Interconversions between different sulfhydryl-related kinetic states in glucokinase.
Rat liver glucokinase (EC 2.7.1.2) undergoes two distinct sulfhydryl-related reversible kinetic transitions. During normal assays in the presence of both substrates but without added reducing agents, the activity decays ("kappa" decay) over time to a new steady-state rate. The half-time for this decay is essentially constant at glucose levels from 2 to 200 mM and averages 6.2 +/- 2 min. Glucokinase in this kappa steady state displays an increased Km for glucose but has the same Vmax as normal, sulfhydryl-activated glucokinase. The kappa form does not itself exhibit kinetic cooperativity with glucose. In contrast, glucokinase incubated with neither glucose nor sulfhydryl reagents decays (mu decay) to a form whose Vmax is near zero. The t 1/2 for this transition is about 0.5 min at 0 or very low (0.5 mM) glucose concentrations. For both decays, incubations of enzyme with intermediate levels of reducing agents give steady-state mixtures of activated and either kappa and/or mu forms, depending on conditions during the decay. Enzyme at intermediate stages of the kappa decay displays an unchanged Vmax, intermediate (increased relative to activated enzyme) glucose S0.5 values, and diminished glucose cooperativity. In contrast, enzyme at intermediate steady-state mixtures of activated and mu forms has a normal glucose S0.5 and cooperativity but a diminished Vmax from the activated states. The enzyme at any stage of each decay may be fully reactivated by the addition of sulfhydryl reducing agents such as dithiothreitol, dithioerythritol, glutathione, or mercaptoethanol. A model is proposed to account for this complex behavior in glucokinase kinetics which proposes different enzymatic states (kappa and mu) locked in by sulfhydryl oxidation of different conformations dictated by glucose concentration. These sulfhydryl-related transitions may be important in regulation of glucokinase activity, since glucokinase is very sensitive (at least 20-fold differential activity) to concentrations of glutathione within the physiological range, perhaps allowing the normally variable glutathione levels or cytosolic redox potential to modify the rate of uptake and storage of blood glucose through control of glucokinase activity.
D000818:Animals; D005941:Glucokinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D006207:Half-Life; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D051381:Rats; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D005941:Glucokinase; D005978:Glutathione