Magnetic resonance studies on the interaction of metal-ion and nucleotide ligands with brain hexokinase

Our previous studies have shown that one manganous ion binds tightly to bovine hexokinase, with a Kd = 25 +/- 4 microM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (epsilon b) is 3.5 at 9 MHz and 23 degrees C [Jarori, G.K. Kasturi, S.R., and Kenkare, U.W. (1981) Arch. Biochem. Biophys. 211, 258-268]. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E X Mn binary complex, we now show the formation of a nucleotide-bridge ternary complex, enzyme X nucleotide X Mn. Addition of glucose 6-phosphate to enzyme X ATP X Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E X ADP X Mn X Glc-6-P appears to be formed when both the products are present. Beta, gamma-Bidentate Cr(III)ATP has been used to elucidate the role of direct binding of Mn(II) in catalysis, and the stoichiometry of metal-ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(III)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron-spin resonance studies on the binding of Mn(II) to the enzyme in the presence of Cr(III)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis. Experiments carried out with hexokinase spin-labeled with 3-(2-iodo-acetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl clearly showed that the direct-binding Mn site on the enzyme is distinctly located from its ATP Mn binding site.     

Dual mechanisms for glucose 6-phosphate inhibition of human brain hexokinase.

Brain hexokinase (HKI) is inhibited potently by its product glucose 6-phosphate (G6P); however, the mechanism of inhibition is unsettled. Two hypotheses have been proposed to account for product inhibition of HKI. In one, G6P binds to the active site (the C-terminal half of HKI) and competes directly with ATP, whereas in the alternative suggestion the inhibitor binds to an allosteric site (the N-terminal half of HKI), which indirectly displaces ATP from the active site. Single mutations within G6P binding pockets, as defined by crystal structures, at either the N- or C-terminal half of HKI have no significant effect on G6P inhibition. On the other hand, the corresponding mutations eliminate product inhibition in a truncated form of HKI, consisting only of the C-terminal half of the enzyme. Only through combined mutations at the active and allosteric sites, using residues for which single mutations had little effect, was product inhibition eliminated in HKI. Evidently, potent inhibition of HKI by G6P can occur from both active and allosteric binding sites. Furthermore, kinetic data reported here, in conjunction with published equilibrium binding data, are consistent with inhibitory sites of comparable affinity linked by a mechanism of negative cooperativity.     

Mapping of glucose and glucose-6-phosphate binding sites on bovine brain hexokinase. A 1H- and 31P-NMR investigation

Inhibition of bovine brain hexokinase by its product, glucose 6-phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6-phosphate on the enzyme with respect to a divalent-cation-binding site characterized previously [Jarori, G. K., Kasturi, S. R. & Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258-268]. The paramagnetic effect of enzyme-bound Mn(II) on the spin-lattice relaxation rates (T-1(1] of ligand nuclei (1H and 31P) in E.Mn(II).Glc and E.Mn(II).Glc6P complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1-H alpha, C1-H beta and C2-H beta of glucose in the E.Mn(II).Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5-30 degrees C at 500 MHz. The ligand nuclear relaxation rates in E.Mn(II).Glc are field-dependent and the Arrhenius plot yields an activation energy (delta E) of 16.7-20.9 kJ/mol. Similar measurements have also been carried out on C1-H alpha, C1-H beta and C6-31P at 270 MHz (1H) and 202.5 MHz (31P) for the E.Mn(II).Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5-30 degrees C, which yielded delta E = 9.2 kJ/mol. The electron-nuclear dipolar correlation time (tau c), determined from the field-dependent measurements of proton relaxation rates in the E.Mn(II).Glc complex, is 0.22-1.27 ns. The distances determined between Mn(II) and C1-H of glucose and glucose 6-phosphate are approximately 1.1 nm and approximately 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare, U. W. (1988) J. Biol. Chem. 263, 15492-15498], suggest that glucose and glucose 6-phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P.Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co-ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of approximately 0.5-0.6 nm from the cation.     

Allosteric inhibition of brain hexokinase by glucose 6-phosphate in the reverse reaction

A study of the reverse reaction of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been performed using a photometric method based on a mutarotase-glucose oxidase-peroxidase-chromogen system to trap and visualize glucose, plus a glycerol kinase-glycerol system to trap ATP. Glucose 6-phosphate or 2-deoxyglucose 6-phosphate were used as phosphoryl donors at different concentrations of ADP. Variation of glucose 6-phosphate concentrations resulted in a biphasic curve from which apparent Km and Ki values of ca. 0.2 mM were calculated. In contrast, variation of 2-deoxyglucose 6-phosphate concentrations resulted in Michaelian kinetics with an apparent Km of 2 mM. The Km value for MgADP was 16 mM irrespective of the nature and concentration of the hexose 6-phosphate substrate. These results are fully consistent with an allosteric site for glucose 6-phosphate as an explanation for the inhibition of animal hexokinases by glucose 6-P and further indicate that the maximal rate is the parameter affected. From these observations and previous knowledge, the possible occurrence in animal hexokinases of a regulatory site for ATP to account for the competition between glucose 6-phosphate and ATP in the forward reaction is postulated.     

