Interaction of hexokinase II isoenzyme from rat skeletal muscles with lecithin liposomes

It was found that in the presence of Mg2+ (pH 7.5) rat skeletal muscle hexokinase isozyme II is firmly adsorbed on mitochondrial and artificial phospholipid membranes (lecithin liposomes). In both cases the adsorption isotherm has similar quantitative and qualitative characteristics, which points to the absence of specific binding sites on the membranes. Under these conditions, immobilization of hexokinase on various membranes is concomitant with similar changes in the enzyme stability upon storage as well as with the pH-dependence of the enzyme activity. It was demonstrated that the bound hexokinase form has a greater value of V, an increased affinity for glucose and a decreased sensitivity to the inhibitory action of glucose-6-phosphate as compared to the free form. Besides, this form is in a greater degree subjected to the inhibitory influence of ADP with respect to glucose. In this case, the enzyme affinity for ATP and the Ki value for ADP with respect to ATP is practically the same both for the free and membrane-bound forms. The data obtained suggest that the phospholipid component of mitochondrial membranes participates in the enzyme binding in the presence of Mg2+. It was assumed that the model system used in the present study, i.e., hexokinase-Mg2+-liposomes, may be successfully used for the analysis of an adsorption mechanism of regulation of hexokinase activity in the cell.     

The hexokinase acceptor theory of insulin action — Hormonal control of functional compartmentation

The hexokinase-acceptor theory of insulin action is described and evidence for its validity is discussed. The theory states that insulin acts by connecting hexokinase to mitochondria. The close association of this particular enzyme to specific energy generating sites stimulates energy generation by the process known as respiratory control. The ADP generated when mitochondrial ATP is utilized by hexokinase to phosphorylate glucose acts as a stimulus — substrate — for further ATP generation by the Krebs cycle. The close proximity of the enzymes to the sites of energy generation makes the process of energy generation more efficient, providing more energy for anabolic reactions, all of which are stimulated by insulin. The lack of insulin effect on brain results from the fact that hexokinase in brain is tightly bound to mitochondria. On the other hand, the requirement of glucose for energy production in brain is evidence for the functional significance of this binding of hexokinase. The insulin-like effect of exercise in the diabetic is seen in the light of this theory as an acceptor effect of creatine liberated by muscle contraction.     

The role of bivalent cations in the binding of hexokinase II isoenzyme to mitochondrial membranes

A comparative study of Mg2+ and Ca2+ effects on the ability of rat skeletal muscle hexokinase isozyme II to bind mitochondrial membranes isolated from the same source was carried out. It was found that the binding ability of the enzyme increases in a similar way in the presence of equimolar amounts of both cations. The dependence of binding ability on cation concentration is hyperbolic, which points to the existence of specific and equivalent metal binding sites during hexokinase attachment to the membranes. Substitution of Ca2+ for Mg2+ does not influence the tightness of the enzyme binding to membranes, which can be evidenced from the type of dependence of the bound hexokinase solubilization degree on KCl concentration in the eluting buffer. The enzyme absorption mediated by various cations is accompanied by corresponding changes in its kinetic properties (V, Km for glucose, Ki for ADP). The role of bivalent cations in the formation of the specific hexokinase-membrane binding is discussed.     

Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme-membrane association control

BACKGROUND: Hexokinase I sets the pace of glycolysis in the brain, catalyzing the ATP-dependent phosphorylation of glucose. The catalytic properties of hexokinase I are dependent on product inhibition as well as on the action of phosphate. In vivo, a large fraction of hexokinase I is bound to the mitochondrial outer membrane, where the enzyme adopts a tetrameric assembly. The mitochondrion-bound hexokinase I is believed to optimize the ATP/ADP exchange between glucose phosphorylation and the mitochondrial oxidative phosphorylation reactions. RESULTS: The crystal structure of human hexokinase I has been determined at 2.25 A resolution. The overall structure of the enzyme is in keeping with the closed conformation previously observed in yeast hexokinase. One molecule of the ATP analogue AMP-PNP is bound to each N-terminal domain of the dimeric enzyme in a surface cleft, showing specific interactions with the nucleotide, and localized positive electrostatic potential. The molecular symmetry brings the two bound AMP-PNP molecules, at the centre of two extended surface regions, to a common side of the dimeric hexokinase I molecule. CONCLUSIONS: The binding of AMP-PNP to a protein site separated from the catalytic centre of human hexokinase I can be related to the role played by some nucleotides in dissociating the enzyme from the mitochondrial membrane, and helps in defining the molecular regions of hexokinase I that are expected to be in contact with the mitochondrion. The structural information presented here is in keeping with monoclonal antibody mapping of the free and mitochondrion-bound forms of the enzyme, and with sequence analysis of hexokinases that differ in their mitochondria binding properties.     

