Fructose is a good substrate for rat liver ‘glucokinase’ (hexokinase D)

Rabajille²,

Niemeyer³

1984

Eur J Biochem

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...

Section: Saturation Function Of Hexokinase D With Mannosementioning

confidence: 98%

Section: Specgicitymentioning

confidence: 99%

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

Rabajille²,

Niemeyer³

1984

Eur J Biochem

“…In method A the phosphorylation was coupled to glucose-6-phosphate dehydrogenase and the rate of reduction of NADP was measured at 340 nm in a Gilford spectrophotometer 2400 provided with a thermospacer maintained at 30°C. The assay medium was that of Cardenas et al (18). The same reaction mixture with ATP omitted acted as a blank.…”

mentioning

confidence: 99%

Kinetic, chromatographic and electrophoretic studies on glucose-phosphorylating enzymes of rat intestinal mucosa

Vera

Archives of Biochemistry and Biophysics

Niemeyer

1984

The number and nature of glucose-phosphorylating enzymes of rat intestinal mucosa were investigated by chromatographic, electrophoretic, and kinetic methods. Three fractions with glucose-phosphorylating activity were obtained from the supernatant fluid of mucosa homogenate by means of DEAE-cellulose chromatography, corresponding to hexokinases A and B (EC 2.'7.1.1.), and N-acetyl-D-glucosamine kinase (EC 2.7.1.59). Although the latter uses N-acetylglucosamine as the main substrate, it is also able to phosphorylate glucose. Electrophoresis in polyacrylamide and in cellulose acetate gels showed the same three enzyme activities. None of these procedures revealed the presence of either hexokinase D ("glucokinase") or hexokinase C in the intestinal mucosa. In the sediment fractions hexokinase A and B, but not N-acetylglucosamine kinase, were found. The K, values for glucose of partially purified hexokinases A and B were 0.025 and 0.174 mM, respectively, and their substrate specificity was the same as that of hexokinases A or B from other tissues. Partially purified N-acetylglucosamine kinase showed hyperbolic saturation functions for N-acetylglucosamine and ATP, with Km values of 0.021 and 0.38 mM, respectively. This enzyme also phosphorylated glucose, mannose, fructose, 2-deoxyglucose, and glucosamine. The dependence of velocity on glucose concentrations was complex, mimicking negative cooperativity. The molecular weight of both hexokinases A and B was 98,000 and that of N-acetylglucosamine kinase was 59,000. The kinetic properties, as well as the chromatographic and electrophoretic mobilities, of N-acetylglucosamine kinase may serve to confuse it with hexokinase D, and thus several criteria should be applied for correct identification.Rat liver contains four hexokinase isozymes (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) which were separated by DEAE-cellulose chromatography and were named A, B, C, and D, according to their order of elution (1).

“…As mentioned already, hexokinase D from mammalian liver is an example of an enzyme that is cooperative with respect to glucose, and noncooperative with respect to fructose, 27 though some authors 34 have reported cooperativity. The specificity of this isoenzyme has been a point of contention since it was discovered, 28,35 and the suggestion of the name "glucokinase" 36 was intended to be provisional until "further characterization of the enzyme could make more appropriate some other name".…”

Section: Application To Mammalian Hexokinasesmentioning

confidence: 98%

“…However, it was not initially realized that hexokinase D (also called hexokinase IV), the isoenzyme characteristic of mammalian liver, is cooperative with respect to glucose, and thus has no K m for glucose, though it follows Michaelis-Menten kinetics with respect to fructose. 27 This complication, together with its high half-saturation concentrations for both substrates, led to much confusion about its specificity, a confusion that persists in the misleading name glucokinase that remains common for this isoenzyme. The lack of an established measure of specificity for a cooperative enzyme led one of us 28 to compare substrates in terms of k cat /K 0.5 .…”

Section: Introductionmentioning

confidence: 99%

Specificity of Non-Michaelis−Menten Enzymes: Necessary Information for Analyzing Metabolic Pathways

Cornish‐Bowden

2010

J. Phys. Chem. B

The specificity of an enzyme obeying the Michaelis-Menten equation is normally measured by comparing the k cat /K m for different substrates, but this is inappropriate for enzymes with a Hill coefficient h different from 1. The obvious alternative of generalizing K m in the expression as K 0.5 , the substrate concentration for half-saturation, is better, but it is not entirely satisfactory either, and here we show that k cat /K 0.5 h gives satisfactory results for analyzing the kinetic behavior of metabolic pathways. The importance of using k cat /K 0.5 h increases with the value of h, but even when h is small, it makes an appreciable difference, as illustrated for the mammalian hexokinases. Reinterpretation of data for the specificity of these enzymes in terms of the proposed definition indicates that hexokinase D, often believed highly specific for glucose, and accordingly called "glucokinase", actually has the lowest preference for glucose over fructose of the four isoenzymes found in mammals.