Yeast Hexokinase Research Articles

The inhibitory action of some antimalarial drugs and related compounds on the hexokinase of yeast and of Plasmodium berghei.

Of various antimalarial compounds tested, only proguanil failed to inhibit yeast hexokinase. The metabolite of proguanil, 10,580, was an effective inhibitor. Some compounds tested which were without antimalarial activity were potent inhibitors of yeast hexokinase. The degree of inhibition increased as the time during which the enzyme had been in contact with the drug increased, and the inhibitory action of mepacrine was reduced when the concentration of ATP was raised. The inhibition of yeast hexokinase by 10,732 was independent of the concentration of ATP.The hexokinase of haemolysates of the reticulocytes of mouse or rat blood was not appreciably higher than that of similar haemolysates of normal erythrocytes. Preparations of mouse or rat erythrocytes parasitized with P. berghei possessed a much higher hexokinase activity.The inhibiting action of various compounds on the hexokinase of P. berghei closely resembled those with yeast hexokinase. Again all antimalarial compounds (apart from proguanil) inhibited the enzyme, but some of the most potent inhibitors were devoid of antimalarial action. Amongst the chemotherapeutically active compounds, there appeared to be an approximate parallelism between antimalarial activity and potency as inhibitors of plasmodial hexokinase. The action of mepacrine on plasmodial hexokinase was reduced by raising the concentration of ATP, but, as with yeast hexokinase, the inhibition by 10,732 was independent of the ATP concentration.From a consideration of the results, it seems doubtful whether this type of inhibitory effect plays more than a minor part in the mechanism of antimalarial action in vivo.

Inhibition of Yeast Hexokinase by Fluoride Ion

Inhibition of Yeast Hexokinase by Fluoride Ion

Inhibition of Yeast Hexokinase by Fluoride Ion

Inhibition of Yeast Hexokinase by Fluoride Ion

Some mechanisms of cleavage of adenosine triphosphate and 1,3-diphosphoglyceric acid

The cleavage of ATP has been investigated with 18O in several phosphokinase reactions, namely yeast hexokinase, phosphoglycerate kinase, and adenylate kinase, and in the hydrolysis of ATP catalyzed by a Mg ++-activated ATPase in muscle extract. In all these reactions, the bond was cleaved between oxygen and the terminal P of ATP. The cleavage of 1,3-diphosphoglyceric acid was investigated in the glyceraldehyde-3-phosphate dehydrogenase reaction and the phosphoglycerate kinase reaction. In the first reaction, the CO bond was cleaved and in the second reaction the OP bond was cleaved. The mechanism of these reactions is discussed in relation to the behavior of 1,3 diphosphoglyceric acid as an acyl or phosphate donor. Comparison with other reactions reveals a general pattern in analogous reactions.

Isolation of 32P-Labeled Phosphoserine from a Yeast Hexokinase Preparation, Incubated with Labeled ATP or Glucose-6-Phosphate.

Isolation of 32P-Labeled Phosphoserine from a Yeast Hexokinase Preparation, Incubated with Labeled ATP or Glucose-6-Phosphate.

STUDIES OF THE KINETICS OF THE REVERSE REACTION OF YEAST HEXOKINASE

STUDIES OF THE KINETICS OF THE REVERSE REACTION OF YEAST HEXOKINASE

Phosphorylation of 2-deoxy-D-glucose by yeast hexokinase: Competition between 2-deoxy-d-glucose and glucose

Most malignant cells display increased glucose absorption and metabolism compared to surrounding tissues. This well-described phenomenon results from a metabolic reprogramming occurring during transformation, that provides the building blocks and supports the high energetic cost of proliferation by increasing glycolysis. These features led to the idea that drugs targeting glycolysis might prove efficient in the context of cancer treatment. One of these drugs, 2-deoxyglucose (2-DG), is a synthetic glucose analog that can be imported into cells and interfere with glycolysis and ATP generation. Its preferential targeting to sites of cell proliferation is supported by the observation that a derived molecule, 2-fluoro-2-deoxyglucose (FDG) accumulates in tumors and is used for cancer imaging. Here, we review the toxicity mechanisms of this drug, from the early-described effects on glycolysis to its other cellular consequences, including inhibition of protein glycosylation and endoplasmic reticulum stress, and its interference with signaling pathways. Then, we summarize the current data on the use of 2-DG as an anti-cancer agent, especially in the context of combination therapies, as novel 2-DG-derived drugs are being developed. We also show how the use of 2-DG helped to decipher glucose-signaling pathways in yeast and favored their engineering for biotechnologies. Finally, we discuss the resistance strategies to this inhibitor that have been identified in the course of these studies and which may have important implications regarding a medical use of this drug.

