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 (less than 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 Ki 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.     

Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig

1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.     

Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution.

Hexokinase I governs the rate-limiting step of glycolysis in brain tissue, being inhibited by its product, glucose 6-phosphate, and allosterically relieved of product inhibition by phosphate. On the basis of small-angle X-ray scattering, the wild-type enzyme is a monomer in the presence of glucose and phosphate at protein concentrations up to 10 mg/mL, but in the presence of glucose 6-phosphate, is a dimer down to protein concentrations as low as 1 mg/mL. A mutant form of hexokinase I, specifically engineered by directed mutation to block dimerization, remains monomeric at high protein concentration under all conditions of ligation. This nondimerizing mutant exhibits wild-type activity, potent inhibition by glucose 6-phosphate, and phosphate reversal of product inhibition. Small-angle X-ray scattering data from the mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose 6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike conformation for the monomer similar to that observed in crystal structures of the hexokinase I dimer. Hence, any mechanism for allosteric regulation of hexokinase I should maintain a global conformation of the polypeptide similar to that observed in crystallographic structures.     

Bovine hexokinase type I: Full-length cDNA sequence and characterisation of the recombinant enzyme

This study reports the revised and full-length cDNA sequence of bovine hexokinase type I obtained from bovine brain. Since dissimilarities have been observed between the published bovine hexokinase type I coding sequence (GenBank accession no. M65140) (Genomics 11: 1014-1024, 1991) and an analysed portion of bovine hexokinase type I gene, the entire open reading frame was re-sequenced and the ends of cDNA isolated by rapid amplification of cDNA ends. The coding sequences, when compared with the published bovine hexokinase type I, contained a large number of mismatches that lead to changes in the resulting amino acid sequence. The revisions result in a hexokinase type I cDNA of 3619 bp that encodes a protein of 917 amino acids highly homologous to human hexokinase type I. The expression of the recombinant full-length enzyme demonstrated that it was a catalytically active hexokinase. When characterised for its kinetic and regulatory properties, it displayed the same affinity for glucose and MgATP as the human hexokinase type I and was inhibited by glucose 6-phosphate competitively versus MgATP. The production of the N- and C-terminal recombinant halves of the enzyme followed by comparison with the full-length hexokinase indicated that the catalytic activity is located in the C-terminal domain. (Mol Cell Biochem 268: 9–18, 2005)     

Mitochondrial and cytosolic hexokinase from rat brain: one and the same enzyme?

Rat brain mitochondrial hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was solubilized by treatment of the mitochondria with glucose 6-phosphate and partly purified. The solubilized enzyme was compared with the cytosolic enzyme fraction. The solubilized and cytosolic enzymes were also compared with the enzyme bound to the mitochondrial membrane. The following observations were made. 1. There is no difference in electrophoretic mobility on cellulose-acetate between the cytosolic and the solubilized enzyme. Both fractions are hexokinase isoenzyme I. 2. There is no difference in kinetic parameters between the cytosolic or solubilized enzymes (P less than 0.001). For the cytosolic enzyme Km for glucose was 0.067 mM (S.E. = 0.024, n = 7); Km for MgATP2- was 0.42 mM (S.E. = 0.13, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.084 mM (S.E. = 0.011, n = 5). For the solubilized enzyme Km for glucose was 0.071 mM (S.E. = 0.021, n = 6); Km for MgATP2- was 0.38 mM (S.E. = 0.11, n = 6) and Ki,app for glucose 1,6-diphosphate was 0.074 mM (S.E. = 0.010, n = 5). However when bound to the mitochondrial membrane, the enzyme has higher affinities for its substrates and a lower affinity for the inhibitor glucose 1,6-diphosphate. For the mitochondrial fraction Km for glucose was 0.045 mM (S.E. = 0.013, n = 7); Km for MgATP2- was 0.13 mM (S.E. = 0.02, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.33 mM (S.E. = 0.03, n = 5). 3. The cytosolic and solubilized enzyme could be (re)-bound to depleted mitochondria to the same extent and with the same affinity. Limited proteolysis fully destroyed the enzyme's ability to bind to depleted mitochondria. 4. Our data support the hypothesis that soluble- and solubilizable enzyme from rat brain are one and the same enzyme, and that there is a simple equilibrium between the enzyme in these two pools.     

Rat brain hexokinase: further studies on the specificity of the hexose and hexose 6-phosphate binding sites

Based on the lack of correlation between the ability of various hexoses to serve as substrate and the ability of the corresponding hexose 6-phosphates to inhibit brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), R. K. Crane and A. Sols (1954, J. Biol. Chem. 210, 597-606) proposed that this enzyme possesses two discrete sites capable of binding hexose moieties, one serving as the substrate binding site and a second, regulatory in function, to which inhibitory 6-phosphates bind. Subsequent work has provided further experimental support for this proposal. The pioneering work by Crane and Sols focused primarily on the specificity of these sites with respect to requirements for orientation of hydroxyl substituents at the various positions of the pyranose ring. The present study explores additional aspects of the specificity of these sites, namely, the effect of substitution of a sulfur atom in place of the oxygen in the pyranose ring on ability to serve as substrate or inhibitor, and the effect of modification in charge of the substituent at the 6-position on inhibitory effectiveness. 5-Thioglucose is a linear competitive (versus glucose) inhibitor of rat brain hexokinase, with a Ki of about 0.2 mM, and is a linear mixed inhibitor (versus ATP), with Ki values in this same range. 5-Thioglucose is not, however, readily phosphorylated by brain hexokinase. Thus, although 5-thioglucose binds with moderate affinity to the glucose binding site, it is not effectively used as a substrate of the enzyme. Inhibition of brain hexokinase by glucose 6-phosphate or its analogs has been found to require a dianionic substituent at the 6-position. The 6-fluorophosphate derivative and glucose 6-sulfate are poor inhibitors of the enzyme, and the Ki for inhibition by 1,5-anhydroglucitol 6-phosphate increases markedly at pH values below the pK of the 6-phosphate group, indicating that the monoanionic form is ineffective as an inhibitor. In contrast to the detrimental effect that substitution of the oxygen atom in the pyranose ring with a sulfur has on ability to serve as substrate, 5-thio analogs are considerably more effective as inhibitors, the Ki for inhibition by 5-thioglucose 6-phosphate being 10-fold lower than that seen with glucose 6-phosphate. This effect of the heteroatom substitution can partially offset the decreased inhibition resulting from monoanionic character at the 6-position, but the 6-fluorophosphate derivative of 5-thioglucose 6-phosphate still inhibits with a Ki about 1000-fold greater than that seen with 5-thioglucose 6-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glucose 6-phosphate-dependent binding of hexokinase to membranes of ascites tumor cells.

A pH-dependent, saturable binding of hexokinase isozyme I from Ehrlich ascites carcinoma to plasma membrane and microsome preparations from the same tissue is demonstrated. This binding is enhanced by glucose 6-phosphate and may be considered as the sum of a glucose 6-phosphate-dependent binding and an independent binding. The half saturation concentration of hexokinase is about 0.4 unit per ml for both types of binding, and a maximal binding of 0.5-2.0 units per mg membrane protein is observed for both, although the pH optimum of the independent binding (5.4) is lower than that of the dependent binding (5.9). The half saturation concentration of glucose 6-phosphate required for the dependent binding is 0.05 mM at pH 6.1. 2-Deoxyglucose 6-phosphate competatively reverses the effect of glucose 6-phosphate on binding but does not diminish its inhibition of hexokinase activity.     

Enzymatic properties of overexpressed human hexokinase fragments

Full-length hexokinase (HK; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), a truncate form of the enzyme lacking the first 11 amino acids (HK-11aa) and the 50 kDa C-terminal half (mini-HK) containing the catalytic domain, were overexpressed and purified to homogeneity to investigate the influence of the N-terminal region of human hexokinase type I (HK) on its regulatory properties. All forms of the enzyme are catalytically active with the HK-11aa being the most active. All the forms of HK showed the same affinity for glucose and MgATP and were also inhibited by glucose 6-phosphate (Glc 6-P) competitively vs. MgATP with similar Kis (28.5-37 M). Glucose 1,6-bisphosphate (Glc 1,6-P2) was also a strong inhibitor of all HKs without significant differences among the different truncate forms of the enzyme (Kis 49.5-59 M). At low concentrations (0-3 mM), Pi was able to reverse the sugar phosphate inhibition of the full-length HK and HK-11aa but not of the mini-HK. In contrast, at high concentrations Pi was an inhibitor of all the hexokinases investigated. These findings confirm that Pi has a low affinity binding site on the C-terminal of HK while counteracts glucose 6-phosphate inhibition by binding to or requiring the N-terminal half of the enzyme. The first 11 N-terminal amino acids influence the specific activity of HK but are unable to affect the kinetic properties investigated.     

