Characterization and compartmentation,in green leaves,of hexokinases with different specificities for glucose,fructose, and mannose and for nucleoside triphosphates

When green leaves of spinach (Spinacia oleracea L.) were surveyed for the presence of hexokinases which utilize glucose, fructose and-or mannose as a substrate, four kinases could be distinguished by their order of elution during chromatography on diethylaminoethyl (DEAE)-cellulose: (i) a hexokinase I with a specificity for fructose, glucose, and mannose, (ii) a fructokinase I with a specificity for fructose, (iii) a hexokinase II with a specificity for glucose, fructose and mannose, and (iv) a fructokinase II with a specificity for fructose. Hexokinases I and II had high apparent K_m values for fructose (8 and 15 mM, respectively) and medium or low apparent K_m values for glucose (150 and 18 μM, respectively) and mannose (18 and 15 μM, respectively). Maximal velocities were highest with fructose, medium with glucose and lowest with mannose. That hexokinases I and II used several sugars as substrate was concluded (i) from their identical elution profiles during enzyme separation and (ii) because their activities with two or three sugars at a time was always lower than the sum of activities with one substrate, indicating competition of the sugars for the reaction with the enzymes. Fructokinases I and II were very specific for fructose (85 and 140 μM, respectively) and had only little, if any, activity with glucose or mannose. All kinases showed varying degrees of activity with nucleoside triphosphates other than ATP. In the presence of all three sugars, hexokinases I and II were considerably more active with ATP than with uridine-, cytidine-, and guanosine 5'-triphosphate (UTP, CTP, GTP) except that, in the presence of glucose, hexokinase I was almost as active with UTP as with ATP. In the presence of fructose, fructokinase I exhibited highest activity with GTP and a gradually decreasing level of activity with CTP, UTP, and ATP. The activities in the presence of the other two sugars were highest with ATP. Fructokinase II was most active with ATP and fructose and progressively less active with GTP, UTP, and CTP. Cell fractionation by isopycnic density-gradient centrifugation or differential centrifugation indicated that fructokinase II was associated with chloroplasts, hexokinase II with mitochondria, and the other two kinases with the non-particulate cell fraction. In green leaves of pea (Pisum sativum L.), only a hexokinase (II) and fructokinase (II) were present. Corn (Zea mays L.) leaves exhibited only very low hexokinase activity. Dedicated to Prof. Dr. Hans Mohr on the occasion of his 60th birthday     

Substrate specificity and product inhibition of different forms of fructokinases and hexokinases in developing potato tubers

The substrate dependence and product inhibition of three different fructokinases and three different hexokinases from growing potato (Solanum tuberosum L.) tubers was investigated. The tubers contained three specific fructokinases (FK1, FK2, FK3) which had a high affinity for fructose K _m=64, 90 and 100 (M) and effectively no activity with glucose or other hexose sugars. The affinity for ATP (K _m=26, 25 and 240 M) was at least tenfold higher than for other nucleoside triphosphates. All three fructokinases showed product inhibition by high fructose (K _i=5.7, 6.0 and 21 mM) and were also inhibited by ADP competitively to ATP. Sensitivity to ADP was increased in the presence of high fructose, or fructose-6-phosphate. In certain conditions, the K _i (ADP) was about threefold below the K _m (ATP). All three fructokinase were also inhibited by fructose-6-phosphate acting non-competitively to fructose (K _i=1.3 mM for FK2). FK1 and FK2 showed very similar kinetic properties whereas FK3, which is only present at low activities in the tuber but high activities in the leaf, had a generally lower affinity for ATP, and lower sensitivity to inhibition by ADP and fructose. The tuber also contained three hexokinases (HK1, HK2, HK3) which had a high affinity for glucose (K _m=41, 130 and 35 M) and mannose but a poor affinity for fructose (K _m=11, 22 and 9 mM). All three hexokinases had a tenfold higher affinity for ATP (K _m=90, 280 and 560 M) than for other nucleoside triphosphates. HK1 and HK2 were both inhibited by ADP (K _i=40 and 108 M) acting competitively to ATP. HK1, but not HK2, was inhibited by glucose-6-phosphate, which acted non-competitively to glucose (K _i=4.1 mM). HK1 and HK2 differed, in that HK1 had a narrower pH optimum, a higher affinity for its substrate, and showed inhibition by glucose-6-phosphate. The relevance of these properties for the regulation of hexose metabolism in vivo is discussed.Abbreviations FK fructokinase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - HK hexokinase - NTP nucleoside triphosphate - P_i inorganic phosphate - UDPGlc uridine-5-diphosphoglucose This work was supported by the Deutsche Froschungsgemeinschaft (SFB 137). We are grateful to Professor E. Beck (Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, FRG) for providing laboratory facilities.     

