Rat Brain Hexokinase Research Articles

Interaction of hexokinase with the outer mitochondrial membrane and a hydrophobic matrix.

The major portion of rat brain hexokinase (HK type 1) is bound to the outer membrane of mitochondria and glucose-6-phosphate (G6P) can release the bound enzyme. In an attempt to look at the 'hydrophobic' component of binding, interaction of the enzyme with a purely hydrophobic matrix, palmityl-substituted Sepharose-4B (Sepharose-lipid) was investigated. Hexokinase readily bound to this matrix with retention of its catalytic activity. Glucose-6-phosphate which has a releasing effect on the mitochondrially bound enzyme, enhanced binding of the enzyme on the hydrophobic matrix. Chymotrypsin treatment of hexokinase which causes loss of binding to mitochondria, also results in loss of adsorption to the hydrophobic matrix, thus demonstrating that the 'hydrophobic tail' present at its N-terminal end is essential for binding in both cases. Data presented provide some new information relevant to understanding how hexokinase interacts with its natural binding matrix, the mitochondrion.

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

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

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.

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 (wild type, porinless, or expressing recombinant human porin) was studied in an effort to identify minimal requirements for each component. A short hydrophobic N-terminal peptide of hexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria. Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loops around residues 110 and 200, as determined by proteolytic-fragment identification using antibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysis than wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboring several natural or engineered porins from various sources, bind hexokinase to variable extent with marked preference for the mammalian porin1 isoform. Genetic alteration of this isoform at the C-, but not the N-terminal, results in a significant reduction of hexokinase binding ability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme to all recombinant mitochondria, as well as to proteolytically digested organelles. Consequently, brain hexokinase association with heterologous mitochondria (yeast) in these conditions occurs to an extent comparable to that with homologous (rat) mitochondria. The study, also pertinent to the topology and organization of porin in the membrane, represents a necessary first step in the functional investigation of the physiological role of mammalian hexokinase binding to mitochondria in reconstituted heterologous recombinant systems, as models to cellular metabolism.

Binding of rat brain hexokinase to recombinant yeast mitochondria: effect of environmental factors and the source of porin.

Heterologous binding of rat brain hexokinase to wild type, porinless, and recombinant yeast mitochondria expressing human porin was assessed, partially characterized, and compared to that in the homologous system (rat liver mitochondria). With porin-containing yeast mitochondria it is shown that (i) a significant, saturable association occurs; (ii) its extent and apparent affinity, correlated with the origin of porin, are enhanced in the presence of dextran; (iii) the binding requires Mg ions and apparently follows a complex cooperative mechanism. This heterologous association does not seem to differ fundamentally from that in the homologous system and represents a good basis for molecular studies in yeast. With porinless yeast mitochondria, binding occurs at much lower affinity, but to many more sites per mitochondrion. The results indicating a major but not exclusive role for porin in the binding are discussed in terms of (i) the mode and mechanism of binding, and (ii) the suitability of the rat hexokinase-yeast mitochondria couple for the study of heterogeneous catalysis in reconstituted cellular model systems.

Purification and Properties of Buffalo (Bubalus bubalis) Erythrocyte Hexokinase

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 ± 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purification of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contaminants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (Km = 0.012 ± 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher Km values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates.

Hexokinase present in human sperm is not tyrosine phosphorylated but its antibodies affect fertilizing capacity.

The present study was conducted to investigate the presence of hexokinase in human sperm cell, its subcellular localization, its modulation and role in capacitation/acrosome reaction, and tyrosine kinase activity, if any. These studies were conducted using antibodies (Ab) raised against rat brain hexokinase (type I isozyme) that have significant cross-reaction with various human tissue hexokinases and neutralize the catalytic activity of the enzyme. Hexokinase Ab reacted with acrosomal, mid-piece and tail regions of methanol-fixed (approximately 70-80%) and live (approximately 35-52%) sperm in the indirect immunofluorescence technique (IFT) and the immunobead binding technique (IBT), respectively. Hexokinase Ab specifically recognized a band of 116 kDa on the Western blot of detergent-solubilized human sperm preparation that was different from the 95-kDa phosphotyrosine protein. Hexokinase Ab caused a significant (P < 0.01 to < 0.001) and concentration-dependent inhibition of human sperm penetration of zona-free hamster oocytes in the sperm penetration assay (SPA). These data indicate that the hexokinase of 116-kDa molecular weight is present in acrosomal, mid-piece, and tail regions of human sperm. The sperm hexokinase is a glycoprotein that is different from the 95-kDa phosphotyrosine protein and is not phosphorylated at tyrosine residues; however, its antibodies cause agglutination and a concentration-dependent inhibition of fertilizing capacity of human sperm.

