Biosynthesis and regulation of fructose-1,6-bisphosphatase and phosphofructokinase in Saccharomyces cerevisiae grown in the presence of glucose and gluconeogenic carbon sources.

The mode of synthesis and the regulation of fructose-1,6-bisphosphatase (Fbpase), a gluconeogenic enzyme, and phosphofructokinase (PFK), a glycolytic enzyme, were investigated in Saccharomyces cerevisiae after growth in the presence of different concentrations of glucose or various gluconeogenic carbon sources. The activity of FBPase appeared in the cells after the complete disappearance of glucose from the growth medium with a concomitant increase of the pH and no significant change in the levels of accumulated ethanol. The appearance of FBPase activity following glucose depletion was dependent upon the synthesis of protein. The FBPase PFK were present in glucose-, ethanol-, glycerol-, lactate-, or pyruvate-grown cells; however, the time of appearance and the levels of both these enzymes varied. The FBPase activity was always higher in 1% glucose-grown cells than in cells grown in the presence of gluconeogenic carbon sources. Phosphoglucose isomerase activity did not vary significantly. Addition of glucose to an FBPase and PFK synthesizing culture resulted in a complete loss, followed by a reappearance, of PFK activity. In the presence of cycloheximide the disappearance of glucose and the changes in the levels of FBPase and PFK were decreased significantly. It is concluded that S. cerevisiae exhibits a more efficient synthesis of FBPase after the exhaustion of glucose compared to the activity present in cells grown in the presence of exogenous gluconeogenic carbon sources. Two metabolically antagonistic enzymes, FBPase and PFK, are present during the transition phase, but not during the exponential phase, of growth, and the decay or inactivation of these enzymes in vivo may be dependent upon a glucose-induced protease activity.     

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

菠萝叶片中依赖焦磷酸的磷酸果糖激酶的光/暗调节特性(英文)

植物中D:果糖6—磷酸1—磷酸转移酶(PPi—PFK,EG 2.7.1.90)活性的调节是非常重要的。这主要是因为它能可逆催化糖酵解和生糖两个方向的反应。光照处理菠萝叶片或离体的菠萝叶圆片使PPi—PFK的酶活性增高。与从暗处理的叶片中提取的酶的特性相比,光照处理的叶片中的酶对糖酵解方向催化活性相对增加。暗处理导致酶催化精酵解方向活性的下降。这种反映在酶活性和特性上的变化可为光照重新恢复。结果表明,菠萝叶片的PPi—PFK对体内糖酵解或生糖途径的贡献可能决定于光的状况。     

Identification and biochemical characterization of two novel UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure.     

The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in Bacillus subtilis

The YvcK protein was previously shown to be dispensable when B. subtilis cells are grown on glycolytic carbon sources but essential for growth and normal shape on gluconeogenic carbon sources. Here, we report that YvcK is localized as a helical-like pattern in the cell. This localization seems independent of the actin-like protein, MreB. A YvcK overproduction restores a normal morphology in an mreB mutant strain when bacteria are grown on PAB medium. Reciprocally, an additional copy of mreB restores a normal growth and morphology in a yvcK mutant strain when bacteria are grown on a gluconeogenic carbon source like gluconate. Furthermore, as already observed for the mreB mutant, the deletion of the gene encoding the penicillin-binding protein PBP1 restores growth and normal shape of a yvcK mutant on gluconeogenic carbon sources. The PBP1 is delocalized in an mreB mutant grown in the absence of magnesium and in a yvcK mutant grown on gluconate medium. Interestingly, its proper localization can be rescued by YvcK overproduction. Therefore, in gluconeogenic growth conditions, YvcK is required for the correct localization of PBP1 and hence for displaying a normal rod shape.     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Metabolic adaptation of the renal carbohydrate metabolism. III

