Hexokinase D ('glucokinase') displays positive cooperativity with mannose with the same h values (1.5 -1.6) as with glucose but with higher values (8 mM at pH 8.0 and 12 mM at pH 7.5). In contrast, fructose and 2-deoxyglucose exhibit Michaelian kinetics [Cardenas, M. L., Rabajille, E., and Niemeyer, H. (1979) Arch. Bid. Med. Exp. 12, 571 -580; Cardenas, M. L., Rabajille, E., and Niemeyer, H. (1984) Biochem. J. 222, 363 -3701. Mannose, fructose, 2-deoxyglucose and N-acetylglucosamine acted as competitive inhibitors of glucose phosphorylation and decreased the cooperativity with glucose. Their relative efficiency for reducing the value of h to 1 .O was: fructose > mannose > 2-deoxyglucose > N-acetylglucosamine. Galactose, which is not a substrate nor an inhibitor, was unable to change the cooperativity. The competitive inhibition of glucose phosphorylation by N-acetylglucosamine or mannose was cooperative at very low glucose concentrations (< 0.5 suggesting the interaction of the inhibitors with more than one enzyme form. These and previously reported results are discussed on the basis of a slow transition model, which assumes that hexokinase D exists mainly in one conformation state (E,) in the absence of ligands and that the binding of glucose (or mannose) induces a conformational transition to E1,. This new conformation would have a higher affinity for the sugar substrates and a higher catalytic activity than E,. Cooperativity would emerge from shifts of the steady-state distribution between the two enzyme forms as the sugar concentration increase. The inhibitors would suppress cooperativity with glucose by inducing or trapping the EII conformation. In addition, the model postulates that the different kinetic behaviour of hexokinase D with the different sugar substrates, cooperative with glucose and mannose and Michaelian with 2-deoxyglucose and fructose, is the consequence of differences in the velocities of the conformational transitions induced by the sugar substrates.'Glucokinase' (ATP : D-glucose 6-phosphotransferase) is one of four hexokinase isoenzymes in rat liver and it is also named hexokinase D [l, 21 or hexokinase IV [3]. However, in view of its substrate specificity in relation to the other animal hexokinases either of these latter names, within the classification EC 2.7.1.1, would be more appropriate 141. For this reason we shall use the name of hexokinase D in this report.The enzyme displays a sigmoidal saturation function for glucose with a Hill coefficient of 1.5 -1.6 and a half-saturation concentration of about 7.5 mM at pH 7.5 [5 -71. This cooperative behaviour accords well with the presumed function of the enzyme [8] and has also been observed in isolated hepatocytes [9]. The molecular interpretation of hexokinase D cooperativity poses some problems, as it is a monomeric protein with only one active site [lo-131 to which classical equilibrium models of cooperativity [14, 151 cannot be applied. As the cooperativity must be purely kinetic in origin steady-state models have been proposed . In a...

Glucokinases obtained from the liver of several species of mammals and amphibians exhibit sigmoidal saturation functions for glucose. Hill coefficients (n(H)) are about 1.5, and half-saturation values (K(0.5)) lie between 1.5 and 8.5 mmol/1. The nn and K(0.5) values are constant throughout the purification steps of rat glucokinase. A dimeric form of rat glucokinase appearing in aged preparations exhibits michaelian kinetics. Sigmoidal kinetics is considered as an adaptive feature of glucokinases to increase the efficiency of the liver uptake of glucose at the changeable concentrations in the blood resulting from variations in the amount of dietary glucose.

Rat liver 'glucokinase' (hexokinase D) catalyses the phosphorylation of fructose with a maximal velocity about 2.5-fold higher than that for the phosphorylation of glucose. The saturation function is hyperbolic and the half-saturation concentration is about 300 mM. Fructose is a competitive inhibitor of the phosphorylation of glucose with a Ki of 107 mM. Fructose protects hexokinase D against inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid), and the apparent dissociation constants are about 300 mM in the presence of different concentrations of the inhibitor. The co-operativity of the enzyme in the phosphorylation of glucose can be abolished by addition of fructose to the reaction medium. Fructose appears to be no better as a substrate for the other mammalian hexokinases than it is for hexokinase D. It is proposed that the name 'glucokinase' ought to be reserved for enzymes that are truly specific for glucose, such as those of micro-organisms and invertebrates, and that liver glucokinase must be called hexokinase D (or hexokinase IV) within the classification EC 2.7.1.1.

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