Characterization of hexokinase isoenzyme types I and II in ascites tumor cells by an interaction with mitochondrial membrane

Summary A difference was observed in the intracellular distribution between type I and II hexokinases in Ehrlich-Lettre hyperdiploid ascites tumor cells (ELD cells). Experiment of the rebinding to the mitochondria for either each or mixture of the partially purified preparations of the two types of hexokinase indicated that the accepting site on the mitochondrial membrane was common for both types. Mild treatment of the two isoenzymes with chymotrypsin resulted in loss of the binding ability to mitochondria without change in the catalytic activity. It was deduced from these results that the essential region in the two types of hexokinase to interact with mitochondria, which was cleaved by chymotrypsin, was the same or near-similar.Secondly, rebinding to and releasing from mitochondria were examined for the two hexokinase isoenzymes in the presence of various factors affecting the interaction between hexokinase and mitochondria, such as divalent cations, glucose 6-phosphate, and Pi. In the absence of divalent cations, about a half of the type I isoenzyme was bound to mitochondria, whereas almost no type II was bound. A difference was also seen between the two types in the concentration of divalent cations required for the saturation of the binding. A more marked difference was observed in the effect of Pi either alone or in combination with glucose 6-phosphate on the activity and binding ability of the two hexokinases. For type I isoenzyme, Pi relieved both inhibitory and releasing effects of glucose 6-phosphate. On the contrary, for type II, Pi had no such a modulating effect on the releasing action of glucose 6-phosphate, and had the inhibitory effect for itself on the enzyme activity.From these results, it is likely that the difference in the intracellular distribution between type I and II hexokinases in ELD cells is due to the difference in their catalytic regions in the reaction with these ligands, which would induce the structural change in the region responsible for the binding to mitochondria.     

Hexokinase II-deficient mice. Prenatal death of homozygotes without disturbances in glucose tolerance in heterozygotes.

Type 2 diabetes is characterized by decreased rates of insulin-stimulated glucose uptake and utilization, reduced hexokinase II mRNA and enzyme production, and low basal levels of glucose 6-phosphate in insulin-sensitive skeletal muscle and adipose tissues. Hexokinase II is primarily expressed in muscle and adipose tissues where it catalyzes the phosphorylation of glucose to glucose 6-phosphate, a possible rate-limiting step for glucose disposal. To investigate the role of hexokinase II in insulin action and in glucose homeostasis as well as in mouse development, we generated a hexokinase II knock-out mouse. Mice homozygous for hexokinase II deficiency (HKII(-/-)) died at approximately 7.5 days post-fertilization, indicating that hexokinase II is vital for mouse embryogenesis after implantation and before organogenesis. HKII(+/-) mice were viable, fertile, and grew normally. Surprisingly, even though HKII(+/-) mice had significantly reduced (by 50%) hexokinase II mRNA and activity levels in skeletal muscle, heart, and adipose tissue, they did not exhibit impaired insulin action or glucose tolerance even when challenged with a high-fat diet.     