Summary The adaptive response of renal metabolism of glucose was studied in isolated rat proximal and distal renal tubules after a high protein-low carbohydrate diet administration. This nutritional situation significantly stimulated the gluconeogenic activity in the renal proximal tubules (about 1.5 fold at 48 hours) due, in part, to a marked increase in the fructose 1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) activities. In this tubular fragment, FBPase activity increased only at subsaturating fructose 1,6-bisphosphate concentration (30% at 48 hours) which involved a significant decrease in the Km (31%) for its substrate without changes in the Vmax. This enzymatic behaviour is probably related to modifications in the activity of the enzyme already present in the renal cells. Proximal PEPCK activity progressively increased at all substrate concentrations (almost 2 fold at 48h of high protein diet) which brought about changes in Vmax without changes in Km. These changes are in agreement with variations in the cellular concentration of the enzyme. Neither gluconeogenesis nor the gluconeogenic enzymes changed in the distal fractions of the renal tubules. On the other hand, a high protein diet did not apparently modify the glycolytic ability in any fragment of the nephron, although a significant increase in the phosphofructokinase (PFK) and pyruvate kinase (PK) activities was found in the distal renal tubules. This short term regulation involved a significant decrease from 24 hours in the Km value of distal PFK (almost 40%) without changes in Vmax. The kinetic behaviour of distal PK was mixed. In the first 24h after high protein diet a significant decrease in the Km for phosphoenolpyruvate was found (30%) without variation in the Vmax, however during the second 24 hours the activity of this glycolytic enzyme increased significantly (almost 1.3 fold) without modifications in its Km value. On the contrary, this nutritional state did not modify the kinetic behaviour of any glycolytic enzyme in the proximal regions of the renal tubules.     

Direct Measurements of Oscillatory Glycolysis in Pancreatic Islet β-Cells Using Novel Fluorescence Resonance Energy Transfer (FRET) Biosensors for Pyruvate Kinase M2 Activity

Pulses of insulin released from pancreatic β-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables β-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 β-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet β-cells expressing PKAR. These results indicate that PKM2 activity in β-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations.     

Sorting of metabolic pathway flux by the plasma membrane in cerebrovascular smooth muscle cells

We used-escin-permeabilized pig cerebral microvessels (PCMV) to study theorganization of carbohydrate metabolism in the cytoplasm of vascularsmooth muscle (VSM) cells. We have previously demonstrated (Lloyd PGand Hardin CD. Am J Physiol Cell Physiol 277: C1250-C1262,1999) that intact PCMV metabolize the glycolytic intermediate[1-¹³C]fructose 1,6-bisphosphate (FBP) to[1-¹³C]glucose with negligible production of[3-¹³C]lactate, while simultaneouslymetabolizing [2-¹³C]glucose to[2-¹³C]lactate. Thus gluconeogenic andglycolytic intermediates do not mix freely in intact VSM cells(compartmentation). Permeabilized PCMV retained the ability tometabolize [2-¹³C]glucose to[2-¹³C]lactate and to metabolize[1-¹³C]FBP to[1-¹³C]glucose. The continued existence ofglycolytic and gluconeogenic activity in permeabilized cells suggeststhat the intermediates of these pathways are channeled (directlytransferred) between enzymes. Both glycolytic and gluconeogenic flux inpermeabilized PCMV were sensitive to the presence of exogenous ATP andNAD. It was most interesting that a major product of[1-¹³C]FBP metabolism in permeabilized PCMV was[3-¹³C]lactate, in direct contrast to ourprevious findings in intact PCMV. Thus disruption of the plasmamembrane altered the distribution of substrates between the glycolyticand gluconeogenic pathways. These data suggest that organization of theplasma membrane into distinct microdomains plays an important role insorting intermediates between the glycolytic and gluconeogenic pathwaysin intact cells.

