A Kinetic Study of the Effect of α-d-Galactose, α-d-Mannose, α-d-Glucosamine, N-Acetyl-α-d-glucosamine, and α-d-Ribose Diphosphate on the Activity of Phosphoglucomutase

The specificity of phosphoglucomutase for various α-d-sugar diphosphates was studied by examining the relative ability of these compounds to activate the dephosphorylated form of the enzyme prepared from rabbit muscle. The rate of the reaction measured spectrophotometrically at 340 mµ with a coupled assay system containing TPN+ and excess d-glucose 6-phosphate dehydrogenase was linear with time only when the incubation was carried out in the presence of α-d-glucose 1,6-diphosphate. When other sugar diphosphates were used, the rate of disappearance of α-d-glucose 1-phosphate increased with time until a maximum linear rate was attained. The final rate, in each case, was dependent on the concentration of α-d-sugar diphosphate added to the incubation mixture, but it was independent of the order of addition of reactants and the concentration of components of the coupled assay system. The apparent Km values were as follows: α-d-glucose 1,6-diphosphate, 0.1 µm; α-d-mannose 1,6-diphosphate, 3.7 µm; α-d-ribose 1,5-diphosphate, 3.2 µm; α-d-glucosamine 1,6-diphosphate, 11 µm; N-acetyl-α-d-glucosamine 1,6-diphosphate, 1.6 µm; α-d-galactose 1,6-diphosphate, 13 µm. The most effective activator of phosphoglucomutase was α-d-glucose 1,6-diphosphate. The Km values of α-d-galactose 1,6-diphosphate and α-d-glucosamine 1,6-diphosphate, the poorest activators, were about 120-fold greater than that of α-d-glucose 1,6-diphosphate. The maximum velocity varied only 2- to 3-fold when different sugar diphosphates were used to activate the reaction. The following maximum velocities, expressed as micromoles min-1 mg-1, were obtained: α-d-glucose 1,6-diphosphate, 500; α-d-mannose, 1,6-diphosphate, 360; α-d-ribose 1,5-diphosphate, 210; α-d-glucosamine 1,6-diphosphate, 370; N-acetyl-d-glucosamine 1,6-diphosphate, 220; α-d-galactose 1,6-diphosphate, 240. Addition of the appropriate α-d-sugar 1-phosphate or d-sugar 6-phosphate decreased the velocity of the reaction being maintained by either α-d-sugar 1,6-diphosphate or α-d-glucose 1,6-diphosphate. The findings reported are consistent with and support a mechanism in which each of the sugar diphosphates combines with dephosphoenzyme to form a sugar diphosphate-enzyme complex which then further reacts to yield a phosphoenzyme intermediate and free sugar monophosphate.