Initial rate and isotope exchange studies of rat skeletal muscle hexokinase.
The kinetic mechanism of rat skeletal muscle hexokinase (hexokinase II) was investigated in light of a proposal by Cornish-Bowden and his co-workers (Gregoriou, M., Trayer, I. P., and Cornish-Bowden, A. (1983) Eur. J. Biochem. 134, 283-288). These investigators reported that the kinetic mechanism is ordered, with glucose adding before ATP and ADP dissociating from hexokinase before glucose-6-P. In addition, these workers suggest that glucose-6-P and ATP add to allosteric sites on hexokinase. We investigated the mechanism of action of hexokinase II by studying initial rate kinetics in the nonphysiological direction and by isotope exchange at chemical equilibrium. The former experiments were carried out in the absence of inhibitors and then with AMP, which is a competitive inhibitor of ADP, and with glucose 1,6-bisphosphate, a competitive inhibitor of glucose-6-P. The findings from these experiments suggest that the kinetic mechanism is rapid equilibrium Random Bi Bi. Isotope exchange at equilibrium studies also supports the random nature of the muscle hexokinase reaction; however, they also suggest that the mechanism is partially ordered, i.e. there is a preferred pathway associated with the branched mechanism. Approximately two-thirds of the flux through the hexokinase reaction involves the glucose on first glucose-6-P off last branch of the Random Bi Bi mechanism. These results imply that the kinetic mechanism is steady state Random Bi Bi. There is some evidence to suggest that glucose-6-P binds to an allosteric site on muscle hexokinase, but none to suppose that ATP binds allosterically. Analysis of the mechanism of Gregoriou et al. suggests that it is at variance with the findings of this report as well as with data available from other laboratories.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D011865:Radioisotope Dilution Technique; D051381:Rats
D002250:Carbon Radioisotopes; D005958:Glucosephosphates; D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase