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The inhibition of glycogen phosphorylase by uridine diphosphate glucose

1961; Elsevier BV; Volume: 6; Issue: 4 Linguagem: Inglês

10.1016/0006-291x(61)90385-0

1090-2104

Neil B. Madsen,

Biochemical and Molecular Research

The effect of the β-glycosidase inhibitor d-gluconohydroximo-1,5-lactone-N-phenylurethane (PUG) on the kinetic and ultracentrifugation properties of glycogen phosphorylase has been studied. Recent crystallographic work at 2.4 Å resolution [D. Barford et al. (1988) Biochemistry27, 6733–6741] has shown that PUG binds in the catalytic site of phosphorylase b crystals with its gluconohydroximolactone moiety occupying a position similar to that observed for other glucosyl compounds and the N-phenylurethane side chain fitting into an adjacent cavity with little conformational change in the enzyme. In solution, PUG was shown to be a potent inhibitor of phosphorylase b, directly competitive with α-d-glucopyranose 1-phosphate (glucose-1-P) (Ki = 0.40 mM) and noncompetitive with respect to glycogen and AMP. When PUG was tested for synergistic inhibition in the presence of caffeine, the Dixon plots of reciprocal velocity versus PUG concentration at different fixed caffeine concentrations provided intersecting lines with interaction constant (α) values of 0.95–1.38, indicating that the binding of one inhibitor is not significantly affected by the binding of the other. For glycogen phosphorolysis, PUG was noncompetitive with respect to phosphate, suggesting that it can bind to the central enzyme-AMP-glycogen-phosphate complex. PUG was shown to inhibit phosphorylase a (without AMP) activity (Ki = 0.43 mM) in a manner similar to that of the b form. However, in the presence of AMP, PUG exhibited complex kinetics, acting as a noncompetitive inhibitor with respect to glucose-1-P, while a twofold decrease of PUG binding to the enzyme-AMP-glycogen complex was observed. Ultracentrifugation experiments demonstrated that PUG does not cause any significant dissociation of phosphorylase a tetramer. Furthermore the dimerization of phosphorylase a by glucose is completely prevented in the presence of PUG. These observations are consistent with PUG binding to both the R and the T conformations of phosphorylase.