Homologous and Heterologous Interactions between Hexokinase and Mitochondrial Porin: Evolutionary Implications

Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane. Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S. cerevisiae. Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast. Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins. If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase–mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction. Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria. There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined.     

Difference in hydrophobicity between mitochondria-bindable and non-bindable forms of hexokinase purified from rat brain

Hexokinase able to bind to mitochondria was purified to homogeneity from rat brain by two successive DEAE-cellulose chromatographic steps. The enzyme lost only the binding ability with almost undetectable change in molecular weight on mild chymotrypsin digestion. The bindable hexokinase was adsorbed to a Phenyl-Sepharose column and eluted with a Lubrol PX gradient, whereas non-bindable hexokinase and yeast hexokinase were not adsorbed under the similar conditions. These results suggest that mitochondria-bindable hexokinase has a hydrophobic region on its surface, which is responsible for the specific interaction with mitochondria.     

Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Two types of binding sites for hexokinase, designated as Type A or Type B sites, have been shown to coexist on brain mitochondria. The ratio of these sites varies between species.HK1 attaches by reversibly binding to the voltage dependent anion channel (VDAC). Regarding the nature of hexokinase binding sites, we investigated if it was linked to distinct VDAC interactomes. We approached this question by 2D BN/SDS-PAGE of mitochondria, followed by mass spectrometry.Our results are consistent with the possibility that the ratio of Type A/Type B sites is due to differential VDAC interactions in bovine and rat neuronal cells.     

An intact hydrophobic N-terminal sequence is critical for binding of rat brain hexokinase to mitochondria

The N-terminal sequence of rat brain hexokinase (ATP: D-hexose-6-phosphotransferase, EC 2.7.1.1) has been determined to be X-NH-Met-Ile-(Ala, Gln)-Ala-Leu-Leu-Ala-Tyr-, where X is a blocking group on the N-terminal methionine, probably an N-acetyl group. Modification of this hydrophobic N-terminal segment by endogenous proteases in crude brain extracts resulted in loss of the ability to bind to mitochondria, but had no effect on catalytic activity, resulting in the appearance of nonbindable enzyme reported by several previous investigators to be present in purified hexokinase preparations. Similar results can be obtained by deliberate limited digestion with chymotrypsin (cleavage points marked by arrows in sequence above). Both bindable and nonbindable enzyme, the latter generated either by endogenous proteases or with chymotrypsin, have an identical C-terminal dipeptide sequence, Ile-Ala. The great susceptibility of the N-terminus to proteolysis plus the marked effect that its proteolytic modification has on binding of hexokinase to anion exchange or hydrophobic (phenyl-Sepharose) matrices suggest that this N-terminal segment is prominently displayed at the enzyme surface. Epitopes recognized by two monoclonal antibodies which block binding of hexokinase to mitochondria (but have no effect on catalytic activity) have been mapped to a 10K fragment cleaved from the N-terminus by limited tryptic digestion. Thus the binding of hexokinase to mitochondria appears to occur via a "binding domain" constituting the N-terminal region of the molecule, with maintenance of an intact hydrophobic sequence at the extreme N-terminus being critical to this interaction. A resulting specific orientation of the molecule on the mitochondrial surface is considered to be a prerequisite for the observed coupling of hexokinase activity and mitochondrial oxidative phosphorylation.     

Binding of Rat Brain Hexokinase to Recombinant Yeast Mitochondria: Identification of Necessary Molecular Determinants

The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wildtype, porinless, or expressing recombinant human porin) was studied in an effort to identifyminimal requirements for each component. A short hydrophobic N-terminal peptide ofhexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria.Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loopsaround residues 110 and 200, as determined by proteolytic-fragment identification usingantibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysisthan wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboringseveral natural or engineered porins from various sources, bind hexokinase to variable extentwith marked preference for the mammalian porin1 isoform. Genetic alteration of this isoformat the C-, but not the N-terminal, results in a significant reduction of hexokinase bindingability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme toall recombinant mitochondria, as well as to proteolytically digested organelles. Consequently,brain hexokinase association with heterologous mitochondria (yeast) in these conditions occursto an extent comparable to that with homologous (rat) mitochondria. The study, also pertinentto the topology and organization of porin in the membrane, represents a necessary first stepin the functional investigation of the physiological role of mammalian hexokinase binding tomitochondria in reconstituted heterologous recombinant systems, as models to cellularmetabolism.     

Further studies on the role of phospholipids in determining the characteristics of mitochondrial binding sites for type I hexokinase

Previous work has indicated that two types (A and B) of binding sites for hexokinase exist, but in different proportions, on brain mitochondria from various species. Hexokinase is readily solubilized from Type A sites by glucose 6-phosphate (Glc-6-P), while hexokinase bound to Type B sites remains bound even in the presence of Glc-6-P. Type A:Type B ratios are approximately 90:10, 60:40, 40:60, and 20:80 for brain mitochondria from rat, rabbit, bovine and human brain, respectively. The present study has indicated that MgCl2-dependent partitioning of mitochondrially bound hexokinase into a hydrophobic (Triton X-114) phase is generally correlated with the proportion of Type B sites. This partitioning behavior is sensitive to phospholipase C, implying that the factor(s) responsible for conferring hydrophobic character is(are) phospholipid(s). Substantial differences were also seen in the resistance of hexokinase, bound to brain mitochondria from various species, to solubilization by Triton X-100, Triton X-114, or digitonin. This resistance increased with proportion of Type B sites. Enrichment of bovine brain mitochondria in acidic phospholipids (phosphatidylserine or phosphatidylinositol), but not phosphatidylcholine or phosphatidylethanolamine, substantially increased solubilization of the enzyme after incubation at 37 degrees C. Collectively, the results imply that the Type A and Type B sites are located in membrane domains of different lipid composition, the Type A sites being in domains enriched in acidic phospholipids which lead to greater susceptibility to solubilisation by Glc-6-P.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.     

Purification of highly bindable rat brain hexokinase by High Performance Liquid Chromatography (HPLC)

Rat brain hexokinase has been purified by a modification of a previously described procedure in which High Performance Liquid Chromatography (HPLC) on an anion exchange column is substituted for DEAE-cellulose column chromatography. The resulting enzyme is obtained in good yield and is nearly homogeneous based on SDS-gel electrophoresis; the specific activity (about 60 units/mg protein) is comparable to the DEAE-purified enzyme. In contrast to the latter enzyme, however, the HPLC-purified enzyme retains its ability to bind to mitochondria. Excellent resolution of bindable and nonbindable forms of rat brain hexokinase is achieved with HPLC.     

All three isoforms of the voltage-dependent anion channel (VDAC1, VDAC2, and VDAC3) are present in mitochondria from bovine, rabbit, and rat brain

All three isoforms of the voltage-dependent anion channel (VDAC) were detected by immunoblot analysis of mitochondria isolated from rat, rabbit, and bovine brain. All three isoforms were associated with mitochondria after fractionation of rat brain extracts on sucrose density gradients. No VDAC isoforms were detected in non-mitochondrial fractions. Relative levels of the mRNAs coding the VDAC isoforms in rat, rabbit, and bovine brain were determined by RT-PCR. In all three species, the mRNA for VDAC2 was predominant. Relative to the mRNA for VDAC3, mRNAs for both VDAC1 and VDAC2 were more highly expressed in bovine brain than in rat brain. These results are consistent with the possibility that differences in relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of Type A:Type B hexokinase binding sites on brain mitochondria.     

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.     

Magnetic resonance studies on interaction of bovine brain hexokinase with manganous ion, glucose, and glucose 6-phosphate

Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, K_D = 25 ± 4 μM and K′_D = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ?_b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ?_b = 3.5 ± 0.4 for tight binding sites and an average ?_b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s^?1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.     

Intracellular distribution of hexokinase in rabbit brain

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence that this particulate fraction also contains endoplasmic vesicles and synaptosomes. Solubilization of the bound hexokinase by different combinations of detergents and metabolites has proved the existence of different hexokinase binding sites. Electron microscopic examination of hexokinase location by immuno-gold labelling techniques confirmed, that hexokinase is indeed predominantly bound to mitochondria but that a significant proportion is also bound to non-mitochondrial membranes. Attempts to quantify this distribution were unsuccessful since different figures were obtained using anti-hexokinase IgG affinity purified on immobilized native or denatured hexokinase. Binding studies of the purified rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitochondria there is another binding site on microsomes. The N-terminal sequence of hexokinase has been shown to be important for mitochondria binding and also for microsome binding. These results suggest that the intracellular localization of hexokinase in rabbit brain is not exclusively mitochondrial and that the metabolic role of this enzyme should be reconsidered by including a binding site on the endoplasmic reticulum.     

Distribution and regulation of mitochondrial hexokinase in rat submandibular gland

1. In rat submandibular gland, hexokinase was distributed not only in cytosol fraction but also in mitochondrial fraction. 2. Glucose-6-phosphate and ATP were most effective substances on releasing hexokinase from mitochondria. However, all the hexokinase in mitochondria could not be extracted with these substances. 3. Concentrations of glucose-6-phosphate and ATP were decreased with the administration of epinephrine in vivo. 4. Increase of the amount of mitochondria-bound hexokinase was observed for 5 min with epinephrine administration, and it returned to the control level after 10 min. 5. In rat submandibular gland, mitochondrial hexokinase may reversibly bind to and release from mitochondria as observed in brain.     

Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary physico-chemical determinants.

The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild-type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.     

