Inhibition and Inactivation of Glucose-phosphorylating Enzymes from Saccharomyces cerevisiae by D-Xylose

Fernández, Roberto García; Herrero, Pilar; Moreno, Fernando Salvador

doi:10.1099/00221287-131-10-2705

Cited by 22 publications

(22 citation statements)

References 6 publications

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“…Figure 2 shows an example for the analysis of yeast cells expressing the high sensitivity FRET glucose sensor FLII 12 Pglu700 μδ6 trapped in a microfluidic device [40]; shown is the average FRET change from several individual yeast cells during perfusion with square pulses of glucose at three different concentrations. Taken together, these results support four major conclusions: (i) glucose levels can drop several orders of magnitude below the K m of the enzymes responsible for metabolism of glucose, namely hexokinases (∼40 μM [95]); (ii) after resupply of glucose to starved cells, cytosolic glucose and ATP levels increased proportionally with increasing external glucose concentrations; (iii) a steep gradient of glucose was maintained across the cell membrane, most probably due to efficient metabolic conversion; and (iv) significant amounts of 'free' glucose were detectable over the nanomolar to millimolar range, indicating that substrate channelling, if present, is not preventing detection of free glucose [96]. The cytosolic levels of ATP increased upon the addition of glucose and reached saturating levels at external glucose concentrations of 2 mM [94].…”

Section: The Use Of Fret Sensors To Study Sugar Flux In Yeast Cellssupporting

confidence: 76%

In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast

Bermejo

Ewald

Lanquar

et al. 2011

Biochemical Journal

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Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation.

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Section: The Use Of Fret Sensors To Study Sugar Flux In Yeast Cellssupporting

confidence: 76%

In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast

Bermejo

Ewald

Lanquar

et al. 2011

Biochemical Journal

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“…In addition, the possibility of H+ generation through the oxidation of glucose by the pentose phosphate shunt can be discounted since in strain AA28 fructose is as effective as glucose at inducing acidification. This strain requires small amounts of glucose to grow in a medium with fructose as carbon source (Aguilera, 1986), since glucans (Ballou, 1982) and inositol (Henry, 1982), as well as NADPH, are generated in yeasts from glucose 6-phosphate through direct oxidation (see Fraenkel, 1982). The glucose requirement of this strain indicates that fructose 6-phosphate cannot enter the pentose phosphate shunt.…”

Section: Discussionmentioning

confidence: 99%

The Mechanism of Intracellular Acidification Induced by Glucose in Saccharomyces cerevisiae

Ramos¹,

Balbı́n²,

Raposo³

et al. 1989

Microbiology

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Addition of glucose or fructose to cells of Saccharomyces cerevisiae adapted to grow in the absence of glucose induced an acidification of the intracellular medium. This acidification appeared to be due to the phosphorylation of the sugar since : (i) glucose analogues which are not efficiently phosphorylated did not induce internal acidification; (ii) glucose addition did not cause internal acidification in a mutant deficient in all the three sugar-phosphorylating enzymes; (iii) fructose did not affect the intracellular pH in a double mutant having only glucokinase activity; (iv) glucose was as effective as fructose in inducing the internal pH drop in a mutant deficient in phosphoglucose isomerase activity; and (v) in strains deficient in two of the three sugar-phosphorylating activities, there was a good correlation between the specific glucose-or fructose-phosphorylating activity of cell extracts and the sugar-induced internal acidification. In addition, in whole cells any of the three yeast sugar kinases were capable of mediating the internal acidification described. Glucose-induced internal acidification was observed even when yeast cells were suspended in growth medium and in cells suspended in buffer containing K+, which supports the possible signalling function of the glucose-induced internal acidification. Evaluation of internal pH by following fluorescence changes of fluorescein-loaded cells indicated that the change in intracellular pH occurred immediately after addition of sugar. The apparent K , for glucose in this process was 2 mM. Changes in both the internal and external pH were determined and it was found that the internal acidification induced by glucose was followed by a partial alkalinization coincident with the initiation of H+ efflux. This reversal of acidification could be due to the activity of the H+-ATPase, since it was inhibited by diethylstilboestrol. Coincidence between internal alkalinization and the H+ efflux was also observed after addition of ethanol.

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“…For example, hexokinase II might be involved in the generation of a phosphorylated metabolite. Hexokinase is known not to undergo autophosphorylation in normal catalysis but will inactivate itself by autophosphorylation in the presence of certain pentoses, such as xylose (5,11,23).…”

Section: Discussionmentioning

confidence: 99%

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

Ma¹,

Bloom²,

Walsh³

et al. 1989

Mol. Cell. Biol.

100

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Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase H is correlated with glucose repression.Hexokinase II is one of the two hexokinase isoenzymes of the budding yeast Saccharomyces cerevisiae. It functions in vivo both in glycolysis (16,17) and in catabolite repression (6,20). Among the mutations that cause failures in glucose repression (25), some have reduced levels of hexokinase activity; these mutations were determined to be alleles of the structural gene of hexokinase II, HXK2 (6). Biochemical analysis of one of the mutants (10) indicated that hexokinase II was indeed defective. The synthesis of invertase from the SUC3 gene (reviewed in references 3 and 4) was derepressed in these mutants even when cells were grown in medium with a high glucose concentration. These results suggest that hexokinase II mediates glucose repression in yeast cells. Mutations in another gene, HEX2 (which may be the same as REG] [22]), also cause failures in glucose repression, but hex2 strains have levels of hexokinase II activity higher than the wild-type level (7).It is difficult to interpret these results because mutations that either reduce (hxk2) or increase (hex2) hexokinase II catalytic activity can cause failures of glucose repression. To determine whether hexokinase II protein is required for glucose repression or derepression, we constructed null mutations in the HXKI (encoding hexokinase I) and HXK2 genes and studied their effects on glucose repression (20). The result that hxk2 null mutants do not show glucose repression indicates that hexokinase II is indeed required for glucose repression. Although hexokinase I is normally not required, it can partially suppress the hxk2 defect in glucose repression if overproduced. Entian and Frohlich (8) isolated mutations in the HXK2gene that cause defects in glucose repression yet still retain hexose phosphorylation activity. On the basis of these * Corresponding author. results and the conformational change observed in yeast hexokinases upon binding ...

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Inhibition and Inactivation of Glucose-phosphorylating Enzymes from Saccharomyces cerevisiae by D-Xylose

Cited by 22 publications

References 6 publications

In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast

In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast

The Mechanism of Intracellular Acidification Induced by Glucose in Saccharomyces cerevisiae

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

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