A reevaluation of the roles of hexokinase I and II in the heart

Hexokinase is responsible for glucose phosphorylation, a process fundamental to regulating glucose uptake. In some tissues, hexokinase translocates to the mitochondria, thereby increasing its efficiency and decreasing its susceptibility to product inhibition. It may also decrease free radical formation in the mitochondria and prevent apoptosis. Whether hexokinase translocation occurs in the heart is controversial; here, using immunogold labeling for the first time, we provide evidence for this process. Rat hearts (6 groups, n = 6/group), perfused with either glucose- or glucose + oleate (0.4 mmol/l)-containing buffer, were exposed to 30-min insulin stimulation, ischemia, or control perfusion. Hexokinase I (HK I) and hexokinase II (HK II) distributions were then determined. In glucose-perfused hearts, HK I-mitochondrial binding increased from 0.41 +/- 0.04 golds/mm in control hearts to 0.71 +/- 0.10 golds/mm after insulin and to 1.54 +/- 0.38 golds/mm after ischemia (P < 0.05). Similarly, HK II-mitochondrial binding increased from 0.16 +/- 0.02 to 0.53 +/- 0.08 golds/mm with insulin and 0.44 +/- 0.07 golds/mm after ischemia (P < 0.05). Under basal conditions, the fraction of HK I that was mitochondrial bound was five times greater than for HK II; insulin and ischemia caused a fourfold increase in HK II binding but only a doubling in HK I binding. Oleate decreased hexokinase-mitochondrial binding and abolished insulin-mediated translocation of HK I. Our data show that mitochondrial-hexokinase binding increases under insulin or ischemic stimulation and that this translocation is modified by oleate. These events are isoform specific, suggesting that HK I and HK II are independently regulated and implying that they perform different roles in cardiac glucose regulation.     

Stabilization of hexokinases I and II of ELD cells by binding to mitochondria

Significance of the binding of hexokinase to mitochondria was examined with respect to stabilization of the enzyme by the binding. Stability during the incubation of the mitochondria-bound forms of hexokinases I and II, both prepared from Ehrlich-Lettre ascites hyperdiploid tumor cells (ELD cells), were compared with that of the corresponding free forms. During the incubation at pH 7.4 and 37 degrees C up to 60 min, hexokinase activities decreased gradually, and the decrease in the activity of the free form was much more marked than that of the bound form for both hexokinases. Hexokinase II was much less stable than I, and the activity of the free form of the former was almost lost by the incubation for 15 min. But, more than a half of the original activity of hexokinase II was retained even after 60 min of the incubation when the enzyme was bound to mitochondria. Addition of 50 mM glucose increased the stability of hexokinase II, but the stabilizing effect was less marked for hexokinase I. On the other hand, addition of 28 mg/ml of bovine serum albumin markedly stabilized hexokinase I to almost the same extent as was observed with mitochondria. On the contrary, the serum albumin had little stabilizing effect on hexokinase II. These findings indicate that the binding to mitochondria stabilizes the hexokinases of ELD cells, though the stability is different by nature between hexokinases I and II.     

The regulation of mitochondrial-bound hexokinases in the liver

A functional coupling between bound hexokinase and the inner mitochondrial compartment has been shown. It is based structurally on the binding of hexokinase to a pore protein which is present in zones of contact between the two boundary membranes. The latter was observed by electron microscopic localization of antiporin and hexokinase at the mitochondrial surface. The four isoenzymes present in liver differ considerably in their activity after binding to the mitochondrial surface. This was found by binding studies using the four isoenzymes isolated from the supernatant. Isoenzyme IV did not bind at all. Isoenzymes I-III did bind and became activated: I, 5.9-fold; II, 39-fold; and III, 1.3-fold. These results suggest that the in vivo activity of hexokinase in the mitochondrial fraction is much larger than so far observed. Furthermore the binding of isoenzymes was differently affected by metabolites. Glucose-6-phosphate exclusively desorbed isoenzyme I from the mitochondrial membrane whereas free fatty acids predominantly liberated isoenzymes II and III. A reciprocal change of the levels of free fatty acids and glucose 6-phosphate in livers of starved rats therefore, can explain why exclusively mitochondrial-bound isoenzymes II and III decreased 10-fold while at the same time isoenzyme I increased.     