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)     

Rat brain hexokinase: glucose and glucose-6-phosphate binding sites and C-terminal amino acid of the purified enzyme

The binding of glucose and glucose-6-P by pure rat brain hexokinase has been studied by using an ultrafiltration procedure [H. Paulus (1969) Anal. Biochem. 32, 91–100]. Each mole of enzyme (molecular weight 98,000) binds 1 mole of glucose or 1 mole of glucose-6-P. The dissociation constant for the enzyme-glucose complex (0.04 mm) is in excellent agreement with the kinetically determined K_m for this substrate. The dissociation constant for the enzyme-glucose-6-P complex was estimated to be 1.3 μm, substantially lower than values of 7–8 μm obtained by alternative methods. This discrepancy appears to be due to retardation of the passage of the charged glucose-6-P through the ultrafiltration membrane, resulting in an effective increase in the ligand concentration at the membrane surface and thereby a decrease in the apparent dissociation constant. No appreciable retardation of the passage of the uncharged glucose molecule was observed.The binding of glucose-6-P (but not glucose) is prevented in the presence of P_i. This is in accord with a previously suggested model in which binding of P_i is considered to stabilize the enzyme in a conformation having little, if any, affinity for glucose-6-P.Serine was found as a C-terminal amino acid. The method used would not have detected C-terminal proline or tryptophan residues, and thus these cannot be excluded by the present experiments. However, in view of other results indicating that rat brain hexokinase consists of a single polypeptide chain, it seems probable that serine is indeed the only C-terminal amino acid in the molecule.     

Magnetic resonance studies of the spatial arrangement of glucose-6-phosphate and chromium (III)-adenosine diphosphate at the catalytic site of hexokinase.

The interaction of CrADP, an exchange-inert paramagnetic analogue of Mg-ADP, with yeast hexokinase has been studied by measuring the effects of CrADP on the longitudinal nuclear relaxation rate (1/T1) of the protons of water and the protons and phosphorus atom of enzyme-bound glucose-6-P. The paramagnetic effect of CrADP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations measuring this paramagnetic effect at several enzyme concentrations in the presence of glucose-6-P yielded a characteristic enhancement factor for 1/T1 of water protons and the dissociation constant of CrADP from the ternary enzyme . ADPCr . glucose-6-P complex. The latter value (2 mM) is similar to that obtained from kinetic inhibition studies (Danenberg and Cleland [1975]. Biochemistry. 14:28). The presence of glucose-6-P increased the enhancement of the water relaxation rate by enzyme-bound CrADP, suggesting the formation of an enzyme . CrADP . glucose-6-P complex. The existence of such a complex was confirmed by the observation of a paramagnetic effect of enzyme-bound CrADP on the l/T1 of the 31P-nucleus and protons of enzyme-bound glucose-6-P. From the paramagnetic effects of enzyme-bound CrADP on the relaxation rates of the 31P-nucleus and the carbon-bound protons of glucose-6-P in the enzyme . ADPCr . glucose-6-P complex, using the correlation time of approximately 0.7 ns, determined from the magnetic field-dependence of 1/T1 of water protons over the range 24.3-360 MHz, a Cr3+ to phosphorus distance of 6.6 +/- 0.7 A and Cr3+ to alpha- and beta-anomeric proton distances of 8.9 and 9.7 A were calculated. These results imply the absence of a direct coordination of the phosphoryl group of glucose-6-P by the nucleotide-bound metal on hexokinase but indicate van der Waals contact between a phosphoryl oxygen of glucose-6-P and the hydration sphere of the nucleotide-bound metal. The distances are consistent with a model that assumes molecular contact between the phosphorus of glucose-6-P and a beta-phosphoryl oxygen of ADP suggesting an associative phosphoryl transfer. Because after phosphorylation of ADP, the metal ion is coordinated to the transferred phosphoryl group, the overall migration of the phosphoryl group during the phosphoryl transfer is approximately 3.6 A toward the nucleotide-bound metal. Little or no catalysis of phosphoryl transfer from glucose-6-P to alpha, beta-bidentate or beta-monodentate CrADP ( less than or equal to 0.05% of the rate found with MgADP) occurred in the presence of hexokinase, as monitored by glucose formation in a coupled assay system using glucose oxidase and peroxidase. The ability of beta, gamma-bidentate CrATP to act as a substrate (Danenberg and Cleland [1975].     

