Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.     

Rat adipose tissue glycogen synthase. Evidence for multiple discrete kinetic species and their interconversion.

Rat adipose tissue glycogen synthase has been kinetically characterized. The classical D form has an apparent Km for UDP-glucose of 0.7 mM and 0.4 mM in the absence and presence of glucose 6-phosphate, respectively. The apparent Ka for glucose 6-phosphate is 0.6 mM. The effect of glucose 6-phosphate on the D form is to enhance the Vmax 7-fold. The I form is also affected by glucose 6-phosphate (Ka, 0.025 mM) but the Vmax is increased only by 20%; apparent Km values for UDP-glucose are 0.4 mM and 0.045 mM in the absence and presence of glucose 6-phosphate, respectively. In addition, two new kinetically distinguishable forms have been observed. The first, designated glycogen synthase Q, arises from an Mg2+ATP-dependent deactivation of the I form. The apparent Km values of glycogen synthase Q for UDP-glucose are identical with those of the I form; however, the apparent Ka for glucose 6-phosphate (0.2 mM) is 8-fold higher than that for the I form and one-third that for the D form. Preparations from fasted or diabetic rats contain a form of glycogen synthase, designated glycogen synthase X, that has a much lower affinity for glucose 6-phosphate than the D form (apparent Ka, 3 mM); the apparent Km values for UDP-glucose are similar to those of the D form (0.7 mM and 0.3 mM in the absence and presence of glucose 6-phosphate, respectively). In preparations from fasted rats a stepwise Mg2+-dependent conversion was demonstrated of synthase X to D to Q to I; this sequential conversion was reversed on incubation with Mg2+ATP. In preparations from fed rats, synthase Q could be generated either by limited activation (from the D form) or, after conversion to the I form, by deactivation with Mg2+ATP. However, even prolonged incubation with Mg2+ATP failed to generate the D (or X) form.     

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.     

[3H]proline incorporation and hydroxyproline concentration in articular cartilage during the development of osteoarthritis caused by immobilization. A study in vivo with rabbits.

1. Activation of glucose 6-phosphate is one of the unique properties of pyruvate kinase from Mycobacterium smegmatis. 2. Pyruvate kinase, partially purified from ultrasonic extracts of the mycobacteria by (NH4)2SO4 fractionation, exhibited sigmoidal kinetics at various concentrations of phosphoenolpyruvate, with a high degree of co-operativity (Hill coefficient, h = 3.7) and S0.5 value of 1.0 mM. 3. In the presence of glucose 6-phosphate, the degree of co-operativity shown by the phosphoenolpyruvate saturation curve was decreased to h = 2.33 and the S0.5 value was lowered to 0.47 mM. 4. The enzyme was activated by AMP and ribose 5-phosphate also, but the activation constant was lowest with glucose 6-phosphate (0.24 mM). 5. The enzyme was strongly inhibited by ATP at all phosphoenolpyruvate concentrations. The concentrations of ATP required to produce half-maximal inhibition of enzyme activity at non-saturating (0.2 mM) and saturating (2 mM) phosphoenolpyruvate concentrations were 1.1 mM and 3 mM respectively. 6. The inhibition of ATP was partially relieved by glucose 6-phosphate. 7. The enzyme exhibited Michaelis-Menten kinetics with ADP as the variable substrate, with an apparent Km of 0.66 mM. 8. The enzyme required Mg2+ or Mn2+ ions for activity. It was not activated by univalent cations. 9. The kinetic data indicate that under physiological conditions glucose 6-phosphate probably plays a significant role in the regulation of pyruvate kinase activity.     

2-Deoxy-D-glucose uptake in cultured human muscle cells

Hexose uptake was studied with cultured human muscle cells using 2-deoxy-D-[1-3H]glucose. At a concentration of 0.25 and 4 mM, phosphorylation rather than transport was the rate-limiting step in the uptake of 2-deoxy-D-glucose. This was not due to inhibition of the hexokinase activity by either ATP depletion or 2-deoxyglucose 6-phosphate accumulation. In cellular homogenates, hexokinase showed a lower Km value for glucose as compared to 2-deoxyglucose. Intact cells preferentially phosphorylated glucose instead of 2-deoxyglucose. Therefore, transport instead of phosphorylation may be rate limiting in the uptake of glucose by cultured human muscle cells. These data suggest caution in using 2-deoxyglucose for measuring glucose transport.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of 6-deoxyglucose in Saccharomyces cerevisiae.

The uptake of 6-deoxyglucose was measured in wild-type Saccharomyces cerevisiae, in a double mutant strain lacking activity for hexokinases A and B (hxkl hxk2), in a triple mutant strain lacking activity for both hexokinases and glucokinase (hxkl hxk2 glk), and in the triple mutant with high levels of activity of single kinases restored by introduction of the cloned genes. In the wild-type strain, uptake of the glucose analog showed two components, with Km values of ca. 20 mM ("high affinity") and 250 mM ("low affinity"), respectively. The double mutant also had high- and low-affinity components, but the triple mutant showed only low-affinity uptake. Reintroduction of the single kinases to the triple mutant restored high-affinity uptake. (Other experiments on 6-deoxyglucose uptake are also presented, including the apparent use of the galactose transport system when induced.) These results show that the recent implication of the kinases in transport of glucose (L.F. Bisson and D.G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983) applies equally to the nonmetabolized analog 6-deoxyglucose and suggests that the role of the kinases in transport is not merely a consequence of metabolism of the transported compound.     

Radiometric oil well assay for glucokinase in microscopic structures

Glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.1) plays a pivotal role in hepatic glucose metabolism and serves as the glucose sensor in pancreatic islet beta-cells. Biochemical studies of this enzyme are complicated by the cellular heterogeneity of the liver and the pancreas and because the presence of hexokinases (ATP:D-hexose 6-phosphotransferases, EC 2.7.1.1) seriously interferes with currently available analytical procedures. A radiometric assay was designed to deal with these problems. It is based on the liberation of 3H2O from D-[2-3H(N)]glucose 6-phosphate, the product of the glucokinase reaction, using exogenous phosphoglucose isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). Interference by hexokinases was largely eliminated by using glucose 6-phosphate as inhibitor and the sensitivity of the assay was greatly increased by using small volumes with the oil well procedure. The assay was sufficiently sensitive to detect about 1 pg of glucokinase. It thus allowed the application of quantitative histochemical procedures to the study of intralobular hepatic glucokinase profiles and the pancreatic beta-cell glucose sensor. The quantitative histochemical procedures were sufficiently sensitive and reliable for measuring important kinetic constants of glucokinase (i.e., the Km and the Hill number) in microscopic samples of tissue.     

A novel dehydrogenase reaction mechanism for hexose-6-phosphate dehydrogenase isolated from the teleost Fundulus heteroclitus

Hexose-6-phosphate dehydrogenase (refers to hexose-6-phosphate dehydrogenase from any species in general) has been purified to apparent homogeneity from the teleost fish Fundulus heteroclitus. The enzyme was characterized for native (210 kDa) and subunit molecular mass (54 kDa), isoelectric point (6.65), amino acid composition, substrate specificity, and metal dependence. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucose 6-sulfate, glucosamine 6-phosphate, and glucose were found to be substrates in the reaction with NADP+, but only glucose was a substrate when NAD+ was used as coenzyme. A unique reaction mechanism for the forward direction was found for this enzyme when glucose 6-phosphate and NADP+ were used as substrates; ordered with glucose 6-phosphate binding first. NAD+ was found to be a competitive inhibitor toward NADP+ and an uncompetitive inhibitor with regard to glucose 6-phosphate in this reaction; Vmax = 7.56 mumol/min/mg, Km(NADP+) = 1.62 microM, Km(glucose 6-phosphate) = 7.29 microM, Kia(glucose 6-phosphate) = 8.66 microM, and Ki(NAD+) = 0.49 microM. The use of alternative substrates confirmed this result. This type of reaction mechanism has not been previously reported for a dehydrogenase.     

Cloning and biochemical characterization of a novel mouse ADP-dependent glucokinase

Glycolysis, the catabolism of glucose to pyruvate, is an iconic central metabolic pathway and often used as a paradigm for explaining the general principles of the regulation/control of cellular metabolism. The ubiquitous mammalian ATP-dependent hexokinases I-III and hexokinase IV, also termed glucokinase, initiate the process by phosphorylating glucose to glucose-6-phosphate. Despite glycolysis having been studied extensively for over 70 years and the last new mammalian ATP-dependent hexokinase isotype having been described in the 1960s, we report here the biochemical characterization of a recombinant ADP-dependent glucokinase cloned from a full-length Mus musculus cDNA, identified by sequence analysis. The recombinant enzyme is quite specific for glucose, is monomeric, has an apparent Km for glucose and ADP of 96 and 280 microM, respectively, and is inhibited by both high concentrations of glucose and AMP. The metabolic role of this enzyme in cells would be dependent on the relative level of its activity to those of the ATP-dependent hexokinases. The greatest advantage of an ADP-GK would clearly be during ischemia/hypoxia, clinically relevant conditions in multiple major disease states, by decreasing the priming cost for the phosphorylation of glucose, saving ATP.     

Transport-associated phosphorylation of 2-deoxyglucose in rat adipocytes

The relationship between ATP levels and 2-deoxyglucose uptake was investigated. When the concentration in the medium lies between 1 and 10 mM 2-deoxyglucose uptake causes a marked decrease in ATP level. This could partly be explained by an inhibiting effect of 2-deoxyglucose and 2-deoxyglucose 6-phosphate on ATP synthesis in the mitochondria. A good correlation between the various ATP levels induced by 2,4-dinitrophenol and the rate of uptake of 5 microM and 0.5 mM (but not 5 mM) 2-deoxyglucose was observed. The addition of glucose and 2-deoxyglucose to cells incubated in the presence of trace amounts of 2-deoxy-[1-14C]glucose induced marked changes in the uptake of the tracer that were associated with a rapid decline in ATP level. It appeared that the phosphorylation of 2-deoxyglucose is an important step in the uptake of the sugar. It is hypothesized that the processes of transport and phosphorylation of 2-deoxyglucose are coupled in rat adipocytes.     

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.     

Hexokinases of tobacco leaves: Subcellular localization and characterization

Subeellular localization of the enzymes which phosphorylate hexoses was studied in photosynthesizing tobacco leaves by means of differential centrifugation and centrifugation in sucrose gradient. More than 80 % of the total hexokinase activity of leaf tissues were found to be associated with the particulate fraction of mitochondria; however, the ratio of the particulate hexokinase fraction to the soluble fraction was influenced by the extraction medium applied. The particulate hexokinases showed a high affinity to glucose (Km = 26.8 μM) and a relatively low affinity to fructose (Km = 17.6 mM). They had a broad pH optimum, because 81 % of the phosphorylating activity obtained for glucose and 75 % of the activity obtained for fructose occurred in pH range from 7.9 to 9.1 (Tris-HCl buffer). The hexokinases were Mg2+ dependent with the highest activity occurring at equimolar Mg2+ and ATP concentrations. Their activity was enhanced by KC1, NaCl, and (NH4)2SO4 at 30 to 120 mM concentrations.     

Glucose 6-phosphate and hexokinase can be used as an ATP-regenerating system by the Ca(2+)-ATPase of sarcoplasmic reticulum.

In the presence of hexokinase, vesicles derived from the sarcoplasmic reticulum of skeletal muscle are able to accumulate Ca2+ in a medium containing ADP and glucose 6-phosphate. No significant Ca2+ uptake is observed if one of these components is omitted from the assay medium. Due to its high affinity for ATP, the Ca(2+)-ATPase can use the very low concentrations of ATP formed from glucose 6-phosphate and ADP to form a Ca2+ gradient. This finding indicates that glucose 6-phosphate and hexokinase can be used as an ATP-regenerating system. The Ca2+ uptake supported by glucose 6-phosphate and ADP is inhibited by glucose and D-xylose. Half-maximal inhibition is observed in the presence of 0.4 mM glucose and 100 mM D-xylose. The transport ratio (Ca2+ transported:substrate utilized) is the same for glucose 6-phosphate and ATP. The Ca2+ gradient formed when glucose 6-phosphate and ADP are the substrates can be used to synthesize ATP from ADP and Pi. The concentration of ATP formed after reversal of the Ca2+ pump is much higher than that expected from direct equilibration of the reaction between glucose 6-phosphate and ADP.     

Studies of 1-deoxy-D-fructose, 1-deoxy-D-glucitol, and 1-deoxy-D-minnitol as antimetabolites.

1-Deoxy-D-fructose was synthesized in 27% yield from D-glucosamine in a three-step procedure involving Raney nickel desulfurization and oxidative deamination with 3,5-di-tert-butyl- 1,2-benzoquinone applied to appropriate intermediates. 1-Deoxyfructose and its reduction products, 1-deoxyglucitol and 1-deoxymannitol, were tested as substrates and antimetabolites. For sheep liver glucitol dehydrogenase, the Km is 53 mM for 1-deoxymannitol, were tested as substrates and antimetabolites. For sheep liver glucitol dehydrogenase, the Km is 53 mM for 1-deoxyglucitol and 89 mM for 1-deoxymannitol with maximal velocities 33 and 18%, respectively, of that with glucitol as substrate. These results require substantial revision of the long-accepted polyol substrate structural requirements for this enzyme which have been reported to include a 1-hydroxy group and a cis-2,4-dihydroxy configuration. Km is 614 and 280 mM for yeast and muscle hexokinases, respectively, acting on 1-deoxyfructose; maximal velocities are 2 and 5% of those obtained with fructose. 1-Deoxyfructose 6-phosphate is a competitive inhibitor of phosphoglucose isomerase with a Ki of 1.1 mM; this is about the same as Km for the natural substrates. It is also an effective inhibitor of phosphofructokinase but does not alter the cooperativity of the enzyme interaction with fructose 6-phosphate nor exhibit cooperativity in its own interaction therewith. These results suggest that the 1-hydroxy group is not crucial for binding but does play a role in the cooperative interactions of this allosteric protein. At equivalent concentrations, 1-deoxyfructose is somewhat better than 2-deoxyglucose as an inhibitor of erythrocyte glycolysis; the 1-deoxypolyols are ineffective. All three 1-deoxy compounds are readily, though incompletely, absorbed from the intestine of mice; most of the absorbed dose appears in the urine unchanged within 24 h. Whether given by oral or intraperitoneal routes, 2 to 6% of administered deoxypolyol or deoxyketose appears in the urine as ketose or polyol, respectively. No acute toxic effects or growth retardation are noted for any of the 1-deoxy analogues when given to mice at levels where 2-deoxyglucose has such effects. The properties of these 1-deoxy sugar analogues recommend them for further studies of enzyme mechanisms, for metabolic studies, and for testing as therapeutic agents against such organisms as certain mammalian parasites with heavy reliance on glycolysis.     

The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.     

The identification of extrahepatic "glucokinase" as N-acetylglucosamine kinase

Several research groups have reported the presence of a high Km glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) in tissues other than adult liver. As shown in this report, protein fractions catalyzing glucose phosphorylation only at high substrate concentrations (100 mM) are indeed found in bovine spleen, rat kidney, human placenta, and newborn rat liver. However, the study of substrate specificities and Michaelis constant values showed that those fractions could be better described as N-acetylglucosamine kinase (ATP:acetamide-2-deoxy-D-glucose-6-phosphotransferase, EC 2.7.1.9) which, in addition to N-acetylglucosamine (Km = 0.066 mM), can also phosphorylate glucose although with very high Km values (370 mM). Furthermore, a homogeneous preparation from bovine spleen was able to phosphorylate both N-acetylglucosamine and glucose. An immune serum against bovine spleen N-acetylglucosamine kinase did not cross-react with purified hexokinases or with glucokinase from rat. However, it was able to remove the putative "glucokinases" from extracts of rat kidney, newborn rat liver, and one of two electrophoretic bands of liver "glucokinase." It is proposed that any report of extrahepatic glucokinase should explicity rule out N-acetylglucosamine kinase as the enzyme being described.     

Novel mutation in hexokinase 2 confers resistance to 2-deoxyglucose by altering protein dynamics

Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2^G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG). Wild-type hexokinases convert 2DG to 2-deoxyglucose-6-phosphate (2DG-6P), but 2DG-6P cannot support downstream glycolysis, resulting in a cellular starvation-like response. Curiously, though the hxk2^G238V mutation encodes a loss-of-function allele, the affected amino acid does not interact directly with bound glucose, 2DG, or ATP. Molecular dynamics simulations suggest that Hxk2^G238V impedes sugar binding by altering the protein dynamics of the glucose-binding cleft, as well as the large-scale domain-closure motions required for catalysis. These findings shed new light on Hxk2 dynamics and highlight how allosteric changes can influence catalysis, providing new structural insights into this critical regulator of carbohydrate metabolism. Given that hexokinases are upregulated in some cancers and that 2DG and its derivatives have been studied in anti-cancer trials, the present work also provides insights that may apply to cancer biology and drug resistance.     

Glucose dehydrogenase (hexose 6-phosphate dehydrogenase) and the microsomal electron transport system. Evidence supporting their possible functional relationship.

The ability of a microsomal enzyme, glucose dehydrogenase (hexose 6-phosphate dehydrogenease) to supply NADPH to the microsomal electron transport system, was investigated. Microsomes could perform oxidative demethylation of aminopyrine using microsomal glucose dehydrogenase in situ as an NADPH generator. This demethylation reaction had apparent Km values of 2.61 X 10(-5) M for NADP+, 4.93 X 10(-5) m for glucose 6-phosphate, and 2.14 X 10(-4) m for 2-deoxyglucose 6-phosphate, a synthetic substrate for glucose dehydrogenase. Phenobarbital treatment enhanced this demethylation activity more markedly than glucose dehydrogenase activity itself. Latent activity of glucose dehydrogenase in intact microsomes could be detected by using inhibitors of microsomal electron transport, i.e. carbon monoxide and p-chloromercuribenzoate (PCMB), and under anaerobic conditions. These observations indicate that in microsomes the NADPH generated by glucose dehydrogenase is immediately oxidized by NADPH-cytochrome c reductase, and that glucose dehydrogenase may be functioning to supply NADPH.     

Activity of a second Trypanosoma brucei hexokinase is controlled by an 18-amino-acid C-terminal tail

Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2-/- procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 +/- 0.02 mM and 0.28 +/- 0.1 mM, respectively. The k(cat) value for TbHK1 is 2.9 x 10(4) min(-1). TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 microM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.