Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate.

Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the Bacillus subtilis transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of ß-cell glycolytic heterogeneity and dynamics.

Aberrant Intracellular pH Regulation Limiting Glyceraldehyde-3-Phosphate Dehydrogenase Activity in the Glucose-Sensitive Yeast tps1Δ Mutant.

Whereas the yeast Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH4Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant.IMPORTANCE Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast tps1Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor.

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), we transplanted the B. subtilis FBP-binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

Resonance light scattering detection of fructose bisphosphates using uranyl-salophen complex-modified gold nanoparticles as optical probe.

In this paper, we report a resonance light scattering (RLS) method for the determination of fructose bisphosphates (FBPs) in water solution using fructose 1,6-bisphosphate (F-1,6-BP) as a model analyte without the procedure of extracting target analyte. The method used a type of modified gold nanoparticles (GNPs) as optical probe. The modified GNPs are uranyl-salophen-cysteamine-GNPs (U-Sal-Cy-GNPs) which are obtained through the acylation reaction of carboxylated salophen with cysteamine-capped GNPs (Cy-GNPs) to form Sal-Cy-GNPs and then the chelation reaction of uranyl with tetradentate ligand salophen in the Sal-Cy-GNPs. A FBP molecule is used easily to connect two U-Sal-Cy-GNPs to cause the aggregation of the GNPs by utilizing the specific affinity of uranyl-salophen complex to phosphate group, resulting in the production of strong RLS signal from the system. The amount of FBPs can be determined through detecting the RLS intensity change of the system. A linear range was found to be 2.5 to 75 nmol/L with a detection limit of 0.91 nmol/L under optimal conditions. The method has been successfully used to determine FBPs in real samples with the recoveries of 96.5-103.5 %.

Spectroscopic study on the reactions of bis-salophen with uranyl and then with fructose 1,6-bisphosphate and the analytical application.

The chelating reaction of bis-salophen with uranyl to form binuclear complex uranyl-bis-salophen (UBS) was studied by fluorescence spectroscopy. The coordination reaction of UBS with fructose 1,6-bisphosphate (F-1,6-BP) to form supramolecular polymer was then studied by resonance light scattering (RLS) spectroscopy. The reaction of bis-salophen with uranyl results in a remarkable enhancement of fluorescence intensity. The maximum emission wavelength of the fluorescence is at 471nm. The reaction of UBS with F-1,6-BP results in a remarkable enhancement of RLS intensity. The maximum scattering wavelength of the RLS is at 460nm. The two reactions were used to establish fluorescence method for the determination of uranium (VI) and RLS method for the determination of F-1,6-BP, respectively. Under optimum conditions, the linear ranges for the detection of uranium (VI) and F-1,6-BP are 0.003-0.35nmol/mL and 0.05-5.0nmol/mL, respectively. The detection limits are 0.0017nmol/mL and 0.020nmol/mL, respectively. The proposed fluorescence method has been successfully applied for the determination of uranium (VI) in environmental water samples with the recoveries of 97.0-104.0%. The proposed RLS method has also been successfully applied for the determination of F-1,6-BP in medicine injection samples with the recoveries of 98.5-102.3%.

Differences in the fatty acid metabolism of visceral adipose tissue in postmenopausal women.

OBJECTIVE: Visceral fat accumulation and metabolic syndrome incidence among women increase after menopause; therefore, fat metabolic changes and fat redistribution may occur according to menstrual status. The aim of our study was to clarify differences in subcutaneous and visceral adipose tissue metabolism between premenopausal and postmenopausal women, using metabolomics. METHODS: Thirty-nine (16 premenopausal and 23 postmenopausal) women were recruited through elective gynecologic surgery, and both subcutaneous and visceral adipose tissues were collected during surgical operation. Metabolite profiling of adipose tissue was performed by capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry. RESULTS: Sedoheptulose 7-phosphate, a midproduct of the pentose phosphate pathway, was significantly higher (P < 0.05) in visceral adipose tissues of premenopausal women. Dihydroxyacetone phosphate and fructose-1,6-biphosphate, midproducts of glycolysis, were significantly higher (P < 0.05) in subcutaneous adipose tissues of postmenopausal women. The concentrations of fatty acid metabolites-heptanoate (C7:0; premenopausal vs postmenopausal, 4.07 [0.72] vs 2.64 [0.28] nmol/g), octanoate (C8:0; 3.52 [0.29] vs 5.20 [0.29] nmol/g), and pelargonate (C9:0; 8.03 [0.49] vs 10.66 [0.44] nmol/g)-in the visceral fat (but not in subcutaneous fat) of postmenopausal women were significantly higher (P < 0.05) than those in the visceral fat of premenopausal women. CONCLUSIONS: Fatty acid metabolites increase in visceral fat (but not in subcutaneous fat) after menopause. The change in fatty acid metabolism in visceral adipose tissues might be related to metabolic syndrome in postmenopausal women.

Determination of fructose 1,6-bisphosphate using a double-receptor sandwich type fluorescence sensing method based on uranyl-salophen complexes.