The functional compartmentation of mitochondrial hexokinase

These studies examined the functional relationship between rat hepatic mitochondria and associated hexokinase (ATP: d-hexose-6-phosphotransferase, 2.7.1.1) to determine whether the binding of hexokinase to mitochondria might provide a privileged interaction with sites of ATP production.Initial kinetic analysis followed the sequential flow of phosphate through ATP generated by the mitochondria into glucose-6-phosphate catalyzed by the bound hexokinase. Kinetics were compared with an identical bound hexokinase-mitochondrial system using externally supplied ATP. The hexokinase had lower apparent K_m values for ATP generated in the mitochondria from supplied ADP than for ATP provided. Respiratory inhibitors blocked both the ADP- and ATP-mediated reactions. Tracer studies further documented that the mitochondrial hexokinase initially and preferentially utilized the internally generated nucleotide.These studies demonstrate that the active site of bound hexokinase is relatively inaccessible to extramitochondrial ATP. They provide evidence that bound hexokinase can sequentially accept mitochondrially generated ATP in a kinetically advantageous way. Finally, they support the assumption that mitochondrial binding of this acceptor enzyme may play a propitious role in cellular energy economy.     

Development of mitochondrial energy metabolism in rat brain

1. The development of pyruvate dehydrogenase and citrate synthase activity in rat brain mitochondria was studied. Whereas the citrate synthase activity starts to increase at about 8 days after birth, that of pyruvate dehydrogenase starts to increase at about 15 days. Measurements of the active proportion of pyruvate dehydrogenase during development were also made. 2. The ability of rat brain mitochondria to oxidize pyruvate follows a similar developmental pattern to that of the pyruvate dehydrogenase. However, the ability to oxidize 3-hydroxybutyrate shows a different developmental pattern (maximal at 20 days and declining by half in the adult), which is compatible with the developmental pattern of the ketone-body-utilizing enzymes. 3. The developmental pattern of both the soluble and the mitochondrially bound hexokinase of rat brain was studied. The total brain hexokinase activity increases markedly at about 15 days, which is mainly due to an increase in activity of the mitochondrially bound form, and reaches the adult situation (approx. 70% being mitochondrial) at about 30 days after birth. 4. The release of the mitochondrially bound hexokinase under different conditions by glucose 6-phosphate was studied. There was insignificant release of the bound hexokinase in media containing high KCl concentrations by glucose 6-phosphate, but in sucrose media half-maximal release of hexokinase was achieved by 70μm-glucose 6-phosphate 5. The production of glucose 6-phosphate by brain mitochondria in the presence of Mg²⁺+glucose was demonstrated, together with the inhibition of this by atractyloside. 6. The results are discussed with respect to the possible biological significance of the similar developmental patterns of pyruvate dehydrogenase and the mitochondrially bound kinases, particularly hexokinase, in the brain. It is suggested that this association may be a mechanism for maintaining an efficient and active aerobic glycolysis which is necessary for full neural expression.     

Glucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase

In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K.K., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 17422-17428; Parry, D.M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). In extending these studies, we have isolated a cDNA clone of hexokinase from a lambda gt11 library of the highly glycolytic, c37 mouse hepatoma cell line. This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons. This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes. The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme. At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in the rat liver glucokinase, a cytoplasmic enzyme. The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold. The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2. This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E. coli. Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed.     

Hexose metabolism in pancreatic islets: regulation of mitochondrial hexokinase binding

A major fraction of hexokinase was found to be bound, presumably to mitochondria, in both normal and tumoral rat pancreatic islet cells examined after either mechanical disruption or digitonin treatment. Spermidine enhanced the binding and glucose 6-phosphate caused the release of hexokinase to and from islet mitochondria, in a manner comparable to that seen in parotid or brain homogenates. In hepatocytes, some hexokinase, but no glucokinase, was found in the bound form. In islet cells, however, the pattern of glucokinase binding was similar to that of hexokinase. It is speculated that the preferential location of both hexokinase and glucokinase on mitochondria may favor the maintenance of a high cytosolic ATP content in islet cells.     

The Identity of Hexokinase Activities from Mitochondrial and Cytoplasmic Fractions of Rat Brain Homogenates

Cytoplasmic hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from the soluble fraction of a rat brain homogenate by a procedure that included a unique affinity elution of the enzyme from Blue Dextran-Sepharose. The purified enzyme was examined with respect to properties in which the impure cytoplasmic enzyme has been reported to differ from the solubilized mitochondrial enzyme. These included the ability to bind to mitochondria, inhibition by quercetin, effect of pH on activity, and kinetics. In all regards the purified mitochondrial and cytoplasmic enzymes appeared identical. In addition, comparative peptide maps after partial proteolysis showed no detectable differences. These results do not support the view that there exist distinct mitochondrial and cytoplasmic forms of hexokinase, the latter being permanently relegated to a cytoplasmic location and unable to participate in a dynamic equilibrium with the mitochondrially-bound enzyme. Alternatives are proposed to explain previous results that had been interpreted as indirect evidence for the existence of a distinct cytoplasmic hexokinase.     

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.