Regulatory properties of human erythrocyte hexokinase during cell ageing

Human red blood cell hexokinase exists in multiple molecular forms with different isoelectric points but similar kinetic and regulatory properties. All three major isoenzymes (HK Ia, Ib, and Ic) are inhibited competitively with respect to Mg.ATP by glucose 6-phosphate (Ki = 15 microM), glucose 1,6-diphosphate (Ki - 22 microM), 2,3-diphosphoglycerate (Ki = 4 mM), ATP (Ki = 1.5 mM), and reduced glutathione (Ki = 3 mM). All these compounds are present in the human erythrocyte at concentrations able to modify the hexokinase reaction velocity. However, the oxygenation state of hemoglobin significantly modifies their free concentrations and the formation of the Mg complexes. The calculated rate of glucose phosphorylation, in the presence of the mentioned compounds, is practically identical to the measured rate of glucose utilization by intact erythrocytes (1.43 +/- 0.15 mumol h-1 ml red blood cells-1). Hexokinase in young red blood cells is fivefold higher when compared with the old ones, but the concentration of many inhibitors of the enzyme is also cell age-dependent. Glucose 6-phosphate, glucose 1,6-diphosphate, 2,3-diphosphoglycerate, ATP, and Mg all decay during cell ageing but at different rates. The free concentrations and the hemoglobin and Mg complexes of both ATP and 2,3-diphosphoglycerate with hemoglobin in the oxy and deoxy forms have been calculated. This information was utilized in the calculation of glucose phosphorylation rate during cell ageing. The results obtained agree with the measured glycolytic rates and suggest that the decay of hexokinase during cell ageing could play a critical role in the process of cell senescence and destruction.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

A large-scale purification of recombinant histone H1.5 from Escherichia coli

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.     

Early effects of hypoxia/reoxygenation on VEGF,Ang-1, Ang-2 and their receptors in the rat myocardium: Implications for myocardial angiogenesis

The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pK_a and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues.Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.     

Adenine nucleotides and magnesium ions in relation to control of mammalian cerebral-cortex hexokinase

1. The kinetics of inhibition of brain soluble cytoplasmic hexokinase by ADP were examined in relation to variations in the concentrations of Mg(2+) and ATP. The type of inhibition observed was dependent on the Mg(2+)/ATP ratio. 2. ADP at Mg(2+)/ATP ratios 2:1 exhibited inhibition of the ;mixed' type; at Mg(2+)/ATP ratios 1:1 the inhibition appeared to be competitive with regard to ATP. 3. Inhibition by free ATP was observed when the Mg(2+)/ATP ratio was less than 1:1. The inhibition was also of the ;mixed' type with respect to MgATP(2-). 4. The inhibitions due to ADP and to free ATP were not additive. The results suggested that there may be up to four sites in the soluble enzyme: for glucose, glucose 6-phosphate, ADP and MgATP(2-). 5. The ;free' non-particulate intracellular Mg(2+) concentration was measured and concluded to be about 1.5mm. 6. The concentrations in vivo of Mg(2+) and ATP likely to be accessible to a cytoplasmic enzyme are suggested to be below those that yield maximum hexokinase rates in vitro. The enzymic rates were measured at relevant suboptimum concentrations of Mg(2+) and ATP in the presence of ADP. Calculations that included non-competitive inhibition due to glucose 6-phosphate (56-65% at 0.25mm) resulted in net rates very similar to the measured rates for overall glycolysis. This system may therefore provide a basis for effective control of cerebral hexokinase.     

Effect of metabolites and phosphorylase on the D to I conversion of glycogen synthase from human polymorphonuclear leukocytes.