     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

In sycamore cells grown on nitrate as opposed to glutamate there is a higher pentose phosphate pathway carbon flux relative to glycolysis in the early stages of cell growth when nitrate assimilation is most active. The high pentose phosphate pathway activity compared with glycolysis in nitrate grown cells is accompanied by enhanced levels of hexokinase, pyruvate kinase, glucose-6-phosphate de-hydrogenase, 6-phosphogluconate dehydrogenase and transketolase. There is no significant increase in activity of the solely glycolytic enzyme, phosphofructokinase. It is suggested that the increased pentose phosphate pathway activity in nitrate grown cells is correlated with a demand by nitrite assimilation for NADPH.II=Jessup and Fowler, 1976 b     

Cytochrome P-450 and the activation of promutagens in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the cellular content of cytochrome P-450 was investigated and shown to be related to the growth phase of aerobic cultures when glucose was the carbon source. When grown on glucose medium the log-phase cells of the diploid strain D5 contained about 9× more cytochrome P-450 than log-phase cells of the diploid strain D4. The D4 cells grown on medium containing glucose contained about 10× more cytochrome P-450 than D4 cell grown on medium containing galactose as carbon source. Cells of strain D4, harvested from log-phase cultures grown on glucose, were capable of metabolizing aflatoxin B₁, dimethylnitrosamine, β-naphthylamine, ethyl carbamate, cyclophosphamide and dimethylsulphoxide to products active genetically in the same cells. The metabolism of the compounds tested was attributed to cyctochrome P-450-dependent mixed-function oxidation since genetic activity was high in log cells grown on medium containing glucose but negligible in log cells grown on medium containing galactose. However, aflatoxin B₁ differed from the other promutagens tested since the genetic activity of this compound in cells grown on galactose medium was similar to the activity in cells grown on glucose medium. This result is discussed in relation to enzyme systems which could metabolize aflatoxin B₁. The results of treating log-phase cells of the strain D5, grown on medium containing glucose, with aflatoxin B₁ and dimethylnitrosamine are presented and compared with the results from the strain D4.     

Mutated forms of phosphoglycerate mutase in yeast affect reversal of metabolic flux. Effect of reversible and irreversible function of an enzyme on pathway reversal

Phosphoglycerate mutase (GPM) functions reversibly in the glycolytic pathway. Mutations altering the reversibility of GPM have been obtained in the yeast, Saccharomyces cerevisiae. While wild-type cells grow on glycolytic (glucose) or gluconeogenic (ethanol) substrates, cells with altered GPMs fall into three categories based on their phenotypes 1) glucose- ethanol-, 2) glucose+ ethanol-, and 3) glucose- ethanol+. Cells with the first two phenotypes possessed GPMs that functioned irreversibly in the glycolytic direction. Cells that were glucose- ethanol+ possessed an enzyme that functioned reversibly. All of the altered GPMs had maximal velocities that were less than 3% of the wild-type level. The properties of the altered GPMs studied here provide a rationale for the occurrence in the glycolytic pathway of several glycolytic enzymes such as GPM, which function at high velocities in relation to the much smaller metabolic flux that they support. The altered GPMs were purified and estimates of their kinetic constants obtained. Free energy profiles were drawn for catalysis by the wild type and a mutant GPM that functioned irreversibly. The mutant enzyme was very inefficient. It was shown that an enzyme that functions irreversibly at a reaction with a Keq value close to 1 would necessarily be inefficient while it could evolve to be efficient when catalyzing a reaction that has a Keq value much greater than 1. In the glycolytic path this could be the reason for the characteristic presence of enzymes that function irreversibly at reactions with large Keq values.     

Antagonistic effects of hypertrehalosemic neuropeptide on the activities of 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase in cockroach fat body

Hypertrehalosemic neuropeptides from the corpora cardiaca such as the decapeptide Bld HrTH bring about a profound switch in the metabolic activity of cockroach fat body during which production of the blood sugar trehalose is stimulated while the catabolism of carbohydrate (glycolysis) is inhibited. The mechanisms of the metabolic switch are not fully understood.Incubation of isolated fat body from the cockroach Blaptica dubia with 10(-8) M Bld HrTH, for 10-60 min, stimulated glycogen breakdown and increased the content of the substrates of both the glycolytic enzyme 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) in the tissue. The glycolytic signal fructose 2,6-bisphosphate was markedly decreased in fat body on incubation with Bld HrTH. The content of ATP was slightly reduced, while the contents of ADP and AMP were increased after incubation with the hormone.Fructose 2,6-bisphosphate is a potent activator of PFK and a strong inhibitor of FBPase purified from fat body. The activity of PFK was decreased by about 90% when the hormone-dependent changes in effectors and substrates in fat body were simulated in vitro. FBPase, in contrast, was activated about 25-fold under these conditions, suggesting the hormone to stimulate gluconeogenesis in fat body. The data support the view that fructose 2,6-bisphosphate is a pivotal intracellular messenger in the hormone-induced metabolic switch from carbohydrate degradation to trehalose production in cockroach fat body.     