In this paper, we report a double-receptor sandwich type fluorescence sensing method for the determination of fructose bisphosphates (FBPs) using fructose 1,6-bisphosphate (F-1,6-BP) as a model analyte based on uranyl-salophen complexes. The solid phase receptor is an immobilized uranyl-salophen (IUS) complex which is bound on the surface of glass slides by covalent bonds. The labeled receptor is another uranyl-salophen complex containing a fluorescence group, or uranyl-salophen-fluorescein (USF). In the procedure of determining F-1,6-BP in sample solution, F-1,6-BP is first adsorbed on the surface of the glass slide through the coordination reaction of F-1,6-BP with IUS. It then binds USF through another coordination reaction to form a sandwich-type structure of IUS-F-1,6-BP-USF. The amount of F-1,6-BP is detected by the determination of the fluorescence intensity of IUS-F-1,6-BP-USF bound on the glass slide. Under optimal conditions, the linear range for the detection of F-1,6-BP is 0.05-5.0 nmol mL(-1) with a detection limit of 0.027 nmol mL(-1). The proposed method has been successfully applied for the determination of F-1,6-BP in real samples with satisfactory results.

Metabolomics of supragingival plaque and oral bacteria.

Dental caries is initiated by demineralization of the tooth surface through acid production by sugar metabolism of supragingival plaque microflora. To elucidate the sugar metabolic system, we used CE-MS to perform metabolomics of the central carbon metabolism, the EMP pathway, the pentose-phosphate pathway, and the TCA cycle in supra- gingival plaque and representative oral bacteria, Streptococcus and Actinomyces. Supragingival plaque contained all the targeted metabolites in the central carbon metabolism, except erythrose 4-phosphate in the pentose-phosphate pathway. After glucose rinse, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and pyruvate in the EMP pathway and 6-phosphogluconate, ribulose 5-phosphate, and sedoheptulose 7-phosphate in the pentose-phosphate pathway, and acetyl CoA were increased. Meanwhile, 3-phosphoglycerate and phosphoenolpyruvate in the EMP pathway and succinate, fumarate, and malate in the TCA cycle were decreased. These pathways and changes in metabolites observed in supragingival plaque were similar to the integration of metabolite profiles in Streptococcus and Actinomyces.

Pfkfb3 is transcriptionally upregulated in diabetic mouse liver through proliferative signals.

The ubiquitous isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (uPFK-2), a product of the Pfkfb3 gene, plays a crucial role in the control of glycolytic flux. In this study, we demonstrate that Pfkfb3 gene expression is increased in streptozotocin-induced diabetic mouse liver. The Pfkfb3/-3566 promoter construct linked to the luciferase reporter gene was delivered to the liver via hydrodynamic gene transfer. This promoter was upregulated in streptozotocin-induced diabetic mouse liver compared with transfected healthy cohorts. In addition, increases were observed in Pfkfb3 mRNA and uPFK-2 protein levels, and intrahepatic fructose-2,6-bisphosphate concentration. During streptozotocin-induced diabetes, phosphorylation of both p38 mitogen-activated protein kinase and Akt was detected, together with the overexpression of the proliferative markers cyclin D and E2F. These findings indicate that uPFK-2 induction is coupled to enhanced hepatocyte proliferation in streptozotocin-induced diabetic mouse liver. Expression decreased when hepatocytes were treated with either rapamycin or LY 294002. This shows that uPFK-2 regulation is phosphoinositide 3-kinase-Akt-mammalian target of rapamycin dependent. These results indicate that fructose-2,6-bisphosphate is essential to the maintenance of the glycolytic flux necessary for providing energy and biosynthetic precursors to dividing cells.

Control analysis of the role of triosephosphate isomerase in glucose metabolism in Lactococcus lactis.

Triosephosphate isomerase (TPI), which catalyses the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), was studied for its control on glycolysis and mixed acid production in L. lactis subspecies lactis IL1403 and L. lactis subspecies cremoris MG1363. Strains in which the TPI activity was modulated from 3%-225% (IL1403) or 13%-103% (MG1363) of the wild-type level were constructed by changing the expression of the tpiA gene. The enzyme was found to be present in high excess in the wild-type cells and 10% TPI activity still supported more than 70% of the wild-type glycolytic flux in both strains. Homolactic product formation was preserved throughout the range of TPI activities studied, although a slight increase in the amount of acetate and formate production was observed in the strains with strongly reduced TPI activity for both IL1403 and MG1363. The upstream metabolites glucose-6-phosphate, fructose-1,6-bisphosphate and DHAP in the IL1403 derivatives were essentially unchanged for TPI activities from 26% to 225%. At a TPI activity of 3%, the level of DHAP increased four times. The finding that an increased level of DHAP coincides with an increase in formate production is surprising and indicates that pyruvate formate lyase is not inhibited by DHAP under these conditions.

Separation and quantitation of fructose-6-phosphate and fructose-1 ,6-diphosphate by LC-ESI-MS for the evaluation of fructose-1,6-biphosphatase activity.

An LC-ESI-MS method was developed for the identification and quantification of fructose-1,6-biphosphate (F1,6BP) and fructose-6-phosphate (F6P), respectively the substrate and the product of the enzymatic reaction catalysed by fructose-1,6-bisphosphatase (F1,6BPase). F1,6BPase, expressed predominantly in liver and kidney, is one of the rate-limiting enzymes of hepatic gluconeogenesis and has become a target for the development of new drugs for type 2 diabetes. The two sugar phosphates were separated on a Phenomenex Luna NH2 column (150 mm x 2.0 mm id) using the following mobile phase: 5 mM triethylamine acetate buffer/ACN (80:20) v/v in a linear pH gradient (from pH = 9 to 10 in 15 min) at the flow rate of 0.3 mL/min. The detection was performed with an IT mass spectrometer in negative polarity (full scan 100-450 m