Regulation of Yeast Inorganic‐Pyrophosphatase Activity by Divalent Cations

The stability constants of the magnesium and calcium complexes of inorganic pyrophosphate and the manganese complex of ATP were measured with a specific ion electrode at pH 5.6 and 25°C. These data were used to calculate the concentrations of free metals, free substrates, and the metal · substrate complex in their mixtures. The effect of these variables on the activity of the inorganic pyrophosphatase from baker's yeast was investigated and it was concluded that the active enzyme · substrate complex has at least three sites specific to Mg 2+ or Ca 2+ and two sites specific to Mn 2+ per protein molecule. Calcium inhibits the enzymatic hydrolysis of inorganic pyrophosphate due to displacement of three magnesium ions from the enzyme complex with the substrate. A simple semi‐automatic analyzer is described which measures inorganic phosphate in solution at concentrations down to 2 μM.