Application of a Double Isotopic Labeling Method to a Study of the Interaction of Mitochondrially Bound Rat Brain Hexokinase with Intramitochondrial Compartments of ATP Generated by Oxidative Phosphorylation

γ-Labeled ATP was produced by rat brain mitochondria utilizing [32P]Pias substrate for oxidative phosphorylation. The32P/14C ratio of Glc-6-P produced by the endogenous mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) using [U-14C]Glc as substrate was determined as a function of time after initiation of oxidative phosphorylation. This same ratio was determined for Glc-6-P formed by added yeast hexokinase using extramitochondrial ATP as substrate. The specific activity of ATP formed by oxidative phosphorylation was manipulated either by initiating the reaction with labeledPiand subsequently adding excess unlabeledPior by initiating the reaction with unlabeledPiand introducing the labeled substrate at a later time. The32P/14C ratio of Glc-6-P formed by yeast hexokinase, reflecting the specific activity of ATP in the extramitochondrial space, was rapidly responsive to such manipulations, but the corresponding changes in the32P/14C ratio of Glc-6-P produced by the endogenous hexokinase were markedly different. The results are consistent with the view that mitochondrially bound hexokinase does not utilize extramitochondrial ATP as substrate but rather is functionally coupled to a discrete intramitochondrial compartment of ATP produced by oxidative phosphorylation.

Schistosoma mansoni Hexokinase: CDNA Cloning and Immunogenicity Studies

DNA encoding a Schistosoma mansoni hexokinase (SHEX) was amplified from cDNA by the polymerase chain reaction using opposing oligonucleotide primers designed to hybridize with two short segments of hexokinase coding sequences that an well-conserved through evolution. The resulting DNA fragment was then used as a probe to identify a full-length hexokinase cDNA clone. SHEX cDNA encodes a 50-kDa protein that is approximately 46% homologous to rat hexokinase, 40% to rat glucokinase, and 34% to yeast hexokinase A. SHEX coding DNA was expressed within Escherichia coli cells and the 50-kDa recombinant product (rSHEX) was partially purified. Mice repeatedly immunized with rSHEX produced antibodies which recognize rSHEX but this offered no significant protection against subsequent cercarial challenge. On Western blots, rSHEX is weakly recognized by antisera against rat brain hexokinase but not by sera from three strains of mice experimentally infected with S. mansoni parasites or from numerous human schistosomiasis patients. Thus, unlike other reported S. mansoni glycolytic enzymes, hexokinase appears to be poorly immunogenic during schistosome infection and of limited potential as a vaccine candidate.

P95, the major phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with unique properties.

Mouse sperm contain a major phosphotyrosine-containing protein of M(r) 95,000 (nonreducing conditions) which has been implicated as a sperm membrane receptor for the egg zona pellucida glycoprotein, ZP3 (Leyton, L., and Saling, P. (1989) Cell 57, 1123-1130; Leyton, L., LeGuen, P., Bunch, D., and Saling, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11692-11695). This protein was purified and subjected to limited tryptic digestion and subsequent amino acid analysis. Three sequenced peptides revealed 100% amino acid identity to a mouse hepatoma hexokinase (Arora, K. K., Fanciulli, M., and Pederson, P. L. (1990) J. Biol. Chem. 265, 6481-6488). The purified protein, which migrated at M(r) 116,000 under reducing conditions (p95/116), reacted with an antiserum to the purified rat brain hexokinase, type 1, and comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the purified rat brain enzyme under both nonreducing and reducing conditions. Unlike p95/116, the rat brain enzyme was not a phosphotyrosine-containing protein. The p95/116 protein could be immunoprecipitated with the hexokinase antiserum or an O-phosphotyrosine antibody. Limited tryptic digestion of the purified p95/116 and the rat brain enzyme generated subsets of identical peptides which reacted with the hexokinase antiserum. However, p95/116 also contained phosphotyrosine-containing peptides that were not present in the rat brain hexokinase. When different mouse tissues were probed with the hexokinase antiserum all tissues, with the exception of liver, contained immunoreactive protein. In contrast, only sperm and testis possessed a phosphotyrosine-containing form of hexokinase. These data suggest that the germ cell component of the testis possesses a unique tyrosine-phosphorylated form of hexokinase.

