Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

Rabbit heart mitochondrial hexokinase: Solubilization and general properties

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

PH dependence of the alpha-glucose 1,6-diphosphate inhibition of hexokinase II.

α-Glucose 1,6-diphosphate is a much better inhibitor of hexokinase II than 1,5-anhydroglucitol 6-phosphate or glucose 6-phosphate (Glc-6-P) at pH 6–7 and poorer at higher pH. Because the K_i of Glc-6-P is pH independent, the observed pH effects are attributed to the phosphate group at C-1 which is bound as a monoanion to a specific site but which is excluded as a dianion. None of the following kinetic properties of the hexokinase II reaction varies greatly with pH: V, K_m of glucose and K_m of ATP.     

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.     

Ligand-induced conformations of rat brain hexokinase: effects of glucose-6-phosphate and inorganic phosphate

Binding of glucose-6-P induces conformational change in rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) as indicated by decreased susceptibility to digestion by chymotrypsin and an increased sedimentation coefficient on sucrose density gradients. These effects are competitively reversed by P_i, as are solubilization (of the mitochondrial form of hexokinase) and inhibition by glucose-6-P. Thus, the observed conformational changes are likely to be directly related to the effect of these ligands on catalytic activity and the interaction of the hexokinase with the mitochondrial membrane.Both glucose-6-P and P_i stabilize the enzyme against heat inactivation; this effect, as well as the effect of glucose-6-P on inactivation by chymotrypsin, have been used to estimate the dissociation constants for the complexes of hexokinase with glucose-6-P and P_i; the values are 7–8 μm, and 0.25 mm, respectively.These observations are consistent with a model in which brain hexokinase may exist in two distinct conformations, rapidly and reversibly interconvertible. The effect of glucose-6-P and P_i are explained by highly preferential binding to one or the other of these conformations.     

Kinetic properties of two starch phosphorylases from pea seeds

Both of the starch phosphorylase fractions from Victory Freezer pea seeds, that can be separated by DEAE—cellulose chromatography and purified by Sepharose 4B-starch affinity chromatography, contain pyridoxal 5′-phosphate. The addition of further quantities of pyridoxal 5′-phosphate causes inactivation. Both enzymes showed similar bi-substrate kinetics with d-Glc-1-P and varying amounts of amylopectin and also with P_i and varying amounts of amylopectin. In the direction of glucan sythesis the K_m for amylopectin with phosphorylase II was much higher than with phosphorylase I. However, the two enzymes differed in their behaviour on glucan degradation at varying concentrations of P_i. With phosphorylase II the K_m for amylopectin was dependent on the concentration of P_i but that for phosphorylase I was constant. Phosphorylase II was strongly inhibited by ADPG in the direction of glucan degradation but only slightly in the direction of glucan synthesis by both ADPG and UDPG. Phosphorylase I was only slightly inhibited by ADPG in both directions and by UDPG in synthesis. UDPG inhibited both enzymes moderately in glucan degradation,     

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

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

Comparison of type I hexokinases from pig heart and kinetic evaluation of the effects of inhibitors.

Type I hexokinase (ATP:D-hexose 6-phospotransferase, EC 2.7.1.1) of porcine heart exists in two chromatographically distinct forms. These do not differ significantly in size, electrophoretic mobility at pH 8.6 or kinetic properties. Both forms obey a sequential mechanism and are potently inhibited by glucose 6-phosphate. In contrast to observations of type I hexokinase from brain, inhibition by glucose 6-phosphate is not relieved by inorganic phosphate. Under most conditions, low concentrations of phosphate (less than 10 mM) have little effect on the kinetic behaviour of the enzyme but at higher concentrations this ligand is an inhibitor. Mannose 6-phosphate inhibits in a manner analogous to glucose 6-phosphate but the Ki is much greater. In view of the similarity of the kinetic parameters governing phosphorylation of mannose and glucose, this difference in affinity for the inhibitor site is seen as consistent with the existence of a separate regulatory site on the enzyme. MgADP inhibits hexokinase but behaves as a normal product inhibitor and inhibition is competitive with respect to MgATP and non-competitive with respect to glucose.     

Glycogen synthase: the existence of a single demonstrable from in bovine adipose tissue.

Glycogen synthase from bovine adipose tissue has been kinetically characterized. Glucose 6-phosphate increased enzyme activity 50-fold with an activation constant (A_0.5) of 2.6 mm. Mg²⁺ reversibly decreased this A_0.5 to 0.75 mm without changing the amount of stimulation by glucose 6-phosphate. Mg²⁺ did not alter the apparent K_m for UDP-glucose (0.13 mm). The pH optimum was broad and centered at pH 7.6. The glucose 6-phosphate activation of the enzyme was reversible and competitively inhibited by ATP (K_i = 0.6 mm) and P_i(K_i = 2.0 mm). The use of exogenous sources of glycogen synthase and glycogen synthase phosphatase suggests that (i) adipose tissue glycogen synthase phosphatase activity in fed mature steers is low or undetectable, and (ii) endogenous bovine adipose tissue glycogen synthase can be activated to other glucose 6-phosphate-dependent forms by addition of adipose tissue extracts from fasted steers or fed rats.     

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

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

The regulation of mitochondrial-bound hexokinases in the liver

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

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K_m for glucose was 45μm and the K_m for ATP 0·5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K_i 0·16mm for the soluble and 0·33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K_i 80μm for the soluble and 40μm for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K_i for ADP was 0·68mm for the soluble and 0·60mm for the particulate enzyme; K_i for AMP was 0·37mm for the soluble and 0·16mm for the particulate enzyme). P_i reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0·3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21·5μmoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25°. The temperature coefficient Q₁₀ between 22° and 38·5° was 1·93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1·07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.     

