Kinetic mechanism of phosphorylase <i>a</i>. I. Initial velocity studies

Abstract

Initial rate studies on rabbit muscle phosphorylase a were carried out in order to assign a kinetic mechanism to this enzyme, which plays an important role in the control of glycogen metabolism. Initial velocities were measured with varied concentrations of both substrates in each reaction direction, both in the presence and the absence of the modifier AMP. Data were analyzed with double-reciprocal plots and secondary replots of slopes and intercepts to yield kinetically derived dissociation constants which may be compared with dissociation constants determined by independent methods. Inhibition studies using UDP-glucose as a substrate analogue are also reported. Inhibition is competitive with glucose 1-phosphate and P 1 and noncompetitive with glycogen.A suitable rate equation for this system has been derived, and it should apply to other two-substrate enzyme–modifier systems exhibiting similar kinetics. The results of this detailed kinetic analysis, in conjunction with the isotope exchange studies at equilibrium which are reported in the following paper, suggest the kinetic mechanism of phosphorylase a to be rapid equilibrium random Bi Bi, i.e. random addition of substrates to the enzyme, with ternary complex interconversion as the rate-limiting step in the reaction sequence.