The effect of 6-deoxy-6-fluoro- d-glucose on yeast fermentation and on hexokinase

6-Deoxy-6-fluoro- d-glucose (6FG), at molar concentrations comparable to that of glucose or fructose present, produced a marked inhibition of the rate of fermentation of intact yeast. In contrast, 6 FG has a much smaller inhibitory effect on the rate of fermentation by a cell-free yeast extract or on the activity of yeast hexokinase. The inhibition by 6FG of intact yeast fermentation was competitive with glucose or fructose, and was overcome by increasing the concentration of these substrates. The rate of fermentation of glucose or of fructose by intact yeast was decreased by reduction in substrate concentration in a concentration range where the rate of fermentation by a yeast extract or the activity of hexokinase was unaffected. These results support the postulate that 6FG inhibits a specific process, not limited by hexokinase activity, which controls the rate of entry of glucose and of fructose into the yeast cell. The uptake of glucose by rat diaphragm was much less sensitive to the presence of 6FG than was fermentation of glucose by intact yeast. Notatin oxidized 6FG at a rate about 3% of that observed with glucose. 6FG was only moderately toxic to rats and did not affect the behavior of or virus synthesis in a tissue culture preparation.

2-Desoxy-D-glucose as an antagonist of glucose in yeast fermentation

2-Desoxy-D-glucose (2DG) has been found to inhibit strongly the anaerobic fermentation of glucose by living yeast. The degree of inhibition was found to be dependent on the molar ratio of substrate to inhibitor, but independent of the absolute quantities of substrate or yeast and of pH of the medium between 4.5 and 7.5.The effect followed the laws of competitive inhibition of an enzyme. The initial rate of fermentation of glucose by fortified cell-free yeast extract was not inhibited by 2DG. The inhibitory effect of 2DG on yeast fermentation was lost with the removal of the yeast cell wall. The evidence indicates that 2DG, acting as a structural analogue of glucose, competitively inhibits the action of a cell-wall enzyme which is essential for the transport of glucose into the cell, thus inhibiting the overall rate of fermentation by living yeast. A new substrate for yeast hexokinase has been discovered through the observation that the adenosine triphosphate-hexokinase system phosphorylated 2-desoxy-D-glucose.

The phosphorylation of D-(+)-glucosamine by crystalline yeast hexokinase

d-glucosamine has been shown to be converted to a phosphorylated amino sugar by ATP in the presence of crystalline yeast hexokinase and magnesium ions. The rate of the reaction is approximately that observed for d-glucose. The turnover number of the enzyme for glucosamine is 12,000 moles/10 5 grams hexokinase/minute at p H 7.8 and 30°. The enzymatically synthesized ester of glucosamine was isolated as the barium salt and shown to have full reducing power and to have an acid-stable phosphate group. The ester consumes four moles of sodium periodate per mole of compound. These facts, together with its elemental composition, show that it is d-glucosamine-6-phosphate. Aqueous solutions of the ester are not stable; with time, one or more substances are formed which have characteristic absorption spectra; simultaneously, the reducing power disappears.

Mechanism of Glyceraldehyde Inhibition of Glycolysis

THE effect of low concentrations of monomeric glyceraldehyde in inhibiting lactic acid formation in tissues using glucose but not in those using glycogen has been the subject of much experimentation and discussion. Stickland1 used dialysed muscle extract, reinforced with yeast hexokinase, and studied both substrates with this system. 0.01–0.001 M glyceraldehyde caused 80–90 per cent inhibition with glucose but had little or no effect with glycogen. Stickland found (private communication) by special tests that glyceraldehyde was not inhibiting hexokinase in this system.

Influence of some inhibitors of animal hexokinase on yeast hexokinase.

THE observations of Colowick et al.1 that the hexokinase activity of extracts of brain or muscles from normal animals may be inhibited by the addition of some anterior pituitary extracts in the presence of adrenal cortex extract, and that this inhibitory effect is prevented by insulin, has been confirmed by Reid, Smith and Young2.