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.     

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus

Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30°C. The optimum pH ranges for trehalose degradation and synthesis were 6.0–7.5 and 6.0–7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P_i (K_i=2.0 mM). The enzyme was highly specific towards trehalose, P_i, glucose and α-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P_i, glucose and α-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K_m values were 61, 4.7, 24 and 6.3 mM for trehalose, P_i, glucose and α-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.     

Kinetic evidence that the high-affinity glucose 6-phosphate site on hexokinase I Is the active site

Two mechanisms have been suggested to account for the regulation of brain hexokinase by glucose 6-phosphate. One mechanism places glucose-6-P at an allosteric site, remote from the active site, while the second describes glucose-6-P as a simple product inhibitor of the enzyme, binding at the γ phosphate subsite within the ATP locus of the active site. To distinguish between these possibilities, we have undertaken a study of the back reaction of hexokinase I. Our data indicate that glucose-6-P displays classical Michaelis-Menten kinetics with brain hexokinase. This finding is consistent only with the high-affinity glucose-6-P site on the enzyme being the catalytic site. The dissociation constant, estimated from the initial-rate experiments is approximately 25 μm, a value that agrees well with the inhibition constant for glucose-6-P in the forward direction. These findings are consistent with an earlier model (W. R. Ellison, J. D. Lueck and H. J. Fromm, (1975) J. Biol. Chem.250, 1864–1871), which maintains that glucose-6-P inhibition of brain hexokinase is a manifestation of product inhibition. In a recent paper, Lazo et al. (P. A. Lazo, A. Sols, and J. E. Wilson, (1980) J. Biol. Chem.255, 7548–7551) reported data obtained from binding studies with rat brain hexokinase at an elevated (250 μm) level of glucose-6-P. These authors believe that their results indicate multiple binding of glucose-6-P to the enzyme and interpret the data in terms of a high-affinity allosteric site and a low-affinity catalytic site. Our results are at variance with this interpretation and are consistent only with the high-affinity site for glucose-6-P on brain hexokinase being the active site.     

Distribution of hexokinase isoenzymes depending on a carbon source in Saccharomyces cerevisiae.

DEAE cellulose chromatography and agar gel electrophoresis of glucose-phosphorylating enzymes in Saccharomyces cerevisiae showed the existence of glucokinase and two hexokinase isoenzymes ( designated as hexokinase I and II ). The distribution of hexokinase isoenzymes was dependent on a carbon source in the medium, while that of glucokinase was not dependent. The cells grown on 3 % ethanol as carbon source showed the isoenzyme pattern with predominant hexokinase I and a little hexokinase II. The isoenzyme pattern of the cells grown on 6 % glucose, which was differnt from that of the cells grown on ethanol, showed that hexokinase I and II were minor and major parts respectively. When the cells grown on 3 % ethanol were incubated on the medium containing 6 % glucose, hexokinase I was repressed and hexokinase II inducted. These facts suggest that two hexokinase isoenzymes, but not glucokinase, are adaptive enzyme.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution.

Hexokinase I governs the rate-limiting step of glycolysis in brain tissue, being inhibited by its product, glucose 6-phosphate, and allosterically relieved of product inhibition by phosphate. On the basis of small-angle X-ray scattering, the wild-type enzyme is a monomer in the presence of glucose and phosphate at protein concentrations up to 10 mg/mL, but in the presence of glucose 6-phosphate, is a dimer down to protein concentrations as low as 1 mg/mL. A mutant form of hexokinase I, specifically engineered by directed mutation to block dimerization, remains monomeric at high protein concentration under all conditions of ligation. This nondimerizing mutant exhibits wild-type activity, potent inhibition by glucose 6-phosphate, and phosphate reversal of product inhibition. Small-angle X-ray scattering data from the mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose 6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike conformation for the monomer similar to that observed in crystal structures of the hexokinase I dimer. Hence, any mechanism for allosteric regulation of hexokinase I should maintain a global conformation of the polypeptide similar to that observed in crystallographic structures.     

Rat brain hexokinase: glucose and glucose-6-phosphate binding sites and C-terminal amino acid of the purified enzyme

The binding of glucose and glucose-6-P by pure rat brain hexokinase has been studied by using an ultrafiltration procedure [H. Paulus (1969) Anal. Biochem. 32, 91–100]. Each mole of enzyme (molecular weight 98,000) binds 1 mole of glucose or 1 mole of glucose-6-P. The dissociation constant for the enzyme-glucose complex (0.04 mm) is in excellent agreement with the kinetically determined K_m for this substrate. The dissociation constant for the enzyme-glucose-6-P complex was estimated to be 1.3 μm, substantially lower than values of 7–8 μm obtained by alternative methods. This discrepancy appears to be due to retardation of the passage of the charged glucose-6-P through the ultrafiltration membrane, resulting in an effective increase in the ligand concentration at the membrane surface and thereby a decrease in the apparent dissociation constant. No appreciable retardation of the passage of the uncharged glucose molecule was observed.The binding of glucose-6-P (but not glucose) is prevented in the presence of P_i. This is in accord with a previously suggested model in which binding of P_i is considered to stabilize the enzyme in a conformation having little, if any, affinity for glucose-6-P.Serine was found as a C-terminal amino acid. The method used would not have detected C-terminal proline or tryptophan residues, and thus these cannot be excluded by the present experiments. However, in view of other results indicating that rat brain hexokinase consists of a single polypeptide chain, it seems probable that serine is indeed the only C-terminal amino acid in the molecule.     

Competitive inhibition of hexokinase isoenzymes by mercurials.

A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by p-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by p-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.