Evaluation of the Rate-determining Steps and the Relative Magnitude of the Individual Rate Constants for the Hydrolytic and Synthetic Activities of the Catalytic Component of Liver Microsomal Glucose 6-Phosphatase

Abstract

Abstract 1. Steady state kinetic constants were determined at pH 6 for various phosphohydrolase and phosphotransferase activities catalyzed by glucose 6-phosphatase of liver microsomes that were supplemented with 0.4% sodium taurocholate and assayed in the presence of albumin. Km and Vmax values were evaluated for reactions with glucose 6-phosphate, carbamyl phosphate, inorganic pyrophosphate, mannose 6-phosphate, ATP, and phosphoenolpyruvate as phosphoryl donor compounds and d-glucose and d-mannose as phosphoryl acceptor compounds. 2. The order of decreasing reactivity of these substrates was carbamyl phosphate g inorganic pyrophosphate g mannose 6-phosphate = glucose 6-phosphate >> ATP g phosphoenolpyruvate. Phosphotransferase activities with d-mannose were significantly faster than the corresponding glucose phosphotransferase activities. With the exception of phosphoenolpyruvate, values for Vmax and Km for a given phosphate substrate were greater in the phosphotransferase reactions. The Km for a given substrate in the phosphotransferase reaction varied with the specific nature and concentration of the second substrate. Values of Km for the phosphoryl donor substrates were greatest with mannose as the acceptor, while Km values for the phosphoryl acceptor varied directly with the reactivity of the phosphoryl donor compound. 3. These observations were shown to be incompatible with the conclusion from an earlier study that the step in the mechanism was the formation of the phosphoryl enzyme from the binary complex of enzyme and phosphoryl donor compound. Rather, it is concluded that the hydrolysis of phosphoryl enzyme is the slowest step in the hydrolysis of all substrates that possess reactivity equal to or greater than glucose 6-phosphate. Moreover, no individual rate constant could be considered as the rate-limiting factor in these reactions. The earlier conclusions appear to be valid at pH 6 in the cases of phosphoenolpyruvate, ATP, or other nucleotides where additional steric or electrostatic factors restrict the rate of formation of phosphoryl enzyme. 4. An analysis of the data for all activities permitted the assignment of upper and lower limits on the relative magnitude of the individual rate constants for the glucose 6-phosphate phosphohydrolase activity and means by which these limits may be refined are discussed.