Yeast Hexokinase Research Articles

Activators of Yeast Hexokinase

Abstract The rate of the ATP:glucose transphosphorylase reaction catalyzed by yeast hexokinase, isozyme Pii, is inhibited by lowering the pH of the reaction below 7.0, especially at suboptimal concentrations of ATP. This effect of acidity is largely overcome by activators such as orthophosphate, citrate, malate, 3-phosphoglycerate, and riboside triphosphates. Thus, in the acid range, ATP appears to serve both as an activator and a substrate with the result that 1/v versus 1/[ATP] plots are nonlinear. It appears that yeast hexokinase may exist as two conformational isomers, an inactive form which is favored in the acid range and an active form favored by various polyanions or by alkaline pH. The conversion of inactive to active enzyme by citrate is slow, requiring about 1 min at 25° when citrate is added to the reaction mixture after the substrates. When citrate is added to the enzyme simultaneously with both substrates, the reaction begins at the fully activated rate indicating that only the ternary complex can undergo the conversion to inactive enzyme. Equilibrium isotope exchange experiments indicate that citrate activates the glucose ⇄ glucose 6-phosphate, ADP ⇄ ATP, and glucose-6-P ⇄ ATP exchanges equally. This is consistent with the idea that the ternary complex is undergoing the allosteric interconversions. Native hexokinase assayed inside semipermeable yeast cells exhibits the same kinetic properties as the isolated native enzyme, indicating that the activation phenomena may be important in controlling the rate of hexokinase in vivo.

A Study on the Kinetics and Mechanism of d-Lyxose and d-Xylose Activation of the Adenosine Triphosphatase Activity Associated with Yeast Hexokinase

Abstract The activation of the ATPase activity of yeast hexokinase by d-lyxose and d-xylose was investigated. Sugars lacking a carbon 6 activate the ATPase activity while the presence of a carbon 6 inhibits the activity. Xylose and lyxose, which are competitive inhibitors of glucose in the hexokinase reaction, induce substrate inhibition by ATP in the hexokinase and ATPase reactions. Ultracentrifugation studies indicate that the various sugars change the conformation of hexokinase to give lower s20,w values. The pentose activation of the ATPase activity has previously been interpreted as evidence for an ordered mechanism, but the initial rate and competitive inhibition studies of this report support a random addition of pentose and ATP in the reaction. These results are consistent with the proposed Random Bi Bi mechanism suggested for hexokinase.

Photoinactivation of aldolases by pyridoxal phosphate and its analogues.

Pyridoxal phosphate can act as a specific photosensitizer for amino acid residues in rabbit muscle and spinach leaf aldolases, but the residues affected depend on the pH of the reaction. Below pH 8 one histidine residue per enzyme subunit is destroyed; above pH 8.5 there is little loss of histidine, and photoinactivation is associated with the destruction of specific tyrosine residues, particularly the COOH-terminal residues. Pyridoxal and 4-pyridinecarboxaldehyde are much less effective than pyridoxal phosphate at neutral pH, but are similar to pyridoxal phosphate in their photosensitizing activity at the higher pH. Compounds lacking the aldehyde group or the pyridine ring show little or no activity. A number of other enzymes, including alpha-glycerophosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and yeast hexokinase, were also photoinactivated in the presence of pyridoxal phosphate; however, rabbit liver aldolase and two isomerases tested were completely resistant. The results suggest that certain enzymes, including rabbit muscle and spinach aldolases, but not rabbit liver aldolase, contain a specific site which interacts with pyridoxal phosphate, and that the conformation of this site changes in the pH range between 8.0 and 8.5

Control of Glycolytic Enzyme Synthesis in Yeast by Products of the Hexokinase Reaction