Allosteric character of the inhibition of rat-muscle hexokinase B by glucose 6-phosphate

We previously provided evidence from isotope-exchange measurements under non-equilibrium conditions that hexokinase B from rat muscle follows a compulsory-order mechanism with glucose binding before MgATP, and with both glucose 6-phosphate and MgATP capable of binding allosterically [Gregoriou, M., Trayer, I. P. & Cornish-Bowden, A. (1983) Eur. J. Biochem. 134, 283-288]. We have now re-examined this work in the light of recent criticisms [Ganson, N. J. & Fromm, H. J. (1985) J. Biol. Chem. 260, 12099-12105]. There is no difficulty in obtaining valid estimates of initial rates of isotope exchange when the equilibrium constant is unfavourable, if one uses highly radioactive reactants and low enzyme concentrations, as we did in the experiments we reported previously. However, our earlier suggestion that MgADP can be released within the inhibitory pathway, which was made for the sake of consistency with the catalytic pathway rather than because of any compelling experimental evidence, must be revised to avoid predicting that the rate must be zero in the absence of MgADP. Although our mechanism admits the possibility of substrate inhibition by MgATP, calculations show that there is no need for this to be observable under ordinary conditions. Indeed, with plausible values assumed for the kinetic constants one can calculate theoretical behaviour according to our model that closely resembles the experimental inhibition experiments that have been claimed as evidence against it.     

Changes in the levels of catecholamines, hexokinase and glucose 6-phosphate dehydrogenase in red cell aging

The activity of the enzymes hexokinase and glucose 6-phosphate dehydrogenase and the level of catecholamines were measured in isolated rat Red Blood Cells (RBC) during cellular aging. The results clearly showed a linear decline in the two enzyme profiles with corresponding increase in age of RBC. A decrease of 75-85% in the activities were found in the oldest cell fractions as compared to the youngest. The levels of glycosylated haemoglobin and catecholamines were found to increase with aging. A correlation can probably be established between the enzyme activities, the levels of glycosylated haemoglobulin and catecholamines during aging.     

Immunological studies on glucose 6-phosphate dehydrogenase of rat liver

Glucose 6-phosphate dehydrogenase (G6PD) was purified from the supernatant fraction of rat liver to a homogeneous preparation by a specific elution with substrate. A specific antibody against the purified enzyme was prepared in rabbits and was shown to completely inhibit the enzyme activity and precipitate the enzyme protein of liver supernatant. With this antiserum, liver supernatants with varying specific G6PD activities obtained under several experimental conditions and supernatants from other tissues examined all formed single precipitin lines, which fused with each other in the Ouchterlony double-diffusion system. Three interconvertible microheterogeneous forms of G6PD in liver, supernatant were immunologically indistinguishable from each other. The G6PDs in participate fractions of liver were, however, distinct from the supernatant enzyme both in inhibition of the enzyme activity and in formation of precipitation by the specific antiserum. Liver supernatant G6PD, which was inactivated with various reagents or by heating, showed a simultaneous loss of ability to form precipitin line. Aggregation and disaggregation of the dehydrogenase to the tetramer and monomer, respectively, also resulted in loss of immunological reactivity. The increase in G6PD activity in the cytoplasm of carbon tetrachloride-treated or glucose casein-refed rat liver was accompanied by a proportional increase in the quantity of immunologically reactive G6PD protein.     

Studies on the molecular weight and lipoprotein nature of glucose-6-phosphate solubilized rat brain hexokinase

Treatment of brain mitochondria with glucose-6-P releases the hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), normally associated with the outer mitochondrial membrane, in soluble form. The glucose-6-P solubilized enzyme sediments during sucrose density gradient centrifugation at a rate compatible with a molecular weight of approx. 100,000. In contrast, in agreement with the results of Craven and Basford [Biochim. Biophys. Acta255, 620 (1972)], the enzyme is eluted in the void volume when chromatographed on Sephadex G-200 in 0.3 m mannitol-0.1 mm EDTA, suggesting a molecular weight much greater than 100,000. The resolution of this paradox is found in the observation that glucose-6-P solubilized hexokinase and several other proteins behave anomalously when chromatographed under these conditions; thus, elution in the void volume is not a satisfactory basis for estimating molecular weight.The glucose-6-P solubilized enzyme can be rebound to the mitochondria in the presence of added divalent cation. Phospholipase C treatment of the enzyme greatly hinders this reassociation but has no effect on hexokinase activity, suggesting the involvement of phospholipid in the interaction of the enzyme with the mitochondria. Based on the observation that sedimentation through a sucrose density gradient does not decrease binding ability, it is suggested that the required phospholipid is bound to the enzyme. After purification to homogeneity, however, the enzyme does not contain appreciable lipid (<0.7 mole phospholipid per mole enzyme), nor can it be rebound to mitochondria. Apparently the lipid, required for binding, is dissociated during purification. The potential significance of lipid in determining the intracellular distribution of the enzyme is discussed.