Purification, properties, and multiple forms of a manganese-activated inorganic pyrophosphatase from Bacillus subtilis.
The hydrolysis of magnesium pyrophosphate by inorganic pyrophosphatase from Bacillus subtilis required its specific, time-dependent, and prior activation by Mn2+ ions. This was reversed when Mn2+ ions were removed with EDTA. Free Mn2+ ions were not required for catalysis. Pyrophosphatase purified to near homogeneity gave a single main band of apparent M(r) 36,000 by SDS-PAGE, but of M(r) 34,000 by matrix-assisted laser desorption ionization-mass spectrometry. The native enzyme equilibrated at pH 7 between three distinct molecular forms. Exposure to Mn2+ generated a catalytically active trimer of specific activity about 5000 mumol pyrophosphate hydrolyzed/min/mg protein. Exposure to EDTA generated two catalytically inactive forms, a dimer at low ionic strength and a separate form, of uncharacterized multimeric nature, at molar concentrations of Na2SO4 or Li2SO4. The latter form was an intermediate in the dimer-trimer transition caused by addition or removal of manganese ions. Mn2+ reacted with this "intermediate" form, apparently by reversible association with two noninteracting binding sites of Kd approximately 0.005 and 0.35 microM, respectively. The properties of this enzyme may account in part for the unusual manganese requirements of B. subtilis and related species.
Kuhn
N J
NJ
School of Biochemistry, University of Birmingham, United Kingdom. N.J.Kuhn@bham.ac.uk
Ward
S
S
D001412:Bacillus subtilis / Q000201:enzymology*; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D043564:Inorganic Pyrophosphatase; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008345:Manganese / Q000378:metabolism* / Q000494:pharmacology; D008970:Molecular Weight; D011755:Pyrophosphatases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D007527:Isoenzymes; D008345:Manganese; D004492:Edetic Acid; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase