Ligand-induced conformations of rat brain hexokinase: studies with hexoses, hexose 6-phosphates, and nucleoside triphosphates leading to development of a new model for the enzyme

Various ligands of rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) have been found to protect the enzyme against either (or both) chymotryptic digestion or inactivation by glutaraldehyde. Using this protective effect, the K_d for various enzyme-ligand complexes has been estimated: hexokinase-Glc, K_d = 0.24 ± 0.03mM (chymotryptic digestion), K_d = 0.26 ± 0.07mM (glutaraldehyde inactivation); hexokinase-Glc-6- P, K_d = 0.041 ± 0.005m M (glutaraldehyde inactivation); hexokinase-ATP, K_d = 1.01 ± 0.28mM (chymotryptic digestion); hexokinase-ATP-Mg ²⁺, K_d = 0.07-0.08mM (chymotryptic digestion). Other nucleoside triphosphates (UTP, ITP, GTP, and CTP) were much less effective than ATP at protecting against chymotrypsin. Various hexoses were tested for their ability to protect against glutaraldehyde. Only ?good” substrates (mannose, 2-deoxyglucose) protected; nonsubstrates (galactose, arabinose) and N-acetylglucosamine, a competitive inhibitor of Glc binding, were not effective. Various hexose 6-phosphates were tested for their ability to protect against glutaraldehyde inactivation. Glc-6-P was much more effective than were mannose-6-P, galactose-6-P, or fructose-6-P. It was observed that ?good” substrates (Glc, mannose) increased the effectiveness of Glc-6-P at solubilizing the mitochondrial form of the enzyme; galactose and N-acetylglucosamine had no effect on solubilization by Glc-6-P. These results are taken as an indication of enhanced Glc-6-P binding in the presence of Glc, as previously reported by Ellison et al. (J. Biol. Chem., 250, 1864–1871, 1975). Along with previous studies on ligand-induced conformations and kinetics of this enzyme, these results form the basis for a new model for brain hexokinase. This model specifically takes into account the ligand-induced conformations at various points in the catalytic cycle and specifically accounts for the ability of various hexoses to serve as substrates and hexose 6-phosphates to serve as inhibitors in terms of their ability to induce specific conformations of the enzyme. The properties of the various conformations involved in the model are designated by a four-letter code which facilitates comparison and discussion.     

Studies on pituitary follitropin. III. Functional role of the amino groups in subunit and receptor interactions of the ovine hormone.

The effectiveness of Glc, mannose, 2-deoxyglucose, fructose, galactose, arabinose, and N-acetylglucosamine at protecting rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) from inactivation by chymotrypsin, glutaraldehyde, heat, and Ellman's reagent have been compared. The relative effectiveness at protecting against these inactivating agents decreases in the order Glc > mannose > 2-deoxyglucose > fructose, galactose, and arabinose, the last three providing no significant protection at all. The nonphosphorylatable substrate analog, N-acetylglucosamine, provides substantial protection against heat inactivation, but is ineffective against inactivation by the other agents. Similar inactivation studies were conducted using several hexose 6-phosphates. Glc-6-P and 1,5-anhydroglucitol-6-P provided substantial protection while 2-deoxyglucose-6-P, fructose-6-P, mannose-6-P, and galactose-6-P were all relatively ineffective at protecting hexokinase activity. The protective effect of these ligands is taken as an indication of ligand-induced conformational changes in brain hexokinase. The results are interpreted in terms of, and considered to support, a recently proposed model (J. E. Wilson, 1978, Arch. Biochem. Biophys.185, 88–99) in which the suitability of a carbohydrate as a substrate depends directly on its ability to induce specific conformational changes prerequisite for subsequent catalytic events while the inhibitory effectiveness of a hexose 6-phosphate is likewise related to its ability to evoke appropriate conformational change in the enzyme. Synergistic interactions between hexose and hexose-6-P binding sites, first reported for Glc and Glc-6-P by Ellison et al. (1975, J. Biol. Chem.250, 1864–1871), have been confirmed. Thus, although fructose and galactose were themselves quite ineffective at providing protection against inactivation of hexokinase by chymotrypsin or glutaraldehyde, they greatly increased the protection afforded by suboptimal (with respect to degree of protection in the absence of added hexose) levels of Glc-6-P. Conversely, the 6-phosphates of fructose, galactose, mannose, and 2-deoxyglucose, which were themselves ineffective at protecting the enzyme activity, markedly enhanced the protection provided by suboptimal levels of Glc or mannose. Based on the relationship between this enhancement of Glc-dependent protection and the hexose-6-P concentration, the dissociation constants for the complexes of hexokinase with 2-deoxyglucose-6-P and mannose-6-P were estimated to be ?0.5 mm.     

