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Kinetics of Purified Liver Phosphorylase

From the ¹ From the Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

Glycogen phosphorylase has been purified from rabbit liver by a simplified procedure which depends on the initial separation of the enzyme from a crude extract by centrifuging it as a complex with particulate glycogen.

Initial rates have been measured with varied concentrations of both substrates in each reaction direction. The data were analyzed with double reciprocal plots and secondary plots of intercepts and slopes to yield the eight Dalziel kinetic coefficients.

Inhibition by uridine diphosphoglucose is competitive with glucose 1-phosphate and inorganic orthophosphate. With the latter substrate the K_i at high glycogen concentration is 0.4 mm and at low glycogen concentration it is 0.2 mm.

Consideration of the relationships among the Dalziel kinetic coefficients, various related kinetic constants, the Haldane relationship, and the patterns of inhibition by UDP-glucose has led to a tentative assignment of a kinetic mechanism which involves random order of substrate addition to enzyme, with rapid equilibria compared to a rate-limiting interconversion of ternary complexes.

On the basis of this tentative kinetic mechanism, the kinetically derived dissociation constants for glycogen, P_i, and glucose-1-P from the free enzyme are 14, 11, and 1.3 mm, respectively. Dissociation constants were also deduced for glycogen and the enzyme-P_i complex (0.93 mm); glycogen and the enzyme-glucose-1-P complex (2.9 mm); P_i and the enzyme-glycogen complex (0.82 mm); and glucose-1-P and the enzyme-glycogen complex (0.28 mm). Some of these differences were rationalized by suggesting a glucose-binding site adjacent to a phosphate-binding site, both of which are occupied by glucose-1-P in one case or by the terminal glucose of glycogen and by P_i.

Increasing the concentration of P_i or glucose-1-P decreases the apparent K_m for glycogen, while increasing the concentration of glycogen decreases the apparent K_m values for either of the other two substrates.

Adenosine 5'-phosphate decreases the apparent K_m values for all three substrates. The apparent K_m for AMP is 6 x 10^-5 m at 24 mm glucose-1-P and 10 x 10^-5 m at 8 mm glucose-1-P.

The apparent K_m values and dissociation constants for glycogen and liver phosphorylase are of the same order of magnitude as those commonly found for the muscle phosphorylases. The apparent K_m values of glucose-1-P and P_i are less than those found for the muscle enzymes, and this is paralleled by smaller K_i values for inhibition by a variety of naturally occurring organic phosphates and pyrophosphates. Alteration of the groups on the glucose moiety of various phosphate compounds decreases binding to the active center with an order of importance of C-4 > C-6 > C-2.

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