THE INHIBITION OF d-GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE BY SPECIFIC ANTISERUM

Antibodies to yeast d-glyceraldehyde 3-phosphate dehydrogenase have been produced in rabbits and chickens. The antiserum from either animal inhibits the activity of the yeast enzyme with the formation of a precipitate that has a low residual activity. One combining site of antibody on the enzyme appears to be the point of DPN attachment. The rate of antigen-antibody formation has been studied with varying concentrations of antibody. The activity of yeast hexokinase is not inhibited by the antiserum to the dehydrogenase. Five of six antisera to the yeast enzyme showed no inhibition of the analogous enzyme from rabbit muscle, but one serum strongly inhibited the muscle dehydrogenase in the absence of DPN. No evidence was obtained that the inhibition by this one antiserum was due to an immune reaction, because the inhibition persisted after absorption with the homologous antigen.

Purification of yeast hexokinase and its reaction with ββ′-dichlorodiethyl sulphide

Research Article| January 01 1948 Purification of yeast hexokinase and its reaction with ββ′-dichlorodiethyl sulphide* K. Bailey; K. Bailey 1Biochemical Laboratory, Cambridge Search for other works by this author on: This Site PubMed Google Scholar E. C. Webb E. C. Webb 1Biochemical Laboratory, Cambridge Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1948) 42 (1): 60–68. https://doi.org/10.1042/bj0420060 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation K. Bailey, E. C. Webb; Purification of yeast hexokinase and its reaction with ββ′-dichlorodiethyl sulphide. Biochem J 1 January 1948; 42 (1): 60–68. doi: https://doi.org/10.1042/bj0420060 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1948 CAMBRIDGE UNIVERSITY PRESS1948 Article PDF first page preview Close Modal You do not currently have access to this content.

REACTION OF ENZYMES AND PROTEINS WITH MUSTARD GAS (BIS(ß-CHLOROETHYL)SULFIDE)

1. The rate of reaction of mustard gas (H) with thirteen proteins has been determined. The extreme variation in reaction rates is about 100:1. 2. No qualitative difference in the results was observed when the treatment with H was carried out by the Dixon or stirring methods. 3. The kinetics have been analyzed and a bimolecular equation derived which fits the facts. 4. The carboxyl groups of all proteins reacted when the reaction with H was carried out at pH 6.0 in M/25 acetate buffer. In most cases the number of carboxyl groups covered was approximately equal to the number of H residues bound. 5. The amino groups of proteins failed to react with the possible exception of yeast hexokinase. 6. The color obtained when proteins were mixed with Folin's phenol reagent at pH 8.0 decreased as the protein was treated with H. The color returned on treatment of the H-protein with alkali and many of the combined H groups were hydrolyzed. Similar results were observed when a concentrated glycyltyrosine solution was treated with H.

ISOLATION OF HEXOKINASE FROM BAKER'S YEAST

1. A method is described for the isolation of hexokinase from baker's yeast. The method is based mainly on fractionation with alcohol and results See PDF for Structure in a 30-fold increase in specific activity. The final product could be crystallized from ammonium sulfate without change in specific activity. 2. The enzyme catalyzes a transfer of phosphate from adenosinetriphosphate to glucose, fructose, or mannose, the relative rates with these three sugars being 1:1.4:0.3. 3. With glucose as substrate, the turnover number for the crystalline enzyme is 13,000 moles of substrate per 105 gm. of protein per minute at 30° and pH 7.5. The temperature coefficient (Q10°) between 0 and 30° is 1.9. 4. Magnesium ions are necessary for the activity, the dissociation constant for the Mg++ -protein complex being 2.6 x 10–3. Fluoride in concentrations as high as 0.125 M has no inhibitory effect on the enzyme when the Mg++ and orthophosphate concentrations are 6.5 x 10–3 M and 1 x 10–3 M, respectively. 5. The crystalline enzyme shows a loss in activity when highly diluted. This loss in activity can be prevented by diluting in the presence of small amounts of other proteins. Of the various protective proteins tested, insulin was the most effective, providing complete protection in a concentration of 6 micrograms per cc.; with serum albumin, a concentration of 60 micrograms per cc. was necessary. Thiol compounds (cysteine, glutathione) exerted no protective action. 6. The inactivation of the crystalline enzyme on incubation with trypsin can be prevented to a marked degree by the presence of glucose. The instability of crude preparations of yeast hexokinase may be attributed to the presence of proteolytic enzymes, since glucose or fructose has a remarkable protective effect on such preparations.