Regulation of ATP-dependent P-(Ser)-HPr formation in Streptococcus mutans and Streptococcus salivarius.

Sugar transport via the phosphoenolpyruvate (PEP) phosphotransferase system involves PEP-dependent phosphorylation of the general phosphotransferase system protein, HPr, at histidine 15. However, gram-positive bacteria can also carry out ATP-dependent phosphorylation of HPr at serine 46 by means of (Ser)HPr kinase. In this study, we demonstrate that (Ser)HPr kinase in crude preparations of Streptococcus mutans Ingbritt and Streptococcus salivarius ATCC 25975 is membrane associated, with pH optima of 7.0 and 7.5, respectively. The latter organism possessed 7- to 27-fold-higher activity than S. mutans NCTC 10449, GS-5, and Ingbritt strains. The enzyme in S. salivarius was activated by fructose-1,6-bisphosphate (FBP) twofold with 0.05 mM ATP, but this intermediate was slightly inhibitory with 1.0 mM ATP at FBP concentrations up to 10 mM. Similar inhibition was observed with the enzyme from S. mutans Ingbritt. A variety of other glycolytic intermediates had no effect on kinase activity under these conditions. The activity and regulation of (Ser)HPr kinase were assessed in vivo by monitoring P-(Ser)-HPr formation in steady-state cells of S. mutans Ingbritt grown in continuous culture with limiting glucose (10 and 50 mM) and with excess glucose (100 and 200 mM). All four forms of HPr [free HPr, P approximately (His)-HPr, P-(Ser)-HPr, and P approximately (His)-P-(Ser)-HPr] could be detected in the cells; however, significant differences in the intracellular levels of the forms were apparent during growth at different glucose concentrations. The total HPr pool increased with increasing concentrations of glucose in the medium, with significant increases in the P-(Ser)-HPr and P approximately HHis)-P-(Ser)-HPr concentrations. For example, while total PEP-dependent phosphorylation [P approximately(His)-HPr plus P approximately (His)-P-(Ser)-HPr] varied only from 21.5 to 52.5 microgram mg of cell protein (-1) in cells grown at the four glucose concentrations, the total ATP-dependent phosphorylation [P-(Ser)-HPr plus P approximately (His)-P-(Ser)-HPr] increased 12-fold from the 10 mM glucose-grown cells (9.1 microgram mg of cell protein (-1) to 106 and 105 microgram mg(-1) in the 100 and 200 mM glucose-grown cultures, respectively. (Ser)HPr kinase activity in membrane preparations of the cells varied little between the 10, 50, and 100 mM glucose-grown cells but increased threefold in the 200 mM glucose-grown cells. The intracellular levels of ATP, glucose-6-phosphate, and FBP increased with external glucose concentration, with the level of FBP being 3.8-fold higher for cells grown with 200 mM glucose than for those grown with 10 mM glucose. However, the variation in the intracellular levels of FBP, particularly between cells grown with 100 and 200 mM glucose, did not correlate with the extent of P-(Ser)-HPr formation, suggesting that the activity of (Ser)HPr kinase is not critically dependent on the availability of intracellular FBP.     