The D to I conversion of glycogen synthase from human polymorphonuclear leukocytes was examined both in a gel-filtered homogenate and in a preparation of glycogen particles with adhering enzymes, purified by chromatography on concanavalin A bound to Sepharose. It was found that glucose 6-phosphate as well as mannose 6-phosphate, glucosamine 6-phosphate, and 2-deoxy-glucose 6-phosphate activated the reaction, whereas the corresponding sugars were without effect. Mn2+ and Ca2+ increased the conversion rate by 51% and 27%, respectively, whereas Mg2+ and inorganic phosphate were without effect. Sodium fluoride inhibited the reaction completely. Glycogen inhibited the reaction in physiological concentrations and 0.5 mM glucose 6-phosphate was able to overcome this inhibition. MgATP greatly augmented the inhibition caused by glycogen in the glycogen particle preparation. This combined effect could be overcome by glucose 6-phosphate in concentrations from 0.1 to 1 mM. Phosphorylase alpha purified from human polymorphonuclear leukocytes inhibited the D to I conversion in a glycogen particle preparation. The inhibition was counteracted by glucose 6-phosphate and to a lesser degree by AMP. Phosphorylase beta was also inhibitory, but only at higher concentrations than phosphorylase alpha. No phosphorylase phosphatase activity was found in the glycogen particle preparation, which may indicate that chromatography on concanavalin A-Sepharose separates this enzyme from the synthase phosphatase or partially destroys the activity of a hypothetical common protein phosphatase.     

In vivo dynamics of the pentose phosphate pathway in Saccharomyces cerevisiae

The in vivo dynamics of the pentose phosphate pathway has been studied with transient experiments in continuous culture of Saccharomyces cerevisiae. Rapid sampling was performed with a special sampling device after disturbing the steady state with a pulse of glucose. The time span of observation was 120 s after the pulse. During this short time period the dynamic effect of protein biosynthesis can be neglected. The metabolites of interest (glucose 6-phosphate, NADP, NADPH, 6-phosphogluconate, and MgATP2-) we determined with enzymatic assays and HPLC. The experimental observations were then used for the identification of kinetic rate equations and parameters under in vivo conditions. In accordance with results from in vitro studies the in vivo diagnosis supports an ordered Bi-Bi mechanism with noncompetitive inhibition by MgATP2- for the enzyme glucose-6-phosphate dehydrogenase. In the case of 6-phosphogluconate dehydrogenase an ordered Bi-Ter mechanism with a competitive inhibition by MgATP2- has been found. Because the MgATP2- concentration decreases abruptly after the pulse of glucose the inhibitory effect vanishes and the flux through the pentose phosphate pathway increases. This regulation phenomenon guarantees the balance of fluxes through glycolysis and pentose phosphate pathway during the dynamic time period.     

Brain hexokinase has no preexisting allosteric site for glucose 6-phosphate

Difference spectroscopic investigations on the interaction of brain hexokinase with glucose and glucose 6-phosphate (Glc-6-P) show that the binary complexes E-glucose and E-Glc-6-P give very similar UV difference spectra. However, the spectrum of the ternary E-glucose-Glc-6-P complex differs markedly from the spectra of the binary complexes, but resembles that produced by the E-glucose-Pi complex. Direct binding studies of the interaction of Glc-6-P with brain hexokinase detect only a single high-affinity binding site for Glc-6-P (KD = 2.8 microM). In the ternary E-glucose-Glc-6-P complex, Glc-6-P has a much higher affinity for the enzyme (KD = 0.9 microM) and a single binding site. Ribose 5-phosphate displaces Glc-6-P from E-glucose-Glc-6-P only, but not from E-Glc-6-P complex. It also fails to displace glucose from E-glucose and E-glucose-Glc-6-P complexes. Scatchard plots of the binding of glucose to brain hexokinase reveal only a single binding site but show distinct evidence of positive cooperativity, which is abolished by Glc-6-P and Pi. These ligands, as well as ribose 5-phosphate, substantially increase the binding affinity of glucose for the enzyme. The spectral evidence, as well as the interactive nature of the sites binding glucose and phosphate-bearing ligands, lead us to conclude that an allosteric site for Glc-6-P of physiological relevance occurs on the enzyme only in the presence of glucose, as a common locus where Glc-6-P, Pi, and ribose 5-phosphate bind. In the absence of glucose, Glc-6-P binds to the enzyme at its active site with high affinity. We also discuss the possibility that, in the absence of glucose, Glc-6-P may still bind to the allosteric site, but with very low affinity, as has been observed in studies on the reverse hexokinase reaction.     

