The reaction mechanism of glycogen synthase I from human polymorphonuclear leukocytes is shown to be either a rapid equilibrium random bi-bi mechanism or an ordered sequential mechanism with uridinediphosphoglucose (UDP-Glc) as the first substrate and UDP as the second product. The rate equations are identical at saturating glycogen concentrations. A multisite enzyme model without subunit interaction is proposed. Three sites are distinguishable on the enzyme : the catalytic site, a site for the attachment of glycogen and the allosteric binding site for glucose 6-phosphate (glucose-6-P). It is proposed that the enzyme can undergo allosteric transition between two states, a and p. The a state is induced by glucose-6-P (activation constant 14 pM) and has a low K, for UDP-Glc (21 pM) and a dissociation constant for the product UDP of 12 pM. For the fi state the corresponding values are 5 -800 pM and 4 pM. The influence of modifiers on the kinetic constants of the rate equation is on K, not I/. ATP, ADP and AMP were found to favour the p state by competing with glucose-6-P for the allosteric site and forming a dead-end complex. Pi, PPi, Sot-, and glycerol 2-phosphate also competed with glucose-6-P, but are in themselves activators, which, however, are not able to induce a complete transformation from the 0 to the a state. UTP, UDP, and UMP are competitive inhibitors of the substrate UDP-Glc. In high concentrations small-molecular-weight anions also interfere with the catalytic site. The influence of divalent cations is indirect, depending on affinity to modifiers. Alone, Mg". has no effect, except that it is toxic to the enzyme.Under 'physiological' conditions a Michaelis constant for UDP-Glc of 81 pM and an activation constant for glucose-6-P of 230 pM were estimated. It is concluded that glycogen synthase I is subject to allosteric control and under physiological conditions is not always fully active.The optimal condition for assay of glycogen synthase I activity requires that product inhibition is avoided, which may be achieved by including Mg2+ in the assay mixture. Also, the presence of Na2S04 is advocated, however, at 2 mM.The regulatory enzyme glycogen synthase catalyses the essentially irreversible reaction Glycogen(,) + UDP-Glc -+

Glycogen-free synthase I from human polymorphonuclear leukocytes is activated by its own substrate, glycogen, in a slow, time-dependent proces (hysteretic activation). This lag in response to addition of glycogen depends on the concentration of glycogen, pH and temperature. At pH 7.4 and at a temperature of 30 "C, the half-time of activation, tl:2, decreases from 89 min at 0.004 mg/ml glycogen to 6 min at 25 mg/ml. The activation is accelerated by increasing temperature and pH, but is not influenced by enzyme concentration, glucose 6-phosphate, UDP, high ionic strength, EDTA, mercaptoethanol, glucose, sucrose or amylase limit dextrin. The K,,, for UDP-glucose (0.024 mM) and the activity ratio were unchanged during the activation process.The activation can be described by tit = uf + (vo -or) eCkr where ut, uf and uo are velocities at times t , 0 and c o and k is a complex rate constant. Evidence from ultracentrifugation and kinetic studies is presented to substantiate the hypothesis that the underlying mechanism is a simple bimolecular process : enzyme + glycogeneenzyme-glycogen complex, with the dissociation constant K, = 0.003 mg/ml. The hysteretic activation may become rate-limiting during experiments in vitro with synthase. The possibility of a physiological role in glycogen metabolism, perhaps in the form of a concerted hysteresis with H+ is discussed.The activity of glycogen synthase, the rate-limiting enzyme of glycogen synthesis (glycogen, + UDPglucose+glycogen,+ I + UDP), is controlled by covalent modification and by metabolite (allosteric) control [l]. In human polymorphonuclear leukocytes, the control is exerted at three levels with regard to degree of phosphorylation and value of kinetic constants. The kinetic constants for substrates and modifiers of the fully phosphorylated enzyme form (D for dependent on glucose-6-P for activity) are of such magnitude that this form of the enzyme can be considered inactive under intracellular conditions [2]. A recently discovered partially phosphorylated enzyme form (R for rheostatic) has been found to account for glycogen synthesis under several physiological conditions, the kinetic constants being of a magnitude compatible with control exerted by the intracellular concentration of glucose-6-P 13 -51. The third non-phosphorylated enzyme form, synthase 1, must from preliminary evidence [6] be considered fully active under all conditions. It can be formed in leukocytes under different experimental conditions, in particular when the inhibitory effect of glycogen on the D to 1 conversion is relieved during starvation [7], and/or when the activity of phosphorylase a reaches a critical, low level [S], suggesting the relief of an inhibitory effect on the R to I conversion [4].When we attempted to detail the kinetics of synthase I we became aware that glycogen, besides being both substrate and product in the enzymatic reaction and inhibitor of the D to I conversion, also confers stability on synthase I against inactivation, as described in the preceding paper [9]. In add...

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Glycogen synthase I was purified from human polymorphonuclear leukocytes by a procedure involving affinity chromatography of the glycogen-enzyme complex, digestion of endogenous glycogen by amylase, starch chromatography and gel filtration. The purified enzyme had a specific activity of 7 -11 U/mg protein, or 4-5 U when expressed per mg of residual glycogen. Further purification to 21 U/mg protein could be achieved. The enzyme was inactive in the absence of added glycogen. A subunit molecular weight of 85 000 was determined by polyacrylamide electrophoresis in sodium dodecylsulfate. The molecular weight of the native enzyme was estimated to be 390000 (13.2 S) by sucrose gradient centrifugation and 410000 by gel filtration indicating that the native enzyme is a tetramer. The gel filtration behavior was not affected by enzyme concentration, temperature, or the presence of ligands. The energy of activation was estimated to 13 500 cal/mol (56.5 kJ/mol), corresponding to a Qlo of 2.2. In the presence of glucose 6-phosphate or Na2S04, the enzyme showed a broad pH optimum between pH 6.8-9.2. In the absence of these ligands and in particularly in the presence of Mg', the enzyme is sensitive to small changes of pH in the interval pH 7.4-8.4.During purification, synthase I requires protection by 0.6 mM dithiothreitol, while high concentrations of mercaptoethanol or dithiothreitol inactivates the enzyme, particularly during freezing, During 24-h incubations, synthase I undergoes a spontaneous, temperature-dependent inactivation which is not due to proteolysis, but presumably is caused by irreversible conformational changes. These can be prevented by high concentrations of glucose 6-phosphate, Na2S04, inorganic phosphate, UDP and glycogen. Mgz+ and traces of ethanol inactivates the enzyme. The lyophilized enzyme is stable for years.Glycogen synthase, in most vertebrate tissues, exists in a phosphorylated, physiologically inactive D form [1,2], and a non-phosphorylated I form, which, when present, is always active [2,3]. These enzyme forms are interconvertible by phosphorylation-dephosphorylation reactions, which are subject to hormonal control [1,3]. The existence of both forms of synthase, as well as the enzymes responsible for the interconversion reactions, have been demonstrated in human polymorphonuclear leukocytes in our laboratory [4-91. Recently a third enzyme form, synthase R, with rheostatic properties has been kinetically characterized [ 101.Synthase D has recently been found to follow a rapid-equilibrium random bi-bi mechanism [l 13. A similar exploration of the bisubstrate and modifier kinetics of synthase I has been hampered by the fact that the I enzyme is readily inactivated [12-141 and is also more susceptible to proteolytic degradation Enzyme. Glycogen synthase (EC 2.4.1.11).than the D enzyme [15]. In addition, glycogen, besides being a substrate, also activates synthase I in a timedependent and concentration-dependent manner, as will be described in a following communication [16].The present paper describe...