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.     

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.     

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.     

Specificity for the glucose-6-P inhibition site of hexokinase

Inhibition of the three animal hexokinase isozymes by the following glucose-6-P analogs has been determined: α-glucose-1,6-P₂, α- and β-methyl glucose-6-P, α- and β-glucose-6-P, 2-Cl- and 4F-glucose-6-P, 5-deoxyglucose-6-P, glucose-6-sulfate, and δ-gluconolactone-6-P. Although both anomers of glucose-6-P were about equally active inhibitors, the β-methyl derivative was inactive. Generally the α-methyl and α-PO₃^? derivatives were good inhibitors though weaker than glucose-6-P except in the case of hexokinase II for which α-glucose-1,6-P₂ was an excellent inhibitor.     

Studies on the kinetics and mechanism of orthophosphate activation of bovine brain hexokinase

The binding of glucose to bovine brain hexokinase, isozyme I, exhibited one binding site per 100,000 molecular weight. Glucose-6-P binding was examined in the absence and presence of ATP. ATP and glucose-6-P were shown to compete for the same binding site on the enzyme. A model was proposed to account for these findings and the previously reported data that glucose-6-P and P_i exhibit mutually exclusive, non-cooperative binding to the enzyme. The model shows that brain hexokinase exists in two rapidly interconvertible states, either with or without P_i and that glucose-6-P binding to the phosphate associated enzyme form is relatively very poor. This proposal has been tested kinetically and the data appear to support the suggested model.     

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.     

A study of the sulfhydryl groups of rat brain hexokinase

Rat brain hexokinase (ATP: d-hexose-6-phosphotransferase, EC 2.7.1.1) is rapidly inactivated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). The inactivation follows monophasic first-order kinetics in either the absence of ligands (k = 0.641 min^?1 at 25 °C) or in the presence of saturating levels of ATP (free or complexed with Mg²⁺) or P₁; the inactivation rate is slightly increased ($\text{k ? 0.7}$ min ^?1) in the presence of ATP or P₁. In contrast, glucose and glucose-6-P markedly decrease the inactivation rate; inactivation in the presence of these ligands is biphasic, with two first-order rates ($\text{k ? 0.5}$ min^?1 and 0.01 min^?1) being distinguishable.The enzyme contains 14 sulfhydryl groups which react with 5,5′-dithiobis-(2-nitrobenzoate); reaction of these groups in the native enzyme is complete after 2 hr at 25 °C, or in approx 5 min with the urea or guanidine-denatured enzyme. In the native enzyme, three classes of sulfhydryl groups are distinguishable and are designated as F-, I-, or S-type based on their fast (k ? 0.7 min^?1), intermediate ($\text{k ? 0.5-0.7}$ min^?1), or slow ($\text{k ? 0.02}$ min^?1 rates of reaction with 5,5′-dithiobis-(2-nitrobenzoate). The correlation of inactivation rates with the rates for reaction of the I-type sulfhydryls indicates that the I-type sulfhydryls include residues necessary for catalytic activity. The F-type residues are clearly not required for activity.The effects of ATP, P₁, glucose, and glucose-6-P on the reactivity of the sulfhydryls have been determined. As in the absence of ligands, S-, I-, and F-type sulfhydryls could be distinguished. In the presence of saturating concentrations of these ligands, the F, I, and S classes of sulfhydryls contained respectively: with ATP, 1, 4, and 7 residues; with P₁, 1, 3, and 7 residues; with glucose, 1, 2, and 5 residues; with glucose-6-P, 1, 2, and 1 residues. Comparison with rate constants for inactivation in the presence of these ligands again indicated that I-type sulfhydryls were particularly important in maintenance of enzyme activity. The present results indicate considerable similarity between the reactivity of the sulfhydryl residues in rat brain hexokinase and the sulfhydryls of the bovine brain enzyme [V. D. Redkar and U. W. Kenkare (1972), J. Biol. Chem., 247, 7576–7584].     

