The Inhibition by Citrate of Inorganic Pyrophosphate-Glucose Phosphotransferase and Glucose 6-Phosphatase

Abstract

Abstract Rat liver microsomal glucose 6-phosphatase previously has been shown to catalyze an inorganic pyrophosphate-glucose phosphotransferase reaction. Citrate, which has been employed as a buffer in some earlier studies of this enzyme, recently has been found to inhibit both of these activities. Detailed investigations of the inhibition of both phosphohydrolase and phosphotransferase reactions by this tricarboxylic acid have been carried out with enzyme prepared by fractional ammonium sulfate precipitation of deoxycholate-treated rat liver microsomal preparations. These studies involved (a) kinetic analysis of inhibition of both activities, including an evaluation of Km and Ki values at a variety of hydrogen ion concentration levels, and (b) investigations of inhibitor specificity. Inhibition, which was observed only below pH 7, was found in all instances to be competitive with respect to phosphate substrate and noncompetitive with respect to glucose. Kglucose (0.083 M) was independent of pH. Km values for glucose 6-phosphate and pyrophosphate and Ki (citrate) values were markedly dependent on pH. Minimal values were observed at the following pH values: KPPi = 0.94 mM at pH 6.1, Kglucose-6-P = 0.42 mM at pH 6.5, and Ki, (citrate) ≅ 6 mM at pH 5.6. Good agreement was observed between Ki (citrate) values determined with both the phosphohydrolase and phosphotransferase activities over the entire pH range studied (pH 4.65 to 6.5). This finding lends additional substantiation for the common identity of both enzymic activities. Of a variety of citrate analogues and other chelating agents and buffers tested, only oxalate approached citrate in effectiveness as an inhibitor. Dixon (38) plots of both pKm and pKi values with respect to pH suggest the possible participation of an imidazolium group of a histidine residue of the enzyme in enzyme-substrate complexation. The results also reveal that KPPi values are appreciably smaller than reported earlier; at pH 6.1, KPPi (0.94 mM) is approximately one-fifth of that previously determined with citrate as buffer. A comparison of activity-pH profiles of pyrophosphate-glucose phosphotransferase and glucokinase indicates that, on the basis of units of activity per g of wet liver, the latter is the principal hepatic glucose-phosphorylating activity above pH 7, while the former activity predominates below this hydrogen ion concentration. A possible role for citrate as an agent regulating pyrophosphate-glucose phosphotransferase and hepatic glucose utilization in general is suggested.