Detection of a progression‐linked DNA restriction fragment in rat hepatoma cells probed with a hexokinase CDNA

We have compared genomic DNA isolated from a series of rat hepatomas and from normal rat liver tissue by restriction fragment analysis. Our objective was to identify a tumor-specific restriction fragment that would be present in rapidly growing tumors and missing in normal tissues. Using a cDNA probe specific for the C-terminal half of rat brain hexokinase, we have identified several changes in DNA restriction fragment lengths in rat hepatomas vs. normal rat liver. Most of these involved restriction fragments that were present in normal rat DNA and missing in tumor DNA. However, one HinfI fragment of M(r) = 13.4 kilobase pairs (kb) was found to be present in rapidly growing rat hepatomas, but missing in normal rat tissue and in hepatomas of slow or intermediate growth rate.

Interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation and creatine kinase

Previous work led to the conclusion that, during oxidative phosphorylation, mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from rat brain was dependent on intramitochondrially compartmented ATP as substrate. The present study demonstrated that, when oxidative phosphorylation was functioning concurrently, mitochondrial creatine kinase could also generate intramitochondrial ATP serving as substrate for hexokinase. In the absence of concurrent oxidative phosphorylation, the kinetics of glucose phosphorylation with ATP generated by creatine kinase were not consistent with the supply of ATP from a saturable intramitochondrial compartment as formed during oxidative phosphorylation. Evidence for intramitochondrially compartmented ATP, generated by creatine kinase, was obtained; this was distinct from compartmented ATP generated by oxidative phosphorylation in terms of kinetics of generation of the compartment and its capacity, sensitivity to release by carboxyatractyloside, and sensitivity to disruption by digitonin. That oxidative phosphorylation did induce a dependence on intramitochondrial ATP as a substrate was further indicated by the observation that, although the initial rate of glucose phosphorylation by mitochondrial hexokinase depended on the extramitochondrial concentration of ATP present at the time oxidative phosphorylation was initiated, a final steady state rate of glucose phosphorylation was attained that was independent of extramitochondrial ATP levels. These and previous results emphasize the probable importance of nucleotide compartmentation in regulation of cerebral glycolytic and oxidative metabolism.

Epitopic regions recognized by monoclonal antibodies against rat brain hexokinase: Association with catalytic and regulatory function

Using direct and competitive epitope mapping methods, 23 monoclonal antibodies (Mabs) against rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) were divided into nine groups, each recognizing epitopes within defined surface regions of the N- or C-terminal domains; the latter have been associated with regulatory or catalytic functions, respectively. Reactivity of Mabs with the isolated domains was also studied. Based on the effect of various ligands on immunoreactivity, specific regions involved in ligand-induced conformational changes were identified. Adjacent epitopic regions, designated Regions F and G and located in the N- and C-terminal domains, respectively, were selectively affected by inhibitory hexose 6-phosphates (or analogs), marking these regions as being involved in transmission of the conformational signal from the regulatory N-terminal domain to the catalytic C-terminal domain. Consistent with this, the K i for inhibition of the enzyme by the glucose 6-phosphate analog, 1,5-anhydroglucitol-6-phosphate, was markedly increased by Mabs binding in these regions, but unaffected by Mabs binding elsewhere in the molecule. Reactivity with Mabs recognizing conformationally sensitive epitopes in Region H of the C-terminal domain was greatly decreased by binding of substrate hexoses that induce closure of a cleft in the catalytic domain; selective recognition of the “open cleft” conformation, thereby preventing closure of the cleft required for progression of the catalytic cycle, can account for the marked decrease in V max that results from binding of these Mabs. Reactivity with Mabs binding to Region H was also decreased in the presence of inhibitory hexose 6-phosphates, implying that cleft closure was also induced by the latter; this is consistent with the suggestion that limitation of access to the C-terminal ATP binding site, resulting from cleft closure, is a factor in inhibition of the enzyme.