Involvement of Porin N,N-dicyclohexylcarbodiimide-Reactive Domain in Hexokinase Binding to the Outer Mitochondrial Membrane

The proportion of hexokinase that is bound to the outer mitochondrial membrane is tissue specific and metabolically regulated. This study examined the role of the N,N-dicyclohexylcarbodiimide-binding domain of mitochondrial porin in binding to hexokinase I. Selective proteolytic cleavage of porin protein was performed and peptides were assayed for their, effect on hexokinase I binding to isolated mitochondria. Specificity of DCCD-reactive domain binding to hexokinase I was demonstrated by competition of the peptides for porin binding sites on hexokinase as well as by blockage hexokinase binding by N,N-dicyclohexylcarbodiimide. One of the peptides, designated as 5 kDa (the smallest of the porin peptides, which contains a DCCD-reactive site), totally blocked binding of the enzyme to the mitochondrial membrane, and significantly enhanced the release of the mitochondrially bound enzyme. These experiments demonstrate that there exists a direct and specific interaction between the DCCD-reactive domain of VDAC and hexokinase I. The peptides were further characterized with respect to their effects on certain functional properties of hexokinase I. None had any detectable effect on catalytic properties, including inhibition by glucose 6-phosphate. To evaluate further the outer mitochondrial membranes role in the hexokinase binding, insertion of VDAC was examined using isolated rat mitochondria. Pre-incubation of mitochondria with purified porin strongly increases hexokinase I binding to rat liver mitochondria. Collectively, the results imply that the high hexokinase-binding capability of porin-enriched mitochondria was due to a quantitative difference in binding sites.     

Tetrameric structure of mitochondrially bound rat brain hexokinase: a crosslinking study

Rat brain hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) was derivatized with sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopro pionate (SAND), a photosensitive and cleavable crosslinking agent. The catalytic activity and mitochondrial binding properties of the enzyme were only marginally affected by reaction with SAND. When the derivatized enzyme was bound to liver mitochondria, photolysis resulted in extensive formation of a single crosslinked species with estimated molecular mass 460 kDa. This was determined to contain only hexokinase and thus represents a tetramer of the 116 kDa (apparent molecular mass in gel system used) monomeric enzyme. Although small amounts of tetramer were detected after photolysis of relatively high concentrations of derivatized enzyme in free solution, tetramer formation was greatly enhanced when the enzyme was bound to mitochondria. No evidence of dimeric or trimeric structures was seen even when only a small fraction of the available binding sites on the mitochondrial membrane were occupied. It is thus concluded that tetramer formation is closely linked with binding of the enzyme to the outer mitochondrial membrane and, more specifically, to the pore structure through which metabolites traverse this membrane. It is speculated that a tetrameric structure surrounding the mitochondrial pores may facilitate interactions between the hexokinase reaction and oxidative phosphorylation, mediated by the adenine nucleotides which are common intermediates in these reactions.     

The localization of hexokinase isoenzymes in red and white skeletal muscles of the rat

Summary Maximum assayable hexokinase activities vary with the proportion of red, fast-twitch, oxidative-glycolytic and intermediate, slow-twitch, oxidative fibres in different rat skeletal muscles. The major isoenzymic form, type II hexokinase, is present throughout the intermyofibrillar sarcoplasm in all fibres but a proportion of the total activity appears to be weakly associated with mitochondria. Variations in the histochemical staining intensity between fibre types correlate with their mitochondrial content and seem to be due mainly to differences in mitochondrially-associated hexokinase activity. Changes in the strength of this association may be important in controlling increases in glucose metabolism in response to prolonged increased muscular activity while regulation of the equilibrium between free and loosely-bound forms may be an important control feature in all skeletal muscle. Type I hexokinase is a minor isoenzymic component of skeletal muscle and occurs mainly in blood vessels and nerves in the perimysia and endomysia. The majority of this isoenzyme is tightly bound to mitochondria and is not detectable in homogenates prepared in the absence of Triton X-100.     

Ascaris suum hexokinase: purification and possible function in compartmentation of glucose 6-phosphate in muscle.