Inhibition of phosphoglucomutase activity by lithium alters cellular calcium homeostasis and signaling in Saccharomyces cerevisiae

Phosphoglucomutase is a key enzyme of glucose metabolism that interconverts glucose-1-phosphate and glucose-6-phosphate. Loss of the major isoform of phosphoglucomutase in Saccharomyces cerevisiae results in a significant increase in the cellular glucose-1-phosphate-to-glucose-6-phosphate ratio when cells are grown in medium containing galactose as carbon source. This imbalance in glucose metabolites was recently shown to also cause a six- to ninefold increase in cellular Ca²⁺ accumulation. We found that Li⁺ inhibition of phosphoglucomutase causes a similar elevation of total cellular Ca²⁺ and an increase in ⁴⁵Ca²⁺ uptake in a wild-type yeast strain grown in medium containing galactose, but not glucose, as sole carbon source. Li⁺ treatment also reduced the transient elevation of cytosolic Ca²⁺ response that is triggered by exposure to external CaCl₂ or by the addition of galactose to yeast cells starved of a carbon source. Finally, we found that the Ca²⁺ overaccumulation induced by Li⁺ exposure was significantly reduced in a strain lacking the vacuolar Ca²⁺-ATPase Pmc1p. These observations suggest that Li⁺ inhibition of phosphoglucomutase results in an increased glucose-1-phosphate-to-glucose-6-phosphate ratio, which results in an accelerated rate of vacuolar Ca²⁺ uptake via the Ca²⁺-ATPase Pmc1p. calcium influx; calcium signal; galactose; glucose phosphate     

Characterization of Phosphofructokinase Activity in Mycobacterium tuberculosis Reveals That a Functional Glycolytic Carbon Flow Is Necessary to Limit the Accumulation of Toxic Metabolic Intermediates under Hypoxia

Metabolic versatility has been increasingly recognized as a major virulence mechanism that enables Mycobacterium tuberculosis to persist in many microenvironments encountered in its host. Glucose is one of the most abundant carbon sources that is exploited by many pathogenic bacteria in the human host. M. tuberculosis has an intact glycolytic pathway that is highly conserved in all clinical isolates sequenced to date suggesting that glucose may represent a non-negligible source of carbon and energy for this pathogen in vivo. Fructose-6-phosphate phosphorylation represents the key-committing step in glycolysis and is catalyzed by a phosphofructokinase (PFK) activity. Two genes, pfkA and pfkB have been annotated to encode putative PFK in M. tuberculosis. Here, we show that PFKA is the sole PFK enzyme in M. tuberculosis with no functional redundancy with PFKB. PFKA is required for growth on glucose as sole carbon source. In co-metabolism experiments, we report that disruption of the glycolytic pathway at the PFK step results in intracellular accumulation of sugar-phosphates that correlated with significant impairment of the cell viability. Concomitantly, we found that the presence of glucose is highly toxic for the long-term survival of hypoxic non-replicating mycobacteria, suggesting that accumulation of glucose-derived toxic metabolites does occur in the absence of sustained aerobic respiration. The culture medium traditionally used to study the physiology of hypoxic mycobacteria is supplemented with glucose. In this medium, M. tuberculosis can survive for only 7–10 days in a true non-replicating state before death is observed. By omitting glucose in the medium this period could be extended for up to at least 40 days without significant viability loss. Therefore, our study suggests that glycolysis leads to accumulation of glucose-derived toxic metabolites that limits long-term survival of hypoxic mycobacteria. Such toxic effect is exacerbated when the glycolytic pathway is disrupted at the PKF step.     

An experimental study on carbon flow in Escherichia coli as a function of kinetic properties and expression levels of the enzyme phosphoglucomutase

Mutants of Escherichia coli deficient in phosphoglucomutase accumulate amylose when the cells are grown on maltose or galactose as carbon source. In the presence of physiological levels of phosphoglucomutase, most of the sugar is catabolized, leading to strongly reduced levels of amylose accumulation. By varying the expression level of heterologous phosphoglucomutase, we show that the minimum level needed to block amylose accumulation corresponds to a phosphoglucomutase activity of 150-600 nmole substrate transformed per min per mg of total soluble protein. Mutant phosphoglucomutases with strongly reduced Vmax values and increased Km values for the substrate glucose-1-phosphate or the co-substrate glucose-1,6-diphosphate, could also reduce amylose accumulation, but much higher enzyme expression levels were required.