Hexokinases of spinach leaves

Two soluble hexokinases and a particulate hexokinase have been separated and partially purified from spinach leaves. One of the soluble hexokinases showed a high affinity for glucose (K_m = 63 μM) which was far greater than that for fructose (K_m = 9.1 mM). However, with saturating fructose the activity was twice that with saturating glucose. The particulate hexokinase showed kinetic properties similar to those of this soluble hexokinase. The second soluble hexokinase was distinct in that it was much more active with fructose than with glucose at all concentrations tested, although the K_m values for these hexoses (210 μM and 71 μM respectively) were similar. The activity of this hexokinase was stimulated by the monovalent cations K⁺ and NH₄⁺.     

Purification and Properties of Purified Hexokinase from the African Trypanosomes and Trypanosoma equiperdum

SYNOPSIS. Hexokinase was in both soluble and particulate fractions of extracts of Trypanosoma brucei, T. gambiense, T. rhodesiense and T. equiperdum. These enzymes have been purified some 350-fold from crude extracts by sonication, (NH₄)₂SO₄ fractionation, and DEAE Sephadex column chromatography. Purified hexokinases had: A) temperature optimum 45-50 C; B) pH optimum 6.5-7.0; C) Mg⁺⁺ and ATP requirements; D) inhibition by ADP, p -hydroxymercuribenzoate, glucose-6-phosphate, and competitive inhibition by mannose, glucosamine, N -acetyl-D-glucosamine and xylose; E) ability to phosphorylate glucose, fructose, mannose, 2-deoxy-D-glucose and glucosamine; F) a glucose requirement for stabilization. T. equiperdum hexokinase was inhibited 33% by ADP as compared to 17-20% with brucei group hexokinase, and showed 22% activity when ITP was substituted for ATP in contrast to 35-40% with brucei group hexokinase.     

Comparative studies on glucose phosphorylating isoenzymes of vertebrates. Identification and characterization of amphibian liver hexokinases

Glucose phosphorylating activities were measured in liver extracts from two urodeles and twenty-six anurans. Fractionation on diethylaminoethyl-cellulose columns of liver extracts from these amphibians permitted the recognition of four hexokinases which are called A, B, C, and D. However, any given amphibian displays only three liver hexokinases and the profiles so far observed are either of the type A-B-D or C-B-D. The distribution of the amphibians in either type of pattern does not show any simple taxonomic relationship. A wide generic and specific, but not individual, variation of the relative proportion of each isoenzyme was observed. Hexokinases A and B were shown to be low K_{m glucose} isoenzymes (0.06 and 0.15 mm glucose, respectively) with normal hyperbolic kinetics. Hexokinase C, also a low K_m isoenzyme (0.05 mm) was found to be inhibited by excess substrate at physiological levels of glucose. Hexokinases A, B, and C were able to phosphorylate fructose, mannose, and 2-deoxyglucose at equal or higher rates than glucose when assayed at saturating sugar levels. Hexokinase D was found to be a high K_m isoenzyme ($\text{K}{}_{0.5}\text{? 2}\text{mM}$) with sigmoidal saturation curves for glucose (Hill coefficient ? 1.6). Fructose and mannose were also phosphorylated by this isoenzyme at about 70% of the glucose rate when studied at saturating sugar concentrations. The properties of the amphibian hexokinases are thus similar, although not identical, to those of mammalian hexokinases.     

Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

Identification,expression and bioactivity of hexokinase in amphioxus: Insights into evolution of vertebrate hexokinase genes

Hexokinase family includes hexokinases I, II, III and IV, that catalyze the phosphorylation of glucose to produce glucose 6-phosphate. Hexokinase IV, also known as glucokinase, is only half size of the other types of hexokinases that contain two hexokinase domains. Despite the enormous progress in the study of hexokinases, the evolutionary relationship between glucokinase and other hexokinases is still uncertain, and the molecular processes leading to the emergence of hexokinases in vertebrates remain controversial. Here we clearly demonstrated the presence of a single hexokinase-like gene in the amphioxus Branchiostoma japonicum, Bjhk, which shows a tissue-specific expression pattern, with the most abundant expression in the hepatic caecum, testis and ovary. The phylogenetic and synteny analyses both reveal that BjHK is the archetype of vertebrate hexokinases IV, i.e. glucokinases. We also found for the first time that recombinant BjHK showed functional enzyme activity resembling vertebrate hexokinases I, II, III and IV. In addition, a native glucokinase activity was detected in the hepatic caecum. Finally, glucokinase activity in the hepatic caecum was markedly reduced by fasting, whereas it was considerably increased by feeding. Altogether, these suggest that Bjhk represents the archetype of glucokinases, from which vertebrate hexokinase gene family was evolved by gene duplication, and that the hepatic caecum plays a role in the control of glucose homeostasis in amphioxus, in favor of the notion that the hepatic caecum is a tissue homologous to liver.     

Differential molecular sieving permits visualization of hexokinase isoenzymes following electrophoretic separation in high resolution acrylamide gels

1. Differential molecular sieving is the concept applied to bring together isoenzymes of ATP:D-hexose-6-phosphotransferase (hexokinases) with glucose 6-phosphate dehydrogenase in acrylamide gels by utilization of their dissimilar electrophoretic mobilities. 2. The hexokinase isoenzymes migrate and separate in gels with pore sizes selected to entrap glucose 6-phosphate dehydrogenase in their interstices. The locations of the bands of specific activity are visualized by fluorescence of NADPH under long wave, ultraviolet radiation. 3. A new discontinuous electrochemical system has been devised to deliver protective thiol groups into the gel. Cysteine (trailing ion) and SO4(2-) (leading ion) form a sharp moving boundary. 4. The high resolution of the system has permitted visualization of a rapidly migrating, high Km hexokinase in murine spleen, fat, kidney and lymph nodes. Hexokinase Types I and II, were observed in all tissues tested, but Type IV was seen only in the liver. 5. The importance of glucose concentration effects on hexokinase activity is emphasized by inactivation of slowly migrating low Km hexokinase Types I and II following exposure to 200 mM glucose during preparation of extracts.     

Comparison of type I hexokinases from pig heart and kinetic evaluation of the effects of inhibitors

Type I hexokinase (ATP:d-hexose 6-phospotransferase, EC 2.7.1.1) of porcine heart exists in two chromatographically distinct forms. These do not differ significantly in size, electrophoretic mobility at pH 8.6 or kinetic properties. Both forms obey a sequential mechanism and are potently inhibited by glucose 6-phosphate. In contrast to observations of type I hexokinase from brain, inhibition by glucose 6-phosphate is not relieved by inorganic phosphate. Under most conditions, low concentrations of phosphate (<10 mM) have little effect on the kinetic behaviour of the enzyme but at higher concentrations this ligand is an inhibitor. Mannose 6-phosphate inhibits in a manner analogous to glucose 6-phosphate but the K_i is much greater. In view of the similarity of the kinetic parameters governing phosphorylation of mannose and glucose, this difference in affinity for the inhibitor site is seen as consistent with the existence of a separate regulatory site on the enzyme. MgADP inhibits hexokinase but behaves as a normal product inhibitor and inhibition is competitive with respect to MgATP and non-competitive with respect to glucose.     

Alterations in carbohydrate metabolism of γ-irradiated cavendish banana

γ-Irradiation of preclimacteric banana resulted in a gradual increase in fructose content, which reached a maximum in 6 days. Although the catabolism of glucose-U-¹⁴C was less in irradiated banana, incorporation of label into fructose was high. Initial fructose accumulation in irradiated banana may be due to a shift in glucose utilization from the glycolytic to the pentose phosphate pathway. The ratio of resporatory CO₂ from glucose-6-¹⁴C and glucose-1-¹⁴C was halved in irradiated bananas indicating predominance of the pentose phosphate pathway. The radioactivity of fructose derived from glucose-6-¹⁴C was almost twice that from glucose-1-¹⁴C in irradiated bananas, whilst in control both fruit the labelled precursors yielded equal amounts. Studies on individual enzymes in these two pathways showed an increase in phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and fructose-6-phosphatase and a decrease in hexokinase in irradiated banana.     

