Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts.

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.

Inhibition of hexokinase activity by a fructose 2,6-bisphosphate-dependent cytosolic protein from liver.

Mammalian and yeast hexokinases were found to be reversibly inhibited by fructose 2,6-bisphosphate, an effect requiring the presence of a cytosolic protein factor. Experimental evidence suggests that this factor (inhibitor) is a regulatory protein, the interactions of which with hexokinases are modulated by fructose 2,6-bisphosphate. The Vmax of hexokinase D was decreased, and no changes on other kinetic parameters were observed. The inhibitor was present in fresh liver cytosol filtered through Sephadex G-25 and was partially isolated by negative absorption on DEAE-cellulose followed by ammonium sulfate fractionation. The inhibitor was also present in brain and kidney, but not in muscle. A molecular mass of 200,000 was determined by gel filtration. The inhibition was dependent on the concentrations of both the inhibitory protein and fructose 2,6-bisphosphate. No delay in fructose 2,6-bisphosphate inhibition was observed. Several other hexose phosphates were tested and were not effective. In the presence of amounts of inhibitor sufficient to produce complete inhibition of hexokinase D, the concentration of fructose 2,6-bisphosphate required to produce 50% inhibition was about 0.5 microM. The inhibitor was unstable and was stabilized by the presence of fructose 2,6-bisphosphate.

Cooperative interactions in hexokinase D ("glucokinase"). Kinetic and fluorescence studies.

Hexokinase D, also called hexokinase IV or glucokinase, is the isoenzyme characteristic of liver. In spite of its common name of glucokinase it phosphorylates also other sugars besides glucose; in particular, it phosphorylates fructose with similar specificity to that shown by the other hexokinases. Although hexokinase D is a monomeric protein it displays positive cooperativity with glucose and mannose. In contrast, the kinetic behaviour with 2-deoxyglucose and fructose is Michaelian. Mannose, fructose, 2-deoxyglucose and N-acetylglucosamine are competitive inhibitors of glucose phosphorylation and suppress the cooperativity. The cooperative behaviour can also be suppressed by the presence of glycerol at the assay medium at concentrations over 20%, with a decrease in the K0.5. Neither glycerol nor the inhibitors affect the monomeric state of the enzyme. Hexokinase D exhibits an intrinsic fluorescence at about 326 nm due to tryptophan residues. The binding of glucose to the enzyme enhances the native fluorescence by about 15%. A dissociation constant for glucose of about 3.5 mM was estimated; this value decreased to about 0.5 mM glucose in the presence of glycerol. These and other results are discussed on the basis of steady-state models which assume 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 a new conformation EII with higher affinity for the sugar substrates. It is postulated that differences in the velocities of the conformational transitions induced by the different sugar substrates give account of the differences in kinetic behaviour with the different sugar substrates.

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

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 greater than mannose greater than 2-deoxyglucose greater than 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 (less than 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 (E1) 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.

Fructose is a good substrate for rat liver 'glucokinase' (hexokinase D).

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.

Stability of hexokinases A, B and C and N-acetylglucosamine kinase in liver cells isolated from rats submitted to diabetes and several dietary conditions.

The effect of dietary and hormonal variations on the specific activities of hexokinase isoenzymes, N-acetylglucosamine kinase and pyruvate kinase isoenzymes in parenchymal and non-parenchymal liver cells was studied. Hexokinase D was markedly decreased in hepatocytes from animals fasted or fed on the carbohydrate-free diet as well as from diabetic rats, attaining a constant low level of about 17% of normal values. Pyruvate kinase L was also diminished in hepatocytes under the same experimental conditions. In contrast, the three high-affinity hexokinase isoenzymes A, B and C remained without variation in total amount or in their relative proportions in hepatocytes and non-parenchymal liver cells isolated from animals under the various conditions studied. N-Acetylglucosamine kinase activities also did not change either in parenchymal or in non-parenchymal liver cells under all conditions. The results are discussed in relation to the significance of N-acetylglucosamine kinase and the various hexokinase isoenzymes for the phosphorylation of glucose after dietary and hormonal manipulations.

Sigmoidal kinetics of glucokinase.

Glucokinases obtained from the liver of several species of mammals and amphibians exhibit sigmoidal saturation functions for glucose. Hill coefficients (nH) are about 1.5, and half-saturation values (K0.5) lie between 1.5 and 8.5 mmol/l. The nH and K0.5 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.