Suppression of kinetic cooperativity of hexokinase D (glucokinase) by competitive inhibitors. A Slow transition model

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Abstract
Hexokinase D (‘glucokinase’) displays positive cooperativity with mannose with the same h values (1.5–1.6) as with glucose but with higher K0.5 values (8 mM at pH 8.0 and 12 mM at pH 7.5). In contrast, fructose and 2‐deoxyglucose exhibit Michaelian kinetics [Cárdenas, M. L., Rabajille, E., and Niemeyer, H. (1979) Arch. Biol. Med. Exp. 12, 571–580; Cárdenas, M. L., Rabajille, E., and Niemeyer, H. (1984) Biochem. J. 222, 363–370]. 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 1.0 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 K0.5), 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 (EI) in the absence of ligands and that the binding of glucose (or mannose) induces a conformational transition to EII. This new conformation would have a higher affinity for the sugar substrates and a higher catalytic activity than EI. 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.