Inactivation and reactivation of hexokinase type II

Hexokinase isozyme II which loses activity rapidly in the absence of glucose ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 10}\text{min}$) is stabilized in the presence of glucose-6-P, P_i and ADP when glucose is also present but not by kinetically inert analogs. Enzyme inactivated by incubation in the absence of glucose is fully and rapidly recovered ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>～-\; 10\; </mtext><mtext>min</mtext>}}$) by addition of both glucose and mercaptoethanol, each at 0.1 m. In the presence of 0.1 mm glucose, both glucose-6-P and P, facilitate the reactivation. Reactivation proceeds in two steps both with unfavorable equilibria: a fast reduction followed by a slow renaturation. Native enzyme is much more resistant to irreversible inactivation by trypsin than is enzyme that has lost its activity by incubation in the absence of glucose. The latter form shows no protection from trypsin action by glucose. Streptozotocin-diabetic rats that have lost hexokinase II preferentially in their insulin-sensitive tissues do not contain an activatable form of hexokinase in at least one of these, heart. The greater sensitivity of inactivated hexokinase to denaturation by trypsin suggests that such a “reservoir” form may be destroyed rapidly in vivo. Glucose may be important in determining the steady-state level of hexokinase II by “guiding” the folding of translation product. In this view insulin would act through its effect on glucose permeability.     

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.     

Inorganic Polyphosphates Regulate Hexokinase Activity and Reactive Oxygen Species Generation in Mitochondria of Rhipicephalus (Boophilus) microplus Embryo

The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase were assayed during embryogenesis of R. microplus. The influence of poly P₃ and poly P₁₅ were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P₃. However, superoxide dismutase was not affected by poly P₁₅, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos.     

Alterations in glucose metabolism in chick embryo cells transformed by Rous sarcoma virus. Transformation-specific changes in the activities of key enzymes of the glycolytic and hexose monophosphate shunt pathways

Effects of transformation by Rous sarcoma virus of Schmidt-Ruppin strain on the activities of key enzymes of the glycolytic and the hexose monophosphate shunt pathways in chick-embryo cells were investigated. Activities of hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, and glucose-6-P dehydrogenase were increased about twofold in the transformed cells, but that of 6-P-gluconate dehydrogenase remained unaltered. The transformation-mediated increase in the activity of hexokinase was confined entirely to the bound form of the enzyme. Cells infected with a temperature-sensitive mutant (Ts-68) of Schmidt-Ruppin strain of Rous sarcoma virus showed the typical increase in the rate of 2-deoxyglucose uptake and the activities of hexokinase, phosphofructokinase, pyruvate kinase, and glucose-6-P dehydrogenase at the permissive temperature (37 °C), but when the infected cells were grown at the nonpermissive temperature (41 °C), the increases in the sugar uptake and activities of these enzymes were abolished. Unlike the regulatory enzymes, lactate dehydrogenase activity was increased at both the permissive and the nonpermissive temperatures.     

A note on stability of purified enzymes to dehydration

Highly purified enzymes from commercial sources were dried from aqueous solutions over CaCl₂ or CaSO₄ at 25° c, 4° c or −4° C. Several heat stable enzymes containing SS groups (chymotrypsin, lipase, pepsin and trypsin) retained most of their activity following drying while papain and ribonuclease retained about one-fourth of their original activity. Most SH containing enzymes lost most if not all activity during drying (catalase, hexokinase, glucose and xanthine oxidases, alcohol, glutamate, isocitrate and glucose-6-phosphate dehydrogenases). Lactic dehydrogenase was the exception in this group retaining 75% of its original activity after drying. Neither sucrose nor mannitol were effective in protecting ribonuclease against inactivation during drying. Temperature during drying had little effect on inactivation.     

