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.     

Adenine nucleotides and magnesium ions in relation to control of mammalian cerebral-cortex hexokinase

1. The kinetics of inhibition of brain soluble cytoplasmic hexokinase by ADP were examined in relation to variations in the concentrations of Mg(2+) and ATP. The type of inhibition observed was dependent on the Mg(2+)/ATP ratio. 2. ADP at Mg(2+)/ATP ratios 2:1 exhibited inhibition of the ;mixed' type; at Mg(2+)/ATP ratios 1:1 the inhibition appeared to be competitive with regard to ATP. 3. Inhibition by free ATP was observed when the Mg(2+)/ATP ratio was less than 1:1. The inhibition was also of the ;mixed' type with respect to MgATP(2-). 4. The inhibitions due to ADP and to free ATP were not additive. The results suggested that there may be up to four sites in the soluble enzyme: for glucose, glucose 6-phosphate, ADP and MgATP(2-). 5. The ;free' non-particulate intracellular Mg(2+) concentration was measured and concluded to be about 1.5mm. 6. The concentrations in vivo of Mg(2+) and ATP likely to be accessible to a cytoplasmic enzyme are suggested to be below those that yield maximum hexokinase rates in vitro. The enzymic rates were measured at relevant suboptimum concentrations of Mg(2+) and ATP in the presence of ADP. Calculations that included non-competitive inhibition due to glucose 6-phosphate (56-65% at 0.25mm) resulted in net rates very similar to the measured rates for overall glycolysis. This system may therefore provide a basis for effective control of cerebral hexokinase.     

Specificity of interaction of hexokinase isozyme II with mitochondrial membranes

Study on the mechanism of hexokinase isozyme II adsorption on mitochondrial membranes in the presence of 10 mM MgCl2 demonstrated that 0.16% of the total proteins of the soluble fraction and the total hexokinase pool are capable of reversible binding to the membrane. The plot for the dependence of the degree of enzyme adsorption on Mg2+ concentration is hyperbolic. Under these conditions, hexokinase competes favourably for the binding sites with lactate dehydrogenase and creatine kinase. Analysis of the adsorption capacity of natural and artificial phospholipid membranes showed that hexokinase isozyme II is adsorbed in much the same way on inner and outer mitochondrial membranes as well as on a mixture of membranes obtained from various sources and on lecithin liposomes. The adsorption properties of hexokinase isozyme II and of its functional analog--isozyme I--point to marked differences in the mechanism of their interaction with the membrane. In contrast with isozyme I, isozyme II of hexokinase undergoes kinetic alterations. Besides, it was found that mild autolysis of isozyme II is accompanied by a loss of the enzyme ability to bind to mitochondrial membranes. The data obtained suggest that the specificity of hexokinase isozyme II adsorption depends on the structural peculiarities of the protein but not on those of the mitochondrial membrane.     

Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig

1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.     

The effect of insulin on the catalytic efficacy of rat skeletal muscle hexokinase isoenzyme II]

The effect of insulin on the intracellular localization of rat skeletal muscle hexokinase isozyme II (hexokinase II) was studied in vivo. It was found that after injection of the hormone the glucose concentration in the muscle gradually increases in parallel with the hexokinase II redistribution between the cytosol and the mitochondrial fraction in the direction of the bound form of the enzyme. This effect of insulin is due to glucose, an indispensable participant of the complex formation between the enzyme and the mitochondrial membrane. It was shown that the effect of glucose as a hexokinase II adsorbing reagent is a highly specific one. The hexokinase II binding to mitochondria in the presence of glucose is accompanied by changes in some kinetic properties of the enzyme. A kinetic analysis of catalytic efficiency of the free and bound hexokinase II forms revealed that the catalytic efficiency of hexokinase II within the composition of the enzyme-membrane complex exceeds by two orders of magnitude that of the free enzyme. The data obtained are discussed in the framework of an adsorption mechanism of hexokinase activity regulation in the cell.     

On the mechanism of oligomycin inhibition of Ca-induced mitochondrial respiration

The addition of oligomycin in the presence of Ca²⁺ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca²⁺-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5–20 μM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca²⁺-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca²⁺ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.     

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.     

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.     

Binding of nucleotides to the ATP-dependent protease La from Escherichia coli

A critical enzyme in protein breakdown in Escherichia coli is the ATP-hydrolyzing protease La, the lon gene product. In order to clarify the role of ATP in proteolysis, we studied ATP and ADP binding to this enzyme using rapid gel filtration to separate free from bound ligands. In the presence of Mg2+ or Mn2+ and 10 microM ATP, two molecules of ATP were bound to the tetrameric enzyme, while at 100 microM ATP (or higher), four ATP molecules were bound, both at 0 and 37 degrees C. Protease La thus has two high affinity sites (S0.5 less than 10(-7) M) for ATP and two lower affinity sites (S0.5 = 12-15 microM). Binding was reversible. In the absence of a divalent ion, ATP bound to only two sites. However, much lower Mg2+ concentrations (50 microM) were required for maximal ATPase binding than for maximal proteolytic and ATPase activity (2 mM). Decavanadate, which is a potent inhibitor of proteolysis, also blocked ATP binding, but orthovanadate had neither effect. Different ATP analogs bind to these sites in distinct ways. Adenyl-5'-yl imidodiphosphate binds to only one high affinity site, while adenyl-5'-yl methylene monophosphonate binds to two. Nevertheless, both non-metabolizable analogs can activate oligopeptide hydrolysis as well as ATP. Although binding of a single nucleotide can activate peptide hydrolysis, occupancy of all four sites appears necessary for maximal protein breakdown. The ATP molecules on all four sites are hydrolyzed rapidly. The Pi is released, but ADP remains on the enzyme. ADP binds to the same four sites, but this process does not require divalent ions. Protease La shows higher affinity for ADP than for ATP. Therefore, in vivo, ADP should inhibit ATP binding and protease La function.     

Effect of ligands on the reactivity of essential sulfhydryls in brain hexokinase. Possible interaction between substrate binding sites.

Inactivation of bovine brain mitochondrial hexokinase by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a sulfhydryl specific reagent, has been investigated. The study shows that the inactivation of the enzyme by DTNB proceeds by way of prior binding of the reagent to the enzyme and involves the reaction of 1 mol of DTNB with a mol of enzyme. At stoichiometric levels of DTNB, the inactivation of the enzyme is accompanied by the formation of a disulfide bond. But it is not clear whether the disulfide bond or the mixed disulfide intermediate formed prior to it causes inactivation. On the basis of considerable protection afforded by glucose against this inactivation it is tentatively concluded that the sulfhydryl residues involved in this inactivation are at the glucose binding site of the enzyme, although other possibilities are not ruled out. An analysis of effects of various substrates and inhibitors on the kinetics of inactivation and sulfhydryl modification by DTNB has led to the proposal that the binding of substrates to the enzyme is interdependent and that glucose and glucose 6-phosphate produce slow conformational changes in the enzyme. Protective effects by ligands have been employed to calculate their dissociation constant with respect to the enzyme. The data also indicate that glucose 6-phosphate and inorganic phosphate share the same locus on the enzyme as the gamma phosphate of ATP and that nucleotides ATP and ADP bind to the enzyme in the absence of Mg2+.     

Modulation of 2-oxoglutarate dehydrogenase complex by inorganic phosphate, Mg(2+), and other effectors

The interplay of inorganic phosphate (Pi) with other ligands such as Mg(2+), ADP, ATP, and Ca(2+) on the activation of 2-oxoglutarate dehydrogenase complex (2-OGDH) in both isolated enzyme complex and mitochondrial extracts was examined. Pi alone activated the enzyme, following biphasic kinetics with high (K(0.5) = 1.96+/-0.42 mM) and low (K(0.5) = 9.8+/-0.4 mM) affinity components for Pi. The activation by Pi was highly pH-dependent; it increased when the pH raised from 7.1 to 7.6, but it was negligible at pH values below 7.1. Mg-Pi and Mg-ADP, but not Mg-ATP, were more potent activators of 2-OGDH than free Pi and free ADP. ATP inhibited the 2-OGDH activity by chelating the free Mg(2+) and also as a Mg-ATP complex. With or without Mg(2+), ADP, and Pi activated the 2-OGDH by increasing the affinity for 2-OG and the V(m) of the reaction; ATP diminished the V(m), but it increased the affinity for 2-OG in the mitochondrial extract. Pi did not modify the 2-OGDH activation by Ca(2+). The results above mentioned were similar for both preparations, except for hyperbolic kinetics in the isolated enzyme and sigmoidal kinetics in the mitochondrial extracts when 2-oxoglutarate was varied. The data of this study indicated that physiological concentrations of Pi may exert a significant activation of 2-OGDH, which was potentiated by Mg(2+) and high pH, but surpassed by ADP.     