Effects of kainate on glucose metabolising enzymes in the brain

Hexokinase and glucose-6-phosphate dehydrogenase activities were studied in brain regions after intraventricular injection of kainic acid. Hexokinase activity was decreased by 10-15% in various regions while glucose-6-phosphate dehydrogenase activity remained unaltered. Soluble hexokinase activity, which remained the smaller fraction of total hexokinase activity, showed slightly more dramatic decreases of 15-35% compared to normal activities in brain regions. This decrease of hexokinase activity in the cytosolic compartment could partly account for the kainate-induced decreases seen in glucose metabolism.     

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.     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Two Uptake Systems for Fructose in Lactococcus lactis subsp. cremoris FD1 Produce Glycolytic and Gluconeogenic Fructose Phosphates and Induce Oscillations in Growth and Lactic Acid Formation

Fructose transport in lactococci is mediated by two phosphotransferase systems (PTS). The constitutive mannose PTS has a broad specificity and may be used for uptake of fructose with a fructose saturation constant (K_Fru) of 0.89 mM, giving intracellular fructose 6-phosphate. The inducible fructose PTS has a very small saturation constant (K_Fru, <17 μM), and the fructose 1-phosphate produced enters the Embden-Meyerhof-Parnas (EMP) pathway as fructose 1,6-diphosphate. Growth in batch cultures of Lactococcus lactis subsp. cremoris FD1 in a yeast extract medium with fructose as the only sugar is poor both with respect to specific growth rate and biomass yield, whereas the specific lactic acid production rate is higher than those in similar fermentations on other sugars metabolized via the EMP pathway, e.g., glucose. In fructose-limited chemostat cultures, the biomass concentration exhibits a strong correlation with the dilution rate, and starting a continuous culture at the end of a batch fermentation leads to large and persistent oscillations in the biomass concentration and specific lactic acid production rate. Two proposed mechanisms underlying this strange growth pattern follow. (i) Fructose transported via the fructose PTS cannot be converted into essential biomass precursors (glucose 6-phosphate or fructose 6-phosphate), because L. lactis subsp. cremoris FD1 is devoid of fructose 1,6-diphosphatase activity. (ii) The fructose PTS apparently produces a metabolite (presumably fructose 1-phosphate) which exerts catabolite repression of both mannose PTS and lactose PTS. Since the repressed mannose PTS and lactose PTS are shown to have identical maximum molar transport rates, the results indicate that it is the general PTS proteins which are repressed.     

Studies on the mode of sugar-phosphate product inhibition of brain hexokinase.

Hexokinase I (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1), a key regulatory glycolytic enzyme in certain tissues, is known to be markedly inhibited under physiological conditions. The action of the primary inhibitory effector, glucose-6-P, is reversed by inorganic orthophosphate (P_i). A molecular model for inhibition and deinhibition of hexokinase was recently proposed [Ellison, W. R., Lueck, J. D., and Fromm, H. J. (1975) J. Biol. Chem.250, 1864–1871]. One of the central assumptions of this model is that glucose-6-P is a normal product inhibitor of hexokinase. It has long been suggested that glucose-6-P is an allosteric inhibitor of hexokinase, whereas other sugar-phosphate products such as mannose-6-P are normal product inhibitors. In this report we investigated the kinetic mechanism of hexokinase action with mannose as substrate and mannose-6-P as an inhibitor. The data obtained show that there are no qualitative differences between glucose and mannose as substrates and glucose-6-P and mannose-6-P as inhibitors. Binding experiments indicate that glucose-6-P and mannose-6-P are competitive binding ligands with hexokinase I. Furthermore, the activation pattern observed with Pi and glucose-6-P inhibited hexokinase is also found with the mannose-6-P inhibited phosphotransferase. These findings suggest that the mechanism of inhibition of glucose-6-P and mannose-6-P represents a difference in degree rather than a difference in kind. An explanation of the results in terms of a stereochemical model is presented.     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

