Kinetic mechanism of the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis: co-operative interactions of AMP and glucose 1-phosphate during catalysis.
We employed our newly developed, continuous, spectrophotometric method [Sergienko and Srivastava (1994) Anal. Biochem. 221, 348-355] for measuring the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis, utilizing varied concentrations of AMP (2-400 microM) and glucose 1-phosphate (G1P; 4 microM to 41 mM). The experimental data revealed that the enzyme catalysis exhibits sigmoidal dependence on both AMP and G1P concentrations, with Hill coefficient and EC50 values (mutually) affected by the concentrations of the above substrates. A detailed kinetic analysis of the substrate-dependent activation, as well as glucose-inhibition data, lead us to propose the following mechanistic features of the glycogen-phosphorylase-catalysed reaction. (1) The enzyme exhibits catalytic activity when two molecules of AMP and two molecules of G1P are bound to the dimeric unit. (2) The binding of one molecule of glucose (the competitive inhibitor of G1P) per dimeric unit results into a complete loss of the enzyme activity. (3) There is no restriction of binding of AMP or G1P when one of the dimeric subunits is already bound with the other ligand. For example, one or two G1P molecules can bind to the enzyme dimer when zero, one or two molecules of AMP are already bound. The magnitudes of rate and equilibrium constants for the glycogen-phosphorylase-catalysed reaction, derived from analyses of the experimental data in the light of a few selected minimal models, are presented.
Sergienko
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EA
Biochemistry Department, North Dakota State University, Fargo 58105, USA.
Srivastava
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DK
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D005947:Glucose / Q000502:physiology; D005958:Glucosephosphates / Q000378:metabolism*; D006003:Glycogen / Q000096:biosynthesis*; D007700:Kinetics; D008956:Models, Chemical; D006005:Phosphorylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013053:Spectrophotometry
D005958:Glucosephosphates; D000249:Adenosine Monophosphate; D006003:Glycogen; C031590:glucose-1-phosphate; D006005:Phosphorylases; D005947:Glucose