Biosynthesis of Bacterial Glycogen: VII. Purification and Properties of the Adenosine Diphosphoglucose Pyrophosphorylase of Rhodospirillum Rubrum

Abstract

Abstract The adenosine diphosphate glucose pyrophosphorylase from Rhodospirillum rubrum has been purified to apparent homogeneity with a heat step, protamine sulfate fractionation, ammonium sulfate fractionation, DEAE-Sephadex chromatography, and two preparative disc gel electrophoresis steps. The molecular weight obtained from this enzyme from sedimentation equilibrium was from 195,000 (for v = 0.70) to 245,000 (for v = 0.75). A molecular weight of 225,000 was obtained by a disc gel electrophoresis technique. The synthesis of ADP-glucose was dependent on the presence of ATP, glucose-1-P, enzyme, and either magnesium or manganous ion. The enzyme was quite specific for ATP. CTP, dGTP, ITP, TTP, and UTP could not substitute for ATP. In the presence of pyruvate, CTP gave 4.4% and dATP 1.9% of the activity found when ATP was the substrate. The enzyme was activated by pyruvate. None of the other glycolytic intermediates tested activated this enzyme. Pyruvate (5 mm) increased the Vmax of ADPglucose synthesis 2.5- to 3-fold, decreased the S0.5 for ATP about 10-fold (from 3.4 mm to 0.36 mm), and shifted the pH optimum of the enzyme from pH 8.5 to a broader pH optimum extending from pH 7.5 to 9.2. The saturation curves for ATP and pyruvate were sigmoidal, and a reciprocal heterotropic interaction exists between these two ligands. Increasing the concentration of one of these ligands decreased the sigmoidicity of the saturation curve for the other, as well as the concentration needed for half-maximal activity. The Hill constant (n) for pyruvate was 1.9 at 0.25 mm ATP, 1.7 at 0.50 mm ATP, and 1.2 at 2.5 mm ATP. n for ATP was 2.97 in the absence of pyruvate and 1.82 in the presence of 5 mm pyruvate. It was also found that pyruvate decreased the amount of metal ion required for half-maximal velocity: from 6.0 mm to 2.7 mm with Mg++, and from 5.4 mm to 1.8 mm with Mn++. When the level of ATP was below 1 mm, and Mg++ below 2 mm, the enzyme was almost totally dependent on pyruvate for activity. At 0.5 mm ATP, pyruvate in the range of concentration of 0.1 mm to 3 mm dramatically increased the activity of the enzyme. The reverse reaction, the pyrophosphorolysis of ADP-glucose, was dependent on the presence of ADP-glucose, enzyme, and Mg++. Pyruvate increased the Vmax of ADP-glucose pyrophosphorolysis from 1.5- to 2-fold, decreased the S0.5 for ADP-glucose more than 5-fold (from 2.0 mm to 0.38 mm), and decreased the Hill constant n with respect to ADP-glucose from n = 2.02 to n = 1.42.