degrees C) and was not influenced by dithiothreitol, sodium dithionite or potassium chloride. The enzyme was very specific for pyrophosphate (PPi) and Mg2+. Magnesium could be partially replaced by Co2+ (15%). The reaction was inhibited for 60% by 1 mM Mn2+ in the presence of 24 mM Mg2+. In addition, the enzyme was inhibited by potassium fluoride (50% at 0.9 mM). Kinetic analysis revealed positive co-operativity for both Mg2+ and PPi with Hill coefficients of 3.3 and 2.0, respectively. Under the experimental conditions at which the enzyme was present as its dimer, the apparent Km of PPi and magnesium were determined and were approx. 0.16 mM and 4.9 mM, respectively; Vmax was estimated at about 570 U/mg.
authors.affiliation: Department of Microbiology, Faculty of Science, University of Nijmegen, The Netherlands.
authors.forename: G J
authors.initials: GJ
authors.lastname: van Alebeek
authors.forename: J T
authors.initials: JT
authors.lastname: Keltjens
authors.forename: C
authors.initials: C
authors.lastname: van der Drift
mesh_terms: D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017014:Methanobacterium / Q000201:enzymology*; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011755:Pyrophosphatases