Abstract Addition of 2-deoxyglucose to a culture of the hybrid yeast Saccharomyces fragilis x Saccharomyces dobzhanskii causes 2- to 200-fold increase in the differential rate of synthesis of all glycolytic enzymes except alcohol dehydrogenase (EC 1.1.1.1). For most enzymes, the stimulation in the rate of synthesis occurs with a delay. The greatest stimulation is observed in the case of glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12), viz. about 200-fold, while P-glycerate mutase (EC 2.7.5.3) and enolase (EC 4.2.1.11) show a 2.5-fold increase in the differential rate of synthesis. Glucosone also stimulates the rate of synthesis of hexokinase (EC 2.7.1.1) and glyceraldehyde-3-P dehydrogenase. Generally, glycolytic enzyme synthesis is elicited by all compounds studied which are substrates of yeast hexokinase, while 6-deoxyglucose fails to induce synthesis either in the hybrid yeast or in Saccharomyces cerevisiae. The loss of fructose-phosphorylating activity in a hexokinaseless mutant of this yeast is associated with the failure of fructose to induce any of the glycolytic enzymes that fructose induces in the wild type. The differential rates of synthesis of all of the glycolytic enzymes between aldolase (EC 4.1.2.7) and pyruvate kinase (EC 2.7.1.40) increase 6- to 150-fold over the basal rates when glucose is added to a P-glucoisomerase (EC 5.3.1.9)-deficient mutant of S. cerevisiae. Experiments with a P-mannoisomerase (EC 5.3.1.8)-deficient mutant of S. cerevisiae indicate that hexokinase and P-fructokinase (EC 2.7.1.11) synthesis is stimulated in response to the presence of mannose. These and other data on the metabolites produced from 2-deoxyglucose have been interpreted to suggest that glucose-6-P is the inducer of the following enzymes: aldolase, triose-P isomerase (EC 5.3.1.1), glyceraldehyde-3-P dehydrogenase, P-glycerate kinase (EC 2.7.2.3), P-glycerate mutase, enolase, and pyruvate kinase. Mannose-6-P is the inducer of hexokinase and, presumably, of P-fructokinase. Pyruvate decarboxylase (EC 4.7.1.1) does not appear to be induced by glucose-6-P. These data are also consistent with P-glucomutase (EC 2.7.5.1) being induced by glucose-1-P.

The preparation of yeast hexokinases.

Abstract Improved methods are described for the preparation of hexokinase from baker's yeast. The isolation procedure is designed to avoid proteolysis, by using mechanical disintegration of the yeast cells, by organophosphate inhibition of the serine-dependent proteases, and by removal of all other proteases by gel filtration. Three isoenzymes, A, B and C, can be obtained thus. For hexokinase A, the ratio of activity in phosphorylating fructose as compared to glucose is about twice that of B or C. Hexokinase C is very similar in properties to B, but is separable by ion-exchange chromatography and appears to be a conformational isoenzyme of B. In the final purified state, the specific activity on glucose (27 mM, pH 8.3, 25.0) is 275 international units per mg for A, 900 for B and 750 for C, these values being higher than those for previously reported forms.

Specific inactivation of yeast hexokinase induced by xylose in the presence of a phosphoryl donor substrate.

Yeast hexokinase can be inactivated by xylose in the presence of MgATP. The effect is highly specific for xylose. The nucleotide and divalent metal specificities are similar to those of the hexokinase reaction. The dissociation constants, as deduced from the effect of the concentration of the inactivating agents on the rate of inactivation, are about 0.2 mM for MgATP and about 10 mM for xylose. These values are similar to the Michaelis constant and Ki value for these compounds as substrate and competitive inhibitor, respectively, in the hexokinase reaction. Sugars capable of binding at the sugar substrate subsite protect against xylose‐induced inactivation with efficiencies closely related to their respective Ks or Ki values. The dissociation constant of glucose, as deduced from its protective effect, is about 0.04 mM.When resting yeast is incubated aerobically with 0.1 M xylose and 1% ethanol, a marked loss of hexokinase activity takes place.Among the possible mechanisms of this irreversible inactivation there is evidence against the involvement of phosphorylation or glycosylation of the protein as well as of a change in its degree of polymerization.

Induced fit in yeast hexokinase.

In the nucleoside triphosphatase activity that yeast hexokinase shows in the absence of acceptor substrate the Km values for MgATP and for MgITP, as well as the Ki for MgGTP, are markedly different from those in the phosphotransferase reaction. In the presence of lyxose or xylose, two non‐phosphorylable analogues of the sugar substrates, these Km or Ki values change to those corresponding to the hexokinase reaction. Besides this effect on the apparent affinities, lyxose and xylose can enhance by about 20 times the maximal rate of hydrolysis of the terminal phosphoryl group of the nucleoside triphosphates. In addition, xylose slowly induces a specific, apparently irreversible, inactivation of the enzyme, both as hexokinase and as nucleoside triphosphatase. The relative hydrolytic activities on ATP, ITP, and GTP resemble the relative efficiencies of these nucleotides as phosphate donors, as well as those in the inactivation of the enzyme in the presence of xylose. The presence of a phosphate group on carbon 6 of glucose does not prevent the sugar effects on the Km values of hexokinase for the nucleoside triphosphates, although it reduces their rates of hydrolysis. These effects indicate the occurrence of an “induced fit” by which certain sugars specifically modify the binding of the nucleoside triphosphate substrates and enhance the labilizing action of the enzyme on their terminal phosphoryl bond. Moreover, hexokinase seems to induce a peculiar configuration of the pyranose ring of the sugar substrate, probably a boat form. When bulky substituents prevent the latter to be formed, sugars, even if they are still able to bind to the enzyme, do not induce an increased labilization of the nucleoside triphosphates as glucose or lyxose do.