Hexokinase (EC 2.7.1.1) is present in a soluble and a bound form in homogenates of Ascaris suum muscle. Cellulose acetate electrophoresis, isoelectric focusing, and ion exchange chromatography confirmed the presence of only one molecular form of hexokinase in this muscle. A procedure for purifying hexokinase from Ascaris muscle has been developed utilizing ion-exchange chromatography, ammonium sulfate fractionation and gel filtration. The enzyme is a monomer with a molecular weight of 100 000 as determined by sodium dodecyl sulfate gel filtration. The Stokes' radius, diffusion coefficient, and frictional ratio have been determined. The apparent Michaelis constants for glucose and ATP are 4.7-10(-3) M and 2.2-10(-4) M, respectively. Ascaris hexokinase also exhibits end-product inhibition by glucose 6-phosphate and ADP. It is postulated that the kinetic parameters of the enzyme are the results of its function, that of generating glucose 6-phosphate primarily for glycogen synthesis.     

Sugar phosphorylation modulates ADP inhibition of maize mitochondrial hexokinase

The preference of maize ( Zea mays L.) mitochondrial hexokinase (EC 2.7.1.1.) for glucose and fructose and the ADP regulation were evaluated. The Michaelis-Menten constants (K_m) varied between 0.02 and 0.09 m M for glucose and from 2 to 6 m M for fructose as substrates. The value of V_max was five times higher in the presence of glucose as compared with fructose in membrane-bound enzyme preparations. It was shown that ADP produced from the reaction inhibits the hexokinase activity (K_i=20–50 μ M ). However, the inhibition was very specific for adenine nucleotide. Only a small inhibition was observed when 1 m M of UDP, CDP or GDP was included in the assay medium. Nevertheless, the ADP inhibition was observed only when glucose was phosphorylated. In assay conditions where fructose serves as substrate, the affinity for ADP decreased by 10-fold (K_i varied between 500 and 1  000 μ M ). These kinetics properties were also observed in partially purified soluble enzyme preparations. These data suggest that the type of hexose bound to the catalytic site modulates the ADP control of maize mitochondrial hexokinase.     

EFFECT OF FATTY ACIDS ON RAT BRAIN PARTICULATE HEXOKINASE

Abstract— The effect of free fatty acids on rat brain particulate hexokinase was studied in vitro. Hexokinase bound with brain mitochondrial fraction was found to be sensitive to the action of free fatty acids, resulting in the solubilization of at least part of bound enzyme activity into the supernatant. The decrease of total enzyme activity observed at the highest free fatty acid concentration was probably due to the inhibition of hexokinase. The physiological consequence of hexokinase solubilization by low concentrations of free fatty acids, similar to that observed in vivo , is discussed in relation to activity changes of soluble and particulate enzyme forms demonstrated previously under hypoxic conditions.     

Adsorption mechanism of hexokinase activity regulation in rat sarcoma M-1

The hexokinase interaction with mitochondrial membranes in rat sarcoma M-1 cells was studied. The conditions of formation of the enzyme complex with mitochondrial membranes and its stability were elaborated. The kinetic parameters of free and membrane-bound hexokinases were determined. The data obtained are discussed within the frame-work of the adsorption mechanism of hexokinase activity regulation in neoplastic tissues.     

Polyamines stimulate the binding of hexokinase type II to mitochondria

Spermine and spermidine enhanced the binding of hexokinase isoenzyme type II to mitochondria, both of which were prepared from Ehrlich-Lettre hyperdiploid ascites tumor cells, at much lower concentrations than Mg2+. Chymotrypsin-treated hexokinase II could not bind to the mitochondrial membrane in the presence of either spermine or Mg2+, indicating that the effect of spermine is not a nonspecific action, since the treatment of chymotrypsin cleaves only the region essential for the binding without any significant effect of the catalytic activity. Both spermine and Mg2+ antagonized the glucose 6-phosphate-induced release of mitochondria-bound hexokinase, and promoted the binding of the solubilized hexokinase II even in the presence of glucose 6-phosphate. However, inhibition of the activity of soluble hexokinase by glucose 6-phosphate was not reversed by spermine and Mg2+. Hexokinase II rebound to mitochondria with spermine and Mg2+ produced glucose 6-phosphate using ATP generated inside the mitochondria, and no difference was observed between the spermine- and Mg2+-rebound systems. Significance of the binding of hexokinase to mitochondria, especially with polyamines, is discussed with reference to high glycolytic rate in tumor cells.     

