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J Biol Chem. 1975 Jun 10;250(11):4081-6.

Effects of magnesium on the kinetic properties of bovine heart glycogen synthase D.


Highly purified glycogen synthase D, free of synthase kinase and phosphatase activities, was prepared from bovine heart. The enzyme had no activity without glucose 6-phosphate. Kinetic studies of this enzyme at various concentrations of UDP-glucose demonstrated that there was no cooperativity with respect to the substrate at any concentration of glucose-6-P with or without Mg2+. Glucose 6-phosphate increased the maximum velocity (Vmax) of the enzyme, but had very little or no effect on the Michaelis constant for UDP-glucose (Km equals 0.33 mM). Free Mg2+ gave a high Vmax at all glucose 6-phosphate concentrations without affecting the Km for the substrate. The double reciprocal plots of reaction rates versus glucose 6-phosphate concentration were biphasic and were interpreted as evidence for two kinetic forms, each with a glucose 6-phosphate binding site of different affinity (A1/2 values equals 0.31 and 1.1 mM). High Mg2+ concentrations nearly abolished the biphasic kinetic behavior of glucose 6-phosphate, suggesting that the Vmax of both enzyme forms was the same at saturating concentration of Mg2+ and glucose 6-phosphate and that magnesium ion might have no effect on the binding of glucose 6-phosphate or on the state of the equilibrium between two forms. Plots of reaction velocity versus Mg2+ concentration showed no cooperativity of Mg2+ activation in the presence or absence of glucose 6-phosphate. Both kinetic forms of glycogen synthase D had the same affinity for Mg2+ (A1/2 is approximately equal to 4 mM). Studies on the inhibition of the enzyme by Pi, ATP, and UTP were carried out with assays specific for the form of synthase with A1/2 for glucose 6-phosphate equals 0.31 mM (high affinity form) and the form with A1/2 for glucose 6-phosphate equals 1.1 mM (LOW AFFINITY FORM) BY ASSAYING WITH AND WITHOUT 5.0 MM free Mg2+, respectively. Both forms of synthase exhibited positive cooperativity with respect to UDP-glucose when inhibited by UTP, but not with Pi or ATP. Thus, each form of the enzyme had more than one UDP-glucose site, and these sites showed cooperativity only in the presence of a uridine nucleotide inhibitor. In the absence of Mg2+ (low affinity form), the inhibitors, Pi, ATP, and UTP, all induced positive cooperativity with respect to glucose 6-phosphate binding to this enzyme. The positive cooperativity induced by ATP was obliterated by adding free Mg2+ (high affinity form), but that induced by other inhibitors was affected slightly or not at all by the cation. These results indicate that each of the enzyme forms (high or low affinity forms) has more than one glucose 6-phosphate site and that these may function in a cooperative manner. The preceding findings are interpreted in relation to the importance of Mg2+ in the regulation of glycogen synthase D activity as well as the regulation of glycogen synthase phosphatase activity in heart.

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