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

Abstract

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#−, and the reaction follows a ternary-complex mechanism with Km¯19.2³2.3 mM for 2- deoxyglucose and 0.56³0.05 mM for MgATP#−. Product inhibition by MgADP− was mixed with respect to MgATP#− 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 INTRODUCTION Hexokinase D (ATP:-glucose 6-phosphotransferase, EC 2.7.1.1), often known as glucokinase or hexokinase IV, is one of the four glucose-phosphorylating isoenzymes present in vertebrates and plays the role of a glucose sensor in pancreatic β-cells and in hepatocytes [1,2]. Mutations in its gene in humans are correlated with a type of diabetes, namely maturity onset diabetes of the young (‘MODY’) [3]. The enzyme shows a sigmoidal dependence of rate on the glucose concentration [4–6], a property of kinetic origin that makes the enzyme sensitive to changes in the blood glucose level. Although the main models for explaining the cooperative behaviour of hexokinase D have postulated an ordered addition of substrates, with glucose as first substrate [7–10], the subject is not closed, and it remains unclear whether the kinetic mechanism is totally ordered. Isotope-exchange studies at equilibrium suggested a small degree of randomness, but no binary EE MgATP#− complex could be detected [11]. The order of product release has not been clearly established either, on account of the co-operative behaviour, which complicates the data analysis. Some kinetic studies [7] suggested MgADP− as the last product, Abbreviations used: CrATP, the complex of ATP with chromium in the 3­ oxidation state, i.e. Cr(III)–ATP; dGlc (when used in the name of a complex or as part of a kinetic symbol), 2-deoxyglucose; GlcNAc (when used in the name of a complex), N-acetylglucosamine; Glc6P (when used in the name of a complex), glucose 6-phosphate; Nbs2, 5,5«-dithiobis-(2-nitrobenzoic acid). 1 This paper is dedicated to the memory of Professor Hermann Niemeyer, whose influence on hexokinase research remains strong. 2 To whom correspondence should be addressed (e-mail cardenas!ibsm.cnrs-mrs.fr). the EE dGlc complex. Experiments involving protection against inactivation by 5,5«-dithiobis-(2-nitrobenzoic acid) support the existence of the EE MgADP− and EE 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#− 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 sugarbinding site in hexokinase D.