Purification, Properties, and Multiple Forms of a Manganese-Activated Inorganic Pyrophosphatase fromBacillus subtilis

The hydrolysis of magnesium pyrophosphate by inorganic pyrophosphatase fromBacillus subtilisrequired 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 apparentMr36,000 by SDS–PAGE, but ofMr34,000 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 μmol 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 Na2SO4or 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 ofKdapproximately 0.005 and 0.35 μM, respectively. The properties of this enzyme may account in part for the unusual manganese requirements ofB. subtilisand related species.