Inorganic pyrophosphatase activity in oral streptococci: purification and properties of the enzyme from Streptococcus salivarius.
Inorganic pyrophosphatase has been purified from the soluble fraction of Streptococcus salivarius by protamine sulfate treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was purified approximately 500-fold with a 33% yield. The purified enzyme was homogeneous since it showed a single band when examined by nondenaturing polyacrylamide gel electrophoresis. It was rich in acidic (glutamic and aspartic) amino acids, as well as serine and glycine. The enzyme was devoid of sulfur-containing amino acids. The purified enzyme was specific for the hydrolysis of inorganic pyrophosphate and did not hydrolyze any other phosphate-ester compound examined. Inorganic pyrophosphatase activity was completely dependent on a divalent cation. Activity was maximum in the presence of Mg2+ while activity in the presence of Mn2+ and Co2+ was significantly lower. In the presence of Mg2+, a number of divalent cations, however, inhibited the enzyme activity. The true substrates for S. salivarius inorganic pyrophosphatase were magnesium-pyrophosphate complexes, i.e., MgPPi and Mg2PPi, while free Mg2+ had no effect on the enzyme activity and free PPi inhibited the hydrolysis of inorganic pyrophosphate. Km value for magnesium-pyrophosphate complexes was 16.4 microM. Km value for total Mg2+ was similar ranging between 14.4 and 20 microM. Analysis of data by Hill plots indicated one binding site for Mg2+ and two for PPi. Among various nucleotides and glycolytic intermediates examined, GDP, GMP, and fructose-1, 6-P2 showed significant inhibitory effect on enzyme activity.
D000596:Amino Acids / Q000032:analysis; D002412:Cations / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D006019:Glycolysis; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*; D013291:Streptococcus / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D002412:Cations; D011755:Pyrophosphatases