Binding and exchange of nucleotides on the chloroplast coupling factor CF1. The role of magnesium

On the soluble part of the coupling factor (CF1), extracted from spinach chloroplasts, three nucleotide-binding sites are identified. Three ADP are bound per CF1 when the enzyme is incubated with ADP either with or without Mg2+. Two ADP and one ATP are bound per CF1 when the enzyme is incubated with a limiting concentration of ATP, in the presence of Mg2+. At high ATP concentration, in the presence of Mg2+, one free ATP exchanges with one bound ADP and two ATP and one ADP remain bound per CF1. When Mg2+ is omitted from the incubation medium of ATP and CF1, only two ADP and around 0.5 ATP are bound per CF1. The three nucleotide binding sites of CF1 fall into two different and independent categories according to the ability of the bound nucleotides to be exchanged with free nucleotides. On one site the bound ADP is difficult to exchange. On the other two sites, the bound nucleotides. ADP or ATP, are readily exchangable. We propose that the two exchangeable sites form the catalytic part of the enzyme where ATP is hydrolyzed. When ATP concentration is high enough, in the presence of Mg2+, one ATP displaces one bound ADP and allows the ATP hydrolysis to proceed. We propose too that the site where ADP is difficult to exchange may represent the 'tight' ADP-binding site, different from the catalytic ones, which becomes exchangeable on the CF1 in vivo when the thylakoid membranes are energized by light, as stressed by Bickel-Sandk?tter and Strotman [(1976) FEBS Lett. 65, 102-106].     

Functioning of mitochondria-bound hexokinase in rat brain in accordance with generation of ATP inside the organelle.

The function of mitochondria-bound hexokinase, the enzymatic form peculiar to the brain, in utilization of ATP generated inside the organelles, was examined by incubating rat brain mitochondrial fraction with [14C]glucose under various conditions. Addition of succinate and ADP to the incubation medium increased glucose 6-phosphate formation by the mitochondrial hexokinase and caused a smaller increase in ATP concentration in the mitochondria. The glucose phosphorylation was markedly inhibited by the addition of dinitrophenol, potassium cyanide, and oligomycin, and the ATP concentration was decreased. On the other hand, addition of atractyloside suppressed the glucose phosphorylation without affecting the mitochondrial hexokinase activity, whereas addition of antiserum against the mitochondrial hexokinase inhibited both glucose 6-phosphate formation and hexokinase activity. A part of both the glucose phosphorylation and hexokinase activities, however, remained even in the presence of the maximum dose of the anti-hexokinase serum and atractyloside. These results indicate the active utilization of intrinsically generated ATP by the mitochondria-bound hexokinase, a part of which may be located away from the surface of the mitochondrial membrane.     

Bioflavonoid regulation of ATPase and hexokinase activity in Ehrlich ascites cell mitochondria.

(1) The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.     

Catalytic sites of mitochondrial ATPase with different properties. Effect of citrate, free ATP and ADP in the presence of dithionite

The extent of stimulation of the hydrolytic activity of mitochondrial ATPase by the reducing agent dithionite has been found to depend on substrate concentration both for the membrane bound enzyme and for the isolated and purified F1ATPase. The results suggest the existence of three catalytic sites differing in their standard reduction potential. The activating effect of free ATP on the hydrolytic activity of rat liver F1-ATPase has been found to be more pronounced on the reduced form of the enzyme. On the contrary, the inhibitory effect of ADP was higher on the oxidized form of F1-ATPase. Citrate has also been found to be an inhibitor of F1-ATPase; its effect was more pronounced on the reduced form of the enzyme, and exhibited a competitive pattern of inhibition with respect to free ATP. The results obtained have been interpreted in the sense that free ATP and ADP may be modifying the standard reduction potential of the enzyme, and suggest the existence of three independent redox cycles in ATPase governed by the exchange of ADP and Pi for the newly synthesized ATP.     

