Allosteric properties of the Mg++-dependent inorganic pyrophosphatase in mouse liver cytoplasm.

Abstract

The Mg++‐dependent inorganic pyrophosphatase of the mouse liver is localized primarily in the cytoplasm. Substrate is the complex ion MgPPi2−; free PPi2− and MgADP are strong inhibitors, while Mg3+‐ions behave as an activator for the enzyme. The [Mg2+]/[substrate] ratio for optimum activity decreases with increasing substrate concentration. At substrate saturation and at pH‐optimum of 8.0, the optimum ratio is 10:1. Under these conditions Vmax= 3.7 μmole × min−1× mg protein−1, and Km= 1.6 × 10−5 M. The enzyme shows allosteric properties of a K‐system with homotropic and heterotropic effects. Homotropic cooperative interactions with Hill coefficients of 2 have been shown for the substrate, the inhibitor MgADP, and the activator Mg2+. Under the influence of the substrate the cooperativity of the activator decreases, while that of the inhibitor increases, the cooperativity of the substrate itself decreases by means of the activator. If the enzyme is preincubated for 5 min at 40°, the allosteric cooperativity of the substrate is lost by desensitization. This effect is accompanied by a partial loss of activity. Pi4− > MgPP2− > Mg2+ > MgADP protect the pyrophosphatase against heat inactivation. The possibility that the pyrophosphatase reaction follows the kinetics of a substrate‐activated enzyme, which is inhibited by free pyrophosphate, could be excluded.