JBC, Vol. 250, Issue 11, 4081-4086, Jun, 1975
Effects of magnesium on the kinetic properties of bovine heart glycogen synthase D
C. Nakai and J. A. Thomas
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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