Effect of ligand binding on the tryptic digestion pattern of rat brain hexokinase: Relationship of ligand-induced conformational changes to catalytic and regulatory functions

The Type I isozyme of rat hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) is comprised of N- and C-terminal domains, associated with regulatory and catalytic functions, respectively. Extensive sequence similarity between the domains is consistent with evolution of the enzyme by gene duplication and fusion. Cleavage at tryptic sites located in the C-terminal domain is markedly sensitive to ligands present during digestion, while analogous sites in the N-terminal domain are either resistant to trypsin or unaffected by the presence of ligands. These results imply a lack of structural equivalence between the N- and C-terminal domains, with the overall structure of the N-terminal domain being “tighter” and with a major component of ligand-induced conformational changes being focused in the C-terminal domain. Based on a previously proposed structure for brain hexokinase, protection by substrate hexoses is attributed to substrate-induced closing of a cleft in the C-terminal domain. Similar protection at C-terminal cleavage sites results from binding of inhibitory hexose-6-phosphates to the N-terminal domain. In addition, hexose-6-phosphates evoke cleavage at a site, T 5, located in a region that has been associated with binding of ATP to the C-terminal domain. Thus, alterations in this region, coupled with reduced accessibility resulting from cleft closure, may account for the mutually exclusive binding of inhibitory hexose-6-phosphates and substrate ATP. In the absence of Mg 2+, all nucleoside triphosphates examined (ATP, UTP, CTP, and GTP) protected against digestion by trypsin. In contrast, ATP-Mg 2+ stabilized the C-terminal domain but destabilized the N-terminal domain, while the chelated forms of the other nucleoside triphosphates were similar to the unchelated forms in their effect on proteolysis; the unique response to ATP-Mg 2+ reflects the specificity for ATP as a substrate.

Disposition of mitochondrially bound hexokinase at the membrane surface, deduced from reactivity with monoclonal antibodies recognizing epitopes of defined location

A panel of monoclonal antibodies against rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) has been employed to investigate the orientation of the mitochondrially bound enzyme on the mitochondrial surface. Based on their ability to immunoprecipitate truncated forms of the protein, obtained by in vitro translation of truncated versions of the mRNA, the epitopes for seven monoclonal antibodies were mapped to regions consisting of 20–50 amino acid residues within the sequence of the N-terminal half of the enzyme. There is extensive sequence similarity between the N- and C-terminal halves of this enzyme, which is thought to have evolved by a process of gene duplication and fusion. However, these antibodies react selectively with epitopes in the N-terminal half, and thus epitopic regions for several of these antibodies could be further defined by eliminating from consideration regions showing substantial sequence similarity with the C-terminal half. The epitope for one of the monoclonal antibodies, designated 4D4, was shown to involve the extreme N-terminus of the enzyme; selective proteolytic modification of this region resulted in loss of immunoreactivity. Relative location of epitopes for three other antibodies, designated 2B, 1C5, and 4C5, within a 20-residue segment was deduced from effects of modifying sulfhydryl residues within this segment on immunoreactivity. Thus, by a combination of sequence analysis and experimental methods, the epitopes for these seven antibodies could be localized to defined regions within the overall sequence. The ability of these antibodies to prevent binding of hexokinase to mitochondria, and their ability to recognize the mitochondrially bound enzyme, provided a basis for assessing the relative proximity of the corresponding epitopes to the mitochondrial surface when the enzyme was bound. The disposition of the bound enzyme on the mitochondrial surface was deduced by relating these results to the proposed structure for brain hexokinase.

Protein kinase activity of rat brain hexokinase

Hexokinase 1 (HK1) purified from rat brain exhibits protein kinase activity, including autophosphorylation and phosphorylation of histone H2A. This protein kinase activity is observed only in the absence of the HK1 carbohydrate substrate, glucose. Analysis of the ATP-binding domains of the mammalian HK1 protein sequences shows significant homology with other mammalian protein kinases.

Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase

Rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) or P i. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P or P i, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog, N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.

