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

Inorganic pyrophosphatase (EC 3.6.1.1.) has been isolated from the archaebacterium Methanobacterium thermoaurotrophicum (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 pyrophosphatase 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 K-m of PPi and magnesium were determined and were approx. 0.16 mM and 4.9 mM, respectively; V-max was estimated at about 570 U/mg.