The kinetics of yeast hexokinase in the light of the induced fit involved in the binding of its sugar substrate.

The kinetics of the initial reaction rates of yeast hexokinase, both in the forward and in the backward direction, have been re‐examined in the light of recent findings concerning the properties of the ATPase activity of hexokinase and its modification by certain non‐phosphorylable analogues of the sugar substrates.A single pattern has been found in the double reciprocal plots even if the concentrations of both the acceptor and the donor substrates are varied over a wide range, with ITP as well as with ATP as donor substrates. A similar pattern has been found for the backward reaction.These results are interpreted as indicative that in the forward as well as in the backward reaction, the interconversion of the ternary complex of the enzyme with both substrates is the limiting step. This fact and the complex effect of glucose 6‐phosphate as an initially‐added product, prevents in principle the distinction between the possible kinetic mechanisms leading to the ternary complex. Nevertheless, taking into account the change in affinities for the nucleotide substrates specifically induced by certain sugars, it becomes possible to formulate a plausible hypothesis. It is postulated that binding of a sugar to an enzyme‐nucleotide complex results in an abortive ternary complex. Accordingly, formation of active ternary complexes in the hexokinase reaction requires the sequential addition of a nucleotide substrate to a binary complex of the enzyme with a sugar substrate.

The alkylation of yeast hexokinase A.

The alkylation of yeast hexokinase A.

Kinetic Studies of the Adenosine 5'-Triphosphatase Activity of Yeast Hexokinase and Its Relationship to the Mechanism of Action of the Enzyme

Abstract Initial reaction velocities of yeast hexokinase and the adenosine 5'-triphosphatase activity associated with hexokinase were studied in the presence and absence of 1.5 m NaCl. At least 500 times more enzyme is required for the latter measurement relative to the determination of hexokinase activity. The Michaelis constants for adenosine 5'-triphosphate for the two activities are essentially the same in the salt environment but differ by a factor of 25 in the absence of NaCl. Gel filtration experiments indicate that the molecular weight of the enzyme is decreased by 10 mm glucose, 1.5 m sodium chloride, or dilution. The sedimentation coefficients were determined for hexokinase with and without 1.5 m NaCl. The s20,w is significantly higher in the absence of the salt. The results from these studies support the view that, when the physical state of the yeast enzyme is the same in both the hexokinase and ATPase assays, the Km values for ATP are the same for both activities. These findings are taken to be evidence in support of a random kinetic mechanism for the yeast phosphotransferase.

A Glucokinase from Saccharomyces cerevisiae

Abstract An ATP:d-glucose 6-phosphotransferase (EC 2.7.1.2) or glucokinase from Saccharomyces cerevisiae is described. The enzyme has been purified about 100-fold from the crude extracts of a hexokinaseless mutant derived from a haploid strain of S. cerevisiae. The Km values for glucose and ATP are 28 and 50 µm, respectively. The maximal velocity of this enzyme toward fructose is 0.4% of that of glucose. The enzyme showed a marked heterogeneity when sedimented in a sucrose density gradient, its molecular weight ranging from 144,000 to a value in excess of 200,000. Yeast glucokinase shares a number of properties with yeast hexokinase (EC 2.7.1.1) in the semiconstitutive nature of its synthesis with glucose, its broad pH optima, and inhibition with ADP and N-acetylglucosamine. Its mode of reaction with glucose and ATP, as indicated by initial velocity patterns, appears to be random.