Differential scanning calorimetric study of rat brain hexokinase: Domain structure and stability

Rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) has been studied by differential scanning calorimetry. In “high-ionic-strength” buffer (50 m m Tris-Cl, 0.5 m m EDTA, 10 m m monothioglycerol, pH 8.5), and assuming two-state behavior with calorimetric enthalpy equal to van't Hoff enthalpy, the endotherm could be deconvoluted into two transitions with T m values of about 48 and 51 °C and enthalpies of about 109 and 112 kcal/mol, respectively. A similar endotherm was seen when glucose or glucose 6-phosphate was present, except that T m values for both transitions were increased. The glucose analog, N-acetylglucosamine, had no observable effect on the endotherm, which is in agreement with previous studies indicating that this ligand, unlike glucose and glucose 6-phosphate, does not induce conformational changes that lead to increased stability of the enzyme. In “low-ionic-strength” buffer (5 m m Tris-Cl, 0.5 m m EDTA, 10 m m monothioglycerol, pH 8.5), the transitions were partially resolved even in the absence of ligands, with T m values of about 49 and 55 °C. Due to difficulties with erratic baseline behavior under the low-ionic-strength conditions, enthalpies were not routinely determined, but these appeared to be similar to those seen in high-ionic-strength buffer. Also similar was the increase in stability, as reflected by the increase in T m for both transitions, when glucose or glucose 6-phosphate was present. Correlation of these transitions with specific regions of the molecule was established by analysis of enzyme in which the domain corresponding to the first transition was selectively denatured by a partial scan in the calorimeter. Subsequent rescanning of these samples showed only the second transition, confirming the selective denaturation of the domain corresponding to the first transition and retention of the folded structure of the second domain. Discrimination between denatured (first transition) and undenatured (second transition) domains was based on the markedly increased susceptibility of the denatured region to tryptic digestion; regions of the molecule that retained their folded structure and resistance to proteolysis were identified by immunoblotting techniques using monoclonal antibodies recognizing epitopes having defined locations within the overall sequence. Based on this analysis, the first transition corresponds to unfolding of the C-terminal half of the molecule, with the second transition resulting from unfolding of the more stable N-terminal half. The order of unfolding could be reversed in the presence of ATP-Mg 2+ and N-acetylglucosamine, conditions which have been shown to result in selective stabilization of the C-terminal domain ( T. K. White and J. E. Wilson, Arch. Biochem. Biophys. 274, 375–393 (1989 ).

Binding of nucleoside triphosphates, inorganic phosphate, and other polyanionic ligands to the N-terminal region of rat brain hexokinase: Relationship to regulation of hexokinase activity by antagonistic interactions between glucose 6-phosphate and inorganic phosphate

Mg 2+-chelates of several nucleoside triphosphates were shown to increase the inactivation of rat brain hexokinase (ATP: d-hexose-6-phosphotransferase, EC 2.7.1.1) by 0.6 m guanidine hydrochloride, with ATP-Mg 2+ having the greatest effect; unchelated forms did not significantly affect inactivation. Since catalytic activity has been associated with the C-terminal half of the molecule, these results were interpreted as indicating a destabilization of this C-terminal region by binding of these chelates to the substrate nucleotide sites, with the particular effectiveness of ATP-Mg 2+ reflecting the specificity for this species as a phosphoryl donor. These compounds were also shown to bind to the N-terminal half of the enzyme, as judged by their ability to protect against denaturation by guanidine hydrochloride and subsequent digestion with trypsin. Both free and Mg 2+-chelated forms afforded protection, with the unchelated nucleotides being most effective; a preference for ATP was seen only with the chelated forms. Thus, it was concluded that the N-terminal half of hexokinase contains a relatively nonspecific nucleotide binding site, distinct from the substrate nucleotide site previously shown to reside in the C-terminal half. On the basis of this same ability to protect the N-terminal half against denaturation and proteolysis, several other polyanionic ligands were shown to bind to this region of the molecule. These included inorganic phosphate, its analogs, sulfate and arsenate, and its homologs, pyrophosphate and tripolyphosphate. All of these anionic ligands were also shown to antagonize inhibition by the glucose 6-phosphate (Glc-6-P) analog, 1,5-anhydroglucitol 6-phosphate. The allosteric site for binding of Glc-6-P has previously been shown to reside in the N-terminal half of the molecule, and it is suggested that the antagonism of inhibition by Glc-6-P (or its analog) by these anionic ligands results from interaction with an anion binding site for which the 6-phosphate group of inhibitory hexose 6-phosphates must compete. A model depicting possible relationships between ligand binding sites on brain hexokinase, and how their interactions might lead to observed regulatory properties, is developed based on these and previous studies of ligand binding as well as evidence that mammalian hexokinases ( M r 100,000) have evolved by duplication and fusion of a gene coding for an ancestral hexokinase with M r 50,000 and which, like the mammalian enzyme, was sensitive to inhibition by Glc-6-P.

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′-dithiopropionate (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.