Determination of the rate of hexokinase-glucose dissociation by the isotope-trapping method

When (14C)glucose is mixed with yeast hexokinase and subsequently diluted with 0.18 mM MgATP and a large amount of unlabeled glucose, half of the enzyme-bound (14C)glucose is converted to glucose-6-P prior to dissociation. At higher concentrations of ATP, all of the bound (14C)glucose is trapped as glucose-6-P indicating that the binary complex is fully functional and that the ternary complex goes to product much more rapidly than it dissociates glucose. An equation is derived that gives the dissociation rate constant of the enzyme glucose complex, k/sub off/, in terms of steady state kinetic parameters, k/sub cat/ and K/sub m/ of ATP, and the concentration of ATP required for half-maximum trapping, K/sub 1/2/: k/sub off/ = K/sub 1/2/ k/sub cat//K/sub m/. It is concluded that glucose dissociates from the binary complex at 29 percent of the net forward reaction rate and that this step is rate determining for the reverse reaction K/sub max/. The forward reaction cannot be viewed as pre-equilibrium, at least with respect to glucose binding. Isotope trapping as applied here provides a new approach for determining the sequence of binding of substrates to enzymes and for estimating the rates of dissociation of substrate from binary and ternary Michaelis complexes in reactions requiring two or more substrates. Slow dissociation of E S complexes must be ruled out in isotope trapping experiments meant to show covalent intermediates. (U.S.)