Kinetic evidence that the high-affinity glucose 6-phosphate site on hexokinase I Is the active site

Two mechanisms have been suggested to account for the regulation of brain hexokinase by glucose 6-phosphate. One mechanism places glucose-6- P at an allosteric site, remote from the active site, while the second describes glucose-6- P as a simple product inhibitor of the enzyme, binding at the γ phosphate subsite within the ATP locus of the active site. To distinguish between these possibilities, we have undertaken a study of the back reaction of hexokinase I. Our data indicate that glucose-6- P displays classical Michaelis-Menten kinetics with brain hexokinase. This finding is consistent only with the high-affinity glucose-6- P site on the enzyme being the catalytic site. The dissociation constant, estimated from the initial-rate experiments is approximately 25 μ m, a value that agrees well with the inhibition constant for glucose-6- P in the forward direction. These findings are consistent with an earlier model (W. R. Ellison, J. D. Lueck and H. J. Fromm, (1975) J. Biol. Chem. 250, 1864–1871), which maintains that glucose-6- P inhibition of brain hexokinase is a manifestation of product inhibition. In a recent paper, Lazo et al. (P. A. Lazo, A. Sols, and J. E. Wilson, (1980) J. Biol. Chem. 255, 7548–7551) reported data obtained from binding studies with rat brain hexokinase at an elevated (250 μ m) level of glucose-6- P. These authors believe that their results indicate multiple binding of glucose-6- P to the enzyme and interpret the data in terms of a high-affinity allosteric site and a low-affinity catalytic site. Our results are at variance with this interpretation and are consistent only with the high-affinity site for glucose-6- P on brain hexokinase being the active site.