The mitochondrial localization of hexokinase in pea leaves

Up to 80% of total cellular hexokinase (EC 2.1.7.4) activity in pea (Pisum sativum L.) leaves was found to be associated with particulate fractions. Fractionation on sucrose density gradients showed this particulate activity to be associated exclusively with mitochondria. In the presence of glucose and ATP, the bound mitochondrial hexokinase could support rates of O₂ uptake of up to 30% of normal ADP-stimulated rates. This stimulation of O₂ uptake by hexokinase was completely sensitive to oligomycin, indicating that it resulted from an increase in the supply of ADP for mitochondrial oxidative phosphorylation. Spectrophotometric measurements of the mitochondrial hexokinase activity showed that ADP could support rapid rates of activity provided oxidizable substrates were also present to support the conversion of ADP to ATP in oxidative phosphorylation. Carboxyatractyloside, an inhibitor of adenine-nucleotide uptake by mitochondria, inhibited this ADP-supported activity, but had no effect on hexokinase activity in the presence of added ATP, demonstrating that the hexokinase enzyme was located external to the inner mitochondrial membrane. Oligomycin also inhibited ADP-supported activity but had no effect on ATP-supported hexokinase activity. Glucose (K_m 53 μM) was the preferred substrate of pea-leaf mitochondrial hexokinase compared with fructose (K_m 5.1 mM). Hexokinase was not solubilised in the presence of glucose-6-phosphate.     

Regulation of mammalian hexokinase: regulatory differences between isoenzyme I and II

Mammalian hexokinase isoenzymes I and II have been shown to differ qualitatively in response to various modifiers. Although both enzymes are inhibited by glucose 6-phosphate, only isoenzyme II exhibits a slow response to the presence of this inhibitor. P_i decreases the affinity of glucose 6-phosphate for Sarcoma 37 hexokinase I, but has no effect on hexokinase II from the same cell. P_i overcomes all of the inhibition of red cell hexokinase by glucose-6-P and hence the two effectors act competitively. At pH 6.5, catecholamines increase the V of isoenzyme I of Sarcoma 37 and brain in the soluble and mitochondrial forms but do not activate these forms of tumor isoenzyme II. Citrate activates brain and tumor isoenzyme I when they are inhibited by tris(hydroxy-methyl)aminomethylethane sulfonate (TES) and ADP; however, tumor isoenzyme II is not activated.     

Kinetic evidence that the high-affinity glucose 6-phosphate site on hexokinase I Is the active site

Two mechanisms have been suggested to account for the regulation of brain hexokinase by glucose 6-phosphate. One mechanism places glucose-6-P at an allosteric site, remote from the active site, while the second describes glucose-6-P as a simple product inhibitor of the enzyme, binding at the γ phosphate subsite within the ATP locus of the active site. To distinguish between these possibilities, we have undertaken a study of the back reaction of hexokinase I. Our data indicate that glucose-6-P displays classical Michaelis-Menten kinetics with brain hexokinase. This finding is consistent only with the high-affinity glucose-6-P site on the enzyme being the catalytic site. The dissociation constant, estimated from the initial-rate experiments is approximately 25 μm, a value that agrees well with the inhibition constant for glucose-6-P in the forward direction. These findings are consistent with an earlier model (W. R. Ellison, J. D. Lueck and H. J. Fromm, (1975) J. Biol. Chem.250, 1864–1871), which maintains that glucose-6-P inhibition of brain hexokinase is a manifestation of product inhibition. In a recent paper, Lazo et al. (P. A. Lazo, A. Sols, and J. E. Wilson, (1980) J. Biol. Chem.255, 7548–7551) reported data obtained from binding studies with rat brain hexokinase at an elevated (250 μm) level of glucose-6-P. These authors believe that their results indicate multiple binding of glucose-6-P to the enzyme and interpret the data in terms of a high-affinity allosteric site and a low-affinity catalytic site. Our results are at variance with this interpretation and are consistent only with the high-affinity site for glucose-6-P on brain hexokinase being the active site.     

Glucose phosphorylation and dephosphorylation in chicken liver.

1. Glucokinase was absent from chicken liver and only the low Km hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The Km of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6-phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efflux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function.