Kinetics of purified liver phosphorylase.

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 l-phosphate and inorganic orthophosphate. With the latter substrate the Ki 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-glucase 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, Pi, and glucose-l-P from the free enzyme are 14, 11, and 1.3 mM, respectively. Dissociation constants were also deduced for glycogen and the enzyme-Pi complex (0.93 mu); glycogen and the enzyme-glucose-l-P complex (2.9 mbr) ; Pi and the enzyme-glycogen complex (0.82 mrd); and glucose-l-P and the enzyme-glycogen complex (0.28 mu). 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-l-P in one case or by the terminal glucose of glycogen and by Pi. Increasing the concentration of Pi or glucose-l-P decreases the apparent K,,, for glycogen, while increasing the concentration of glycogen decreases the apparent Km values for either of the other two substrates. Adenosine 5’-phosphate decreases the apparent K, values for all three substrates. The apparent K,,, for AMP is 6 x