, Volume 12, Issue 1, pp 45-61

Purification and steady state kinetic mechanism of glycogen synthase-D from human polymorpho-nuclear leukocytes

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The authors' work on the purification and steady state kinetic investigation of the enzyme glycogen synthase D (UDP-glucose: glycogen 4-α-glucosyl-transferase, EC from human polymorphonuclear leukocytes is reviewed. The main features of the kinetic mechanism for catalysis of the reaction UDPG + glycogenn ⇌ UDP + glycogen(n+1) are: (i) Lineweaver-Burk plots in both substrates are linear, exhibiting intersecting patterns; (ii) UDP is a competitive, respectively noncompetitive, inhibitor towards the substrates UDPG and glycogen; (iii) the essential activator glucose-6-phosphate (G-6-P) showed an intersecting pattern towards glycogen and an equilibrium ordered pattern towards UDPG. These features identify in this case the mechanism as a rapid equilibrium random bi-bi mechanism, with G-6-P adding to the enzyme prior to the substrate UDPG. New results on the influence of the modifiers NaCl, Ca++, Mn++, Mg++, HPO4 −-, SO4 −-, and ATP on the enzyme are reported. Interpreting the observations in terms of the established mechanism, the following results are obtained: The effect of salt (NaCl) is nonspecific and fairly small, probably reflecting a general action of the electrolyte medium on the conformation of the enzyme. Divalent cations affect only the rate limiting step, i.e. the interconversion of the quaternary enzyme-substrate-activator complexes. The anions interact exclusively with the G-6-P binding site of the enzyme. The dissociation constants for the enzyme-modifier complexes are determined, and a kinetic mechanism for the action of the anions is proposed, leading to activation or inhibition, depending on the concentration of G-6-P.

An invited article