Studies on the binding of nucleotides by rat brain hexokinase

Various nucleoside di- and triphosphates have been compared with respect to their ability to protect rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activity against inactivation by chymotrypsin, glutaraldehyde, heat, and 5,5′-dithiobis(2-nitrobenzoic) acid. ATP could be distinguished from other nucleoside triphosphates in these comparisons, which may be related to the specificity with which ATP is utilized as a substrate. All nucleoside derivatives examined provided substantial protection against two or more of the above inactivating agents, indicating relatively nonspecific binding of nucleotides by brain hexokinase, consistent with a similar lack of specificity in the inhibition of this enzyme by nucleoside derivatives. The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tetraiodofluorescein (TIF) was enhanced by binding to brain hexokinase. TNS binding was not affected by the presence of various relevant metabolites (Glc, glucose 6-phosphate, ATP), nor did TNS inhibit the enzyme. In contrast, substantial (approximately 70%) decreases in the fluorescence of bound TIF resulted from the addition of various nucleoside derivatives, and TIF served as a competitive inhibitor of brain hexokinase. These observations are consistent with the view that TIF binds to a nucleotide binding site of the enzyme. The inability of nucleotides to totally displace TIF was taken to indicate the existence of an additional TIF binding site (or sites) discrete from the catalytic site, and probably identical to the site(s) at which TNS binds with no effect on catalytic activity. The effects of saturating levels of ATP and ADP were not additive indicating that both compounds were displacing TIF from the same site i.e., a common nucleotide binding site. Glc, mannose, and 2-deoxyglucose greatly enhanced the ability of nucleotides to displace TIF, while fructose, galactose, and N-acetylglucosamine did not, indicating the existence of interactions between hexose and nucleotide binding sites; the hexoses themselves were not effective at displacing TIF. The enhanced binding of nucleotides in the presence of the first three hexoses but not the latter three can be directly correlated with the relative ability of these hexoses to induce specific conformational changes in the enzyme. The hexoses themselves were not effective at displacing TIF. Glucose 6-phosphate and 1,5-anhydroglucitol 6-phosphate could also displace TIF, and as with the nucleotides, a maximum of approximately 70% decrease in fluorescence was observed and the effectiveness of glucose 6-phosphate was enhanced in the presence of Glc. Other hexose 6-phosphates tested were not effective at displacing TIF. The specificity with which hexose 6-phosphates displaced TIF could be correlated with their ability to induce specific conformational change in the enzyme. The results are discussed as they relate to the kinetic mechanism and allosteric regulation by nucleotides that have been proposed for this enzyme.     

Rabbit heart mitochondrial hexokinase: Solubilization and general properties

In rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent K_m for glucose (0.065 mm), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent K_m for ATPMg than the solubilized enzyme, whereas the apparent K_m for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968)Proc. Natl. Acad. Sci. USA61, 598–605]. Furthermore, low concentrations (0.1 mm) of glucose 6-phosphate (G6P) or ATP^4? specifically solubilize hexokinase. The solubilizing effect of G6P and ATP^4?, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with P_i or Mg²⁺. In addition, enzyme solubilization by G6P can be reversed by Mg²⁺ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J.37, 18–19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one.     

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.     

Some observations on mitochondrial-bound hexokinase and creatine kinase of the heart

A large part of the hexokinase activity of the rat brain 20,000g supernatant became mitochondrial bound when incubated with rat heart mitochondria which had been pretreated with glucose-6-phosphate. This binding was dependent on small-molecular compounds (as yet unidentified) of the brain supernatant. Divalent cations, spermine, and pentalysine strongly stimulated the binding of brain supernatant hexokinase to heart mitochondria. Inorganic phosphate, alpha-glycerophosphate, and fructose-1,6-diphosphate showed some stimulatory effect. No effect was observed with insulin or glucose. Mitochondria isolated from hearts of fasted rats had less specific hexokinase activity than mitochondria from fasted and then carbohydrate refed rats. This dietary treatment had no significant effect on the total heart hexokinase activity. Oligomycin did not inhibit the formation of creatine phosphate or glucose-6-phosphate by isolated rabbit heart mitochondria incubated in the presence of phosphoenolpyruvate and pyruvate kinase. However, the presence of creatine inhibited the formation of glucose-6-phosphate when the ATP/ADP ratio was low, indicating that creatine kinase has a greater access to ATP/ADP translocation than has hexokinase.