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.

[1]  M. Cárdenas,et al.  Suppression of kinetic cooperativity of hexokinase D (glucokinase) by competitive inhibitors. A slow transition model. , 1984, European journal of biochemistry.

[2]  J. Morrison,et al.  Isotope exchange studies of the mechanism of the reaction catalyzed by adenosine triphosphate: creatine phosphotransferase. , 1966, The Journal of biological chemistry.

[3]  A. Cornish-Bowden,et al.  Isotope-exchange evidence that glucose 6-phosphate inhibits rat-muscle hexokinase II at an allosteric site. , 1983, European journal of biochemistry.

[4]  A. Cornish-Bowden.  A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. , 1974, The Biochemical journal.

[5]  Fructose is a good substrate for rat liver 'glucokinase' (hexokinase D). , 1984, The Biochemical journal.

[6]  A. Cornish-Bowden,et al.  Effect of glycerol on glucokinase activity: loss of cooperative behavior with respect to glucose. , 1985, Archives of biochemistry and biophysics.

[7]  W. Cleland,et al.  Kinetic studies of Escherichia coli galactokinase. , 1968, Biochemistry.

[8]  P. Tippett,et al.  Observation of a kinetic slow transition in monomeric glucokinase. , 1990, Biochemistry.

[9]  I. Weber,et al.  Molecular Model of Human β-Cell Glucokinase Built by Analogy to the Crystal Structure of Yeast Hexokinase B , 1994, Diabetes.

[10]  M. Dixon.  The graphical determination of Km and Ki , 1972 .

[11]  Kinetics of hexokinase D ('glucokinase') with inosine triphosphate as phosphate donor. Loss of kinetic co-operativity with respect to glucose. , 1987, The Biochemical journal.

[12]  Athel Cornish-Bowden,et al.  Analysis of enzyme kinetic data , 1995 .

[13]  M. DePamphilis,et al.  Preparation and properties of chromium (3)-nucleotide complexes for use in the study of enzyme mechanisms. , 1973, Biochemistry.

[14]  F M Matschinsky,et al.  Glucokinase as Glucose Sensor and Metabolic Signal Generator in Pancreatic β-Cells and Hepatocytes , 1990, Diabetes.

[15]  A. Cornish-Bowden,et al.  Concentration of MgATP2- and other ions in solution. Calculation of the true concentrations of species present in mixtures of associating ions. , 1976, The Biochemical journal.

[16]  M. Stoffel,et al.  Human glucokinase gene: isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[17]  A. Cornish-Bowden,et al.  Isotope-exchange evidence for an ordered mechanism for rat-liver glucokinase, a monomeric cooperative enzyme. , 1981, Biochemistry.

[18]  A. Cornish-Bowden.  Fundamentals of Enzyme Kinetics , 1979 .

[19]  A. Cornish-Bowden,et al.  Kinetic evidence for a 'mnemonical' mechanism for rat liver glucokinase. , 1977, The Biochemical journal.

[20]  S. Lenzen,et al.  Importance of cysteine residues for the stability and catalytic activity of human pancreatic beta cell glucokinase. , 2000, Archives of biochemistry and biophysics.

[21]  H. J. Tsai,et al.  Functional organization of mammalian hexokinases: both N- and C-terminal halves of the rat type II isozyme possess catalytic sites. , 1996, Archives of biochemistry and biophysics.

[22]  T. Ureta,et al.  Sigmoidal kinetics of glucokinase. , 1975, Enzyme.

[23]  M Cascante,et al.  Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data. , 2001, The Biochemical journal.

[24]  T. Ureta,et al.  NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties. , 1990, The Biochemical journal.

[25]  A. Cornish-Bowden,et al.  Evolution and regulatory role of the hexokinases. , 1998, Biochimica et biophysica acta.

[26]  M. Cárdenas.  Glucokinase: Its Regulation and Role in Liver Metabolism , 1995 .

[27]  E. Van Schaftingen,et al.  Short‐term control of glucokinase activity: role of a regulatory protein , 1994, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[28]  N. Phillips,et al.  Kinetic mechanisms of polyphosphate glucokinase from Mycobacterium tuberculosis. , 1996, Biochemistry.

[29]  M. Dixon.  The graphical determination of K m and K i . , 1972, The Biochemical journal.

[30]  H. Najafi,et al.  Chromatographic resolution and kinetic characterization of glucokinase from islets of Langerhans. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[31]  L. Agius,et al.  Investigation of the mechanism by which glucose analogues cause translocation of glucokinase in hepatocytes: evidence for two glucose binding sites. , 2000, The Biochemical journal.

[32]  L. C. Schroeter.  CHAPTER 1 – PREPARATION AND PROPERTIES , 1966 .

[33]  A. Cornish-Bowden,et al.  Kinetics of rat liver glucokinase. Co-operative interactions with glucose at physiologically significant concentrations. , 1976, The Biochemical journal.

[34]  H. Fromm,et al.  Kinetic studies of solubilized brain hexokinase with D-fructose as a substrate. , 1968, Biochemical and biophysical research communications.

[35]  E. Schaftingen,et al.  Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose. , 2000, Diabetes.

[36]  E. Van Schaftingen,et al.  Analysis of the Cooperativity of Human β-Cell Glucokinase through the Stimulatory Effect of Glucose on Fructose Phosphorylation* , 2001, The Journal of Biological Chemistry.

[37]  A. Cornish-Bowden,et al.  Robust regression of enzyme kinetic data. , 1986, The Biochemical journal.