Purification and properties of a soluble inorganic pyrophosphatase from Rhodopseudomonas palustris.
A soluble inorganic pyrophosphatase from photolithoautotrophically grown Rhodopseudomonas palustris was purified to a state of apparent homogeneity applying high resolving liquid chromatography steps. Values of 65 500 and 64 500 were calculated for the relative molecular mass under non-dissociating conditions employing gel filtration and high-performance liquid chromatography, respectively. Dissociation sodium dodecyl sulfate gel electrophoresis resulted in a value of 32 000, indicating that the enzyme is composed of two subunits of equal molecular mass. Isoelectric focusing revealed a pI value of 4.7. The purified enzyme was specific for PPi and the activity was modified by divalent cations. Ca2+, Mn2+, Mg2+ and Co2+ were potent activators at a concentration ratio of [Me2+]/[PPi] less than 1. Ca2+ turned out to be the most potent activator. Free Me2+ was inhibitory on the PPiase activity. The (Me-PPi) complex is regarded as the functional substrate. Km and Ki values of the metal activation and inhibition were determined. An activation energy of Ea = 14.4 kJ/mol was derived from Arrhenius plots for the enzymatic reaction.
D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002845:Chromatography; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010788:Photosynthesis; D011755:Pyrophosphatases / Q000302:isolation & purification* / Q000378:metabolism; D012241:Rhodopseudomonas / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity; D013816:Thermodynamics
D002413:Cations, Divalent; D011755:Pyrophosphatases; D002118:Calcium