Monoclonal antibodies against rat brain hexokinase. Utilization in epitope mapping studies and establishment of structure-function relationships

The effects of seven monoclonal antibodies on various functions of rat brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) have been assessed. Specifically, effects on catalytic properties (Km values for substrates, glucose and ATP X Mg2+; Ki for inhibition by glucose 6-phosphate), binding to the outer mitochondrial membrane, and glucose 6-phosphate-induced solubilization of mitochondrially bound hexokinase were examined. Epitope mapping studies with the native enzyme provided information about the relative spatial distribution of the epitopes on the surface of the native molecule. Binding of nucleotides (ATP or ATP X Mg2+) was shown to perturb the epitopes recognized by two of these antibodies. Neither nucleotides nor other ligands (glucose, glucose 6-phosphate, Pi) had detectable effect on epitopes recognized by the other five antibodies. Peptide mapping techniques in conjunction with immunoblotting permitted assignment of the epitopes recognized by several of the antibodies to specific segments within the overall primary structure. These results, together with previous work relating to the organization of structural domains within the molecule, permitted development of a three-dimensional model which provides a useful representation of major structural and immunological features of the enzyme, and depicts the association of those features with specific functions.     

Crystal structures of mutant monomeric hexokinase I reveal multiple ADP binding sites and conformational changes relevant to allosteric regulation

Hexokinase I, the pacemaker of glycolysis in brain tissue, is composed of two structurally similar halves connected by an alpha-helix. The enzyme dimerizes at elevated protein concentrations in solution and in crystal structures; however, almost all published data reflect the properties of a hexokinase I monomer in solution. Crystal structures of mutant forms of recombinant human hexokinase I, presented here, reveal the enzyme monomer for the first time. The mutant hexokinases bind both glucose 6-phosphate and glucose with high affinity to their N and C-terminal halves, and ADP, also with high affinity, to a site near the N terminus of the polypeptide chain. Exposure of the monomer crystals to ADP in the complete absence of glucose 6-phosphate reveals a second binding site for adenine nucleotides at the putative active site (C-half), with conformational changes extending 15 A to the contact interface between the N and C-halves. The structures reveal distinct conformational states for the C-half and a rigid-body rotation of the N-half, as possible elements of a structure-based mechanism for allosteric regulation of catalysis.     

Effect of ligands on the reactivity of essential sulfhydryls in brain hexokinase. Possible interaction between substrate binding sites.

Inactivation of bovine brain mitochondrial hexokinase by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a sulfhydryl specific reagent, has been investigated. The study shows that the inactivation of the enzyme by DTNB proceeds by way of prior binding of the reagent to the enzyme and involves the reaction of 1 mol of DTNB with a mol of enzyme. At stoichiometric levels of DTNB, the inactivation of the enzyme is accompanied by the formation of a disulfide bond. But it is not clear whether the disulfide bond or the mixed disulfide intermediate formed prior to it causes inactivation. On the basis of considerable protection afforded by glucose against this inactivation it is tentatively concluded that the sulfhydryl residues involved in this inactivation are at the glucose binding site of the enzyme, although other possibilities are not ruled out. An analysis of effects of various substrates and inhibitors on the kinetics of inactivation and sulfhydryl modification by DTNB has led to the proposal that the binding of substrates to the enzyme is interdependent and that glucose and glucose 6-phosphate produce slow conformational changes in the enzyme. Protective effects by ligands have been employed to calculate their dissociation constant with respect to the enzyme. The data also indicate that glucose 6-phosphate and inorganic phosphate share the same locus on the enzyme as the gamma phosphate of ATP and that nucleotides ATP and ADP bind to the enzyme in the absence of Mg2+.     

Changes in enzymatic activities involved in glucose metabolism by acyl-CoAs in Trypanosoma cruzi

This study describes the effect of some saturated and unsaturated free fatty acids and acyl-CoA thioesters on Trypanosoma cruzi glucose 6-phosphate dehydrogenase and hexokinase activities. Glucose 6-phosphate dehydrogenase was sensitive to the destabilizing effect provoked by free fatty acids, while hexokinase remained unaltered. Glucose 6-phosphate dehydrogenase inhibition by free fatty acids was dependent on acid concentration and chain length. Both enzymes were inhibited when they were incubated with acyl-CoA thioesters. The acyl-CoA thioesters inhibited glucose 6-phosphate dehydrogenase at a lower concentration than the free fatty acids; the ligands glucose 6-phosphate and NADP+ afforded protection. The inhibition of hexokinase by acyl-CoAs was not reverted when the enzyme was incubated with ATP. The type of inhibition found with acyl-CoAs in relation to glucose 6-phosphate dehydrogenase and hexokinase suggests that this type inhibition may produce an in vivo modulation of these enzymatic activities.