PURIFICATION AND CHARACTERIZATION OF INORGANIC PYROPHOSPHATASE FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM (STRAIN DELTA-H)

Inorganic pyrophosphatase (EC 3.6.1.1.) has been isolated from the arc haebacterium Methanobacterium thermoaurotrophicum (strain Delta H). Th e enzyme was purified 850-fold in three steps to electrophoretic homog eneity. The soluble pyrophosphatase consists of four identical subunit s: 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 pyrophosphatase show ed 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 pota ssium chloride. The enzyme was very specific for pyrophosphate (PPi) a nd Mg2+. Magnesium could be partially replaced by Co2+ (15%). The reac tion 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 t he experimental conditions at which the enzyme was present as its dime r, the apparent K-m of PPi and magnesium were determined and were appr ox. 0.16 mM and 4.9 mM, respectively; V-max was estimated at about 570 U/mg.