Studies on the Allosteric Properties of Glycogen Synthase I

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Abstract

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, α and β. The α state is induced by glucose-6-P (activation constant 14 μM) and has a low Km for UDP-Glc (21 μM) and a dissociation constant for the product UDP of 12 μM. For the β state the corresponding values are 5–800 μM and 4 μM. The influence of modifiers on the kinetic constants of the rate equation is on K, not V. ATP, ADP and AMP were found to favour the β state by competing with glucose-6-P for the allosteric site and forming a dead-end complex. Pi, PPi, SO42−, 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 β to the α 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, Mg2+ has no effect, except that it is toxic to the enzyme.

Under ‘physiological’ conditions a Michaelis constant for UDP-Glc of 81 μM and an activation constant for glucose-6-P of 230 μM 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 Na2SO4 is advocated, however, at 2 mM.

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