Time-dependent 31P saturation transfer in the phosphoglucomutase reaction. Characterization of the spin system for the Cd(II) enzyme and evaluation of rate constants for the transfer process

Time-dependent 31P saturation-transfer studies were conducted with the Cd2+-activated form of muscle phosphoglucomutase to probe the origin of the 100-fold difference between its catalytic efficiency (in terms of kcat) and that of the more efficient Mg2+-activated enzyme. The present paper describes the equilibrium mixture of phosphoglucomutase and its substrate/product pair when the concentration of the Cd2+ enzyme approaches that of the substrate and how the nine-spin 31P NMR system provided by this mixture was treated. It shows that the presence of abortive complexes is not a significant factor in the reduced activity of the Cd2+ enzyme since the complex of the dephosphoenzyme and glucose 1,6-bisphosphate, which accounts for a large majority of the enzyme present at equilibrium, is catalytically competent. It also shows that rate constants for saturation transfer obtained at three different ratios of enzyme to free substrate are mutually compatible. These constants, which were measured at chemical equilibrium, can be used to provide a quantitative kinetic rationale for the reduced steady-state activity elicited by Cd2+ relative to Mg2+ [cf. Ray, W.J., Post, C.B., & Puvathingal, J.M. (1989) Biochemistry (following paper in this issue)]. They also provide minimal estimates of 350 and 150 s-1 for the rate constants describing (PO3-) transfer from the Cd2+ phosphoenzyme to the 6-position of bound glucose 1-phosphate and to the 1-position of bound glucose 6-phosphate, respectively. These minimal estimates are compared with analogous estimates for the Mg2+ and Li+ forms of the enzyme in the accompanying paper.     

Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme

A glucose analog, N-(bromoacetyl)-D-glucosamine (GlcNBrAc), previously used to label the glucose binding sites of rat muscle Type II and bovine brain Type I hexokinases, also inactivates rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) with pseudo-first-order kinetics. Inactivation occurs predominantly via a "specific" pathway involving formation of a complex between hexokinase and GlcNBrAc, but significant nonspecific (i.e., without prior complex formation) inactivation also occurs, and equations to describe this behavior are derived. Inactivation is dependent on deprotonation of a residue with an alkaline pKa, consistent with the modified residue being a sulfhydryl group as reported to be the case with the hexokinase of bovine brain. The affinity label modifies three residues (per molecule of enzyme) at indistinguishable rates, but only one of these residues appears to be critical for activity. Amino acid analysis of the modified enzyme indicates derivatization of three cysteine residues; there was no indication of modification of other residues potentially reactive with haloacetyl derivatives. Kinetic analysis and effects of protective ligands were consistent with location of the critical sulfhydryl at the glucose binding site. Peptide mapping techniques permitted localization of the critical residue, and thus the glucose binding site, in a 40-kDa domain at the C-terminus of the enzyme. This is the same domain recently shown to include the ATP binding site. Thus, catalytic function is assigned to the C-terminal domain of rat brain hexokinase.     

Hexokinase isoenzymes in diabetes

Hexokinase is present in the tissues in four isoenzymic forms. Cerebral tissue contains predominantly Type I hexokinase which is believed to be insulin-insensitive. In cerebral tissue about 60 to 70% of the hexokinase is bound to the particulate fraction. The changes in the distribution of hexokinase Type I and Type II together with the bound and free hexokinase have been studied in control, diabetic and diabetic animals treated with insulin. The results indicate that the presence of insulin is essential for the normal binding of the hexokinase to the particulate fraction. In heart tissue, Type II hexokinase bound to the pellet shows a significant decrease in diabetes, which is reversed on insulin administration.