Kinetic studies of rat liver hexokinase D ('glucokinase') in non-co-operative conditions show an ordered mechanism with MgADP as the last product to be released.

The kinetic mechanism of rat liver hexokinase D ('glucokinase') was studied under non-co-operative conditions with 2-deoxyglucose as substrate, chosen to avoid uncertainties derived from the co-operativity observed with the physiological substrate, glucose. The enzyme shows hyperbolic kinetics with respect to both 2-deoxyglucose and MgATP(2-), and the reaction follows a ternary-complex mechanism with K (m)=19.2+/-2.3 mM for 2-deoxyglucose and 0.56+/-0.05 mM for MgATP(2-). Product inhibition by MgADP(-) was mixed with respect to MgATP(2-) and was largely competitive with respect to 2-deoxyglucose, suggesting an ordered mechanism with 2-deoxyglucose as first substrate and MgADP(-) as last product. Dead-end inhibition by N -acetylglucosamine, AMP and the inert complex CrATP [the complex of ATP with chromium in the 3+ oxidation state, i.e. Cr(III)-ATP], studied with respect to both substrates, also supports an ordered mechanism with 2-deoxyglucose as first substrate. AMP appears to bind both to the free enzyme and to the E*dGlc complex. Experiments involving protection against inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid) support the existence of the E*MgADP(-) and E*AMP complexes suggested by the kinetic studies. MgADP(-), AMP, 2-deoxyglucose, glucose and mannose were strong protectors, supporting the existence of binary complexes with the enzyme. Glucose 6-phosphate failed to protect, even at concentrations as high as 100 mM, and MgATP(2-) protected only slightly (12%). The inactivation results support the postulated ordered mechanism with 2-deoxyglucose as first substrate and MgADP(-) as last product. In addition, the straight-line dependence observed when the reciprocal value of the inactivation constant was plotted against the sugar-ligand concentration supports the view that there is just one sugar-binding site in hexokinase D.