Comparative study of free and bound glycolytic enzymes from sea scorpion brain.

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45 degrees C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.     

Interactions between K+ and ATP binding to the (Na+ + K+)-dependent ATPase.

K+ appears to decrease the affinity of the (Na+ + K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) for its substrate, Mg2+ - ATP, and Mg2+ - ATP, in turn, appears to decrease the affinity of the enzyme for K+. These antagonisms have been investigated in terms of a quantitative model defining the magnitude of the effects as well as identifying the class of K+ sites on the enzyme involved. K+ increased the apparent Km for Mg2+ - ATP, an effect that was antagonized competitively by Na+. The data can be fitted to a model in which Mg2+ - ATP binding is prevented by occupancy of alpha-sites on the enzyme by K+ (i.e. sites of moderate affinity for K+ accessible on the "free" non-phosphorylated enzyme, in situ on the external membrane surface). By contrast, occupancy of these alpha-sites by Na+ has no effect on Mg2+ - ATP binding to the enzyme. On the other hand, Mg2+ - ATP decreased the apparent affinity of the enzyme for K+ at the alpha-sites, in terms of (i) the KD for K+ measured by K+-accelerated inactivation of the enzyme by F-, and (ii) the concentration of K+ for half-maximal activation of the K+-dependent phosphatase reaction (which reflects the terminal hydrolytic steps of the overall ATPase reaction). These data fit the same quantitative model. Although this formulation does not support schemes in which ATP binding effects the release of transported K+ from discharge sites, it is consistent with observations that K+ can inhibit the enzyme at low substrate concentrations, and that Li+, which has poor efficacy when occupying these alpha-sites, can stimulate enzymatic activity at high K+ concentrations by displacing the inhibitory K+.     

Purification and properties of the cytoplasmic hexokinase from rabbit brain

About 90% of the total hexokinase activity in rabbit brain was found to be associated with mitochondria while the remaining part was found in the cytosolic fraction. The soluble enzyme was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The molecular weight of the purified hexokinase was found to be approximately 98,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.2 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses. Mg . ATP2-, among the nucleotide substrates, was the most effective phosphate donor. The properties of the purified cytoplasmatic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in molecular weight, isoelectric point, pH dependence of activity, electrophoretic mobility and affinity for glucose and Mg.ATP2-. However, the temperature dependence of activity, and the specificity for several hexose substrates were markedly different.     

Crystal structures of an ATP-dependent hexokinase with broad substrate specificity from the hyperthermophilic archaeon Sulfolobus tokodaii

Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg(2+), and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg(2+) ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.     

The roles of ADP2- and Mg2+ in control steps of phosphoglycerate kinase

1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide, ADP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ADP binding to the enzyme. A preliminary study of adenosine binding to phosphoglycerate kinase was made in order to be sure of the nature of the adenine site. From the change in chemical shifts of the 'basic patch' histidine resonances (His62, 167 and 170), the nucleotide C8-H, C2-H and C1'-H resonances and resonances 40 and 41 (assigned to Thr373 and Thr375 in the hydrophobic, i.e. catalytic, site), it is apparent that there are at least two ADP binding sites on the enzyme: one at the hydrophobic (catalytic) site and one at the electrostatic site. A comparison of the results for ADP and ATP reveals differences due to the differential binding of the phosphate groups. The presence of Mg2+ results in further differences being observed. The data suggest that the primary binding site of ADP, in the absence of Mg2+, involves electrostatic interactions between the diphosphate chain of the substrate and the 'basic patch' region of the N-terminal domain. In the presence of greater than or equal to 1:1 ratio of Mg2+/ADP, however, the primary binding site involves predominantly hydrophobic interactions between the adenosine moiety and the catalytic site, with secondary binding occurring at the electrostatic site. Addition of Mg2+, therefore, tends to reduce the affinity of the electrostatic site (presumably by competing for ADP). It is suggested that alpha-helix XII, including residues 372, 373 and 375, moves differentially on binding ADP, Mg ADP, ATP or Mg . ATP, consistent with Mg2+ assisting the transfer of the gamma-phosphate of ATP to 3-phosphoglycerate during catalysis.