Kinetic Study of Yeast Hexokinase

The ionic species ATP4− and Mg2+, at high concentrations, decrease the rate of glucose phosphorylation catalyzed by yeast hexokinase. A theoretical study of all the possible mechanisms (271 models) of this inhibition has been made. The comparison of these mechanisms with the experimental results shows that only one model can explain the inhibition of the reaction by ATP, and that only one can also explain the effect of the high magnesium concentrations.It was thus possible to show that the ionic species ATP4− can be bound on hexokinase and on the hexokinase‐glucose complex. Its affinity is much stronger for the second form of the enzyme than for the first. The complexes thus formed are devoid of any activity (“dead‐end” complexes). The inhibition of the hexokinase by the high nucleotide concentrations is therefore due to the formation of these complexes. A ternary chelate of the Mg(ATP)26− type does not exist.Magnesium possesses no affinity for the enzyme. Its role in the catalysis must therefore by indirect. The results presented agree with the idea that the metal polarizes the phosphoryl bond of the ATP that is split when glucose‐6‐phosphate is formed. Magnesium can form with ATP a ternary chelate of the Mg2ATP type, unable to be bound on the hexokinase. The inhibiting effect of high concentrations of the metal can be explained by the formation of this inactive ternary chelate and by the decrease in concentration of the MgATP2− complex.

The melecular weight of the [formula omitted] polypeptide chain of yeast hexokinase

Commercial preparations of yeast hexokinase are contaminated with a trace of at least one proteolytic enzyme. This contaminant will digest the hexokinase in the presence of sodium dodecyl sulfate (SDS), and a reliable molecular weight can be obtained by SDS-polyacrylamide gel electrophoresis only when specific steps are taken to prevent this proteolysis. When appropriate precautions are taken, this technique yields a value of about 51,000 for the polypeptide chain molecular weight of yeast hexokinase. A similar value is obtained by gel filtration in 6M guanidine-HCl.

Product Inhibition of the Hexokinases

ADP3- and ADPMg caused mixed inhibition of purified yeast hexokinase isoenzymes and ascites tumor hexokinases I and II when ATPMg is the varied substrate. Since exchange between ADPMg and ATPMg is observed under steady state conditions in which free glucose-6-P is not available for reaction, the effect of ADPMg on Vmax must result from its reaction at the ADP product site of the enzyme· glucose-6-P complex. Thus, this type of inhibition, which is rare among the kinases, is attributed to a relatively slow release of the second product, glucose-6-P. In the case of yeast hexokinase, the replot of the intercepts of the 1/V against 1/ATPMg plot as a function of ADPMg concentration is hyperbolic. Thus, the alternative sequence of product release, glucose-6-P first, is occurring; however, this sequence is probably of minor significance except in the presence of high concentrations of ADPMg. Under certain conditions, mixed inhibition can be observed by glucose-6-P with ATPMg or ITPMg, the variable substrate. In no case was exchange observed between glucose-6-32P and ATPMg under product-inhibited steady state conditions in which free ADP was kept low by a suitable trapping reaction. This indicates that any inhibition of Vmax by glucose-6-P is not due to action at the glucose-6-P product site of an enzyme·ADP complex which would have to dissociate rapidly, but to action at a particular modifier site. Such a site is therefore indicated for tumor hexokinase II. A simple general kinetic procedure is described for evaluating the inhibition due to ADP3- when other inhibitors such as ADPMg and ATP4- are also present.

Active-site thiol groups of yeast hexokinase.

Research Article| December 01 1969 Active-site thiol groups of yeast hexokinase J G Jones J G Jones 1Department of Biochemistry, University College, Cathays Park, Cardiff Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1969) 115 (5): 41P. https://doi.org/10.1042/bj1150041Pa Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation J G Jones; Active-site thiol groups of yeast hexokinase. Biochem J 1 December 1969; 115 (5): 41P. doi: https://doi.org/10.1042/bj1150041Pa Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1969 The Biochemical Society1969 Article PDF first page preview Close Modal You do not currently have access to this content.

Effect of pH on the kinetics of yeast hexokinase

The kinetics of the yeast hexokinase reaction has been studied in the range of pH 6.3‐9.9 at 25°. Initial velocities were determined by varying the concentrations of glucose and in dependence of ionic species of MgATP complexes, which were calculated from the total concentrations and the known equilibrium constants. Maximum velocity is decreased in the acid region due to formation of an inactive central enzyme‐glucose‐MgATP complex with a pK value of 6.8. In the same range the Michaelis constant for glucose decreases too, indicating that this group is protonated in the enzyme‐glucose complex but not in the state of the free enzyme. In the alkaline region the Michaelis constant for glucose increases. This is caused by dissociation of a group with pK 9.9 in the free enzyme but not in the enzyme‐glucose complex. The Michaelis constant for the MgATP is not altered in the acid and neutral pH regions, but is also increased in the alkaline region. This finding shows the presence of an additional site of the enzyme dissociating with a pK of 9.9. This must be in the nondissociated state for binding of MgATP. Free uncomplexed ATP acts as a competitive inhibitor of MgATP. Due to the fact, that both the uncompetitive and the noncompetitive inhibition of ATP is abolished with increasing pH, it is suggested that inhibition is caused by the ion ATPH3−. The uncompetitive part of the inhibition is also decreased in the acid region. It appears therefore that the enzyme‐glucose complex can combine with the free ATP only when the group with pK 6.8 is dissociated. The pH effects on the kinetics of the yeast hexokinase reaction are consistent with the assumption that the dissociated form of the group with pK 6.8 of the central complex favours as base catalyst the release of a proton from the sugar and increases therefore the nucleophilic attack of the hydroxyl group of the sugar to the phosphoryl group of ATP.

The molecular weight and dissociation properties of yeast hexokinase

The molecular weight and dissociation properties of yeast hexokinase

On the multiple forms of yeast hexokinase

Multiple forms of hexokinase have been detected by agar gel and polyacrylamide gel electro‐phoresis in crude extracts obtained from yeast cells of different strains. Electrophoretic mobility and number of enzyme forms depend on the conditions of cell disruption and vary with the respective yeast strain. Extracts from commercial baker's yeast, prepared by short‐time mechanical disruption contain two or three isozymes of hexokinase. Cell‐lysis by proteolytic procedures (toluene, extraction of dried yeast) changed the electrophoretic pattern profoundly.Between the exponential and the stationary phases of growth characteristic differences in the pattern of the two isozymes of hexokinase have been found.Five different strains of yeast revealed different patterns of hexokinase isozymes.Electrophoresis on polyacrylamide gels, using a concentration gradient of the polymer, indicated that the differences in the mobilities of these enzymes are presumably not caused by major differences in their molecular sizes. Trypsin caused an increase of electrophoretic mobility of one or both forms of the enzyme from baker's yeast.

The Modification of Yeast Hexokinases by Proteases and Its Relationship to the Dissociation of Hexokinase into Subunits

Abstract Crystalline hexokinase prepared by the method of Darrow and Colowick consists largely of enzyme which has been modified in its chromatographic and electrophoretic behavior by yeast protease which is present during the isolation procedure. This proteolytic modification can be prevented by removal of the protease by chromatography on diethylaminoethyl cellulose; it can also be largely inhibited by phenylmethanesulfonyl fluoride. Preparations made by the diethylaminoethyl cellulose method contain two forms of the enzyme (P-I and P-II), both of which are more basic by chromatography and electrophoresis than is protease-modified enzyme (S forms). Form P-II shows the same catalytic activity as the Darrow-Colowick preparation, but Form P-I is less active. The two forms are equally susceptible to modification by trypsin. Forms P-I and P-II exist mainly as tetramers of molecular weight about 100,000 at low ionic strength in the neutral pH region, as measured by ultracentrifuge, light scattering, or gel filtration. They tend to dissociate, without loss of catalytic activity, to dimers as the ionic strength, temperature, or pH is raised or when glucose is added in the presence of phosphate. They can be converted to S forms by trypsin or yeast protease only when glucose or salts are present, suggesting that the enzyme must be in the dimer state in order for modification to occur. The protease-modified enzyme tends to exist mainly as a dimer of molecular weight around 50,000 at neutral pH. Thus, modification by trypsin seems to remove some groups which normally play a role in the association of dimer molecules to form the tetramer structure. Mercaptoethanol causes a striking decrease in sedimentation rate when added to the enzyme at pH 9.0. This may be correlated with the recent finding that mercaptoethanol can cause inactivation of the enzyme at this pH.

Chemical coupling of yeast hexokinase to Sephadex

Yeast hexokinase was coupled to Sephadex by means of cyanogen bromide. The activity of the fixed enzyme was 2% of that before coupling. The activity of the noncoupled part of the enzyme was also decreased to the same degree. This decrease cannot be caused by the coupling reaction itself. The Michaelis-constants and the pH-activity curve of the coupled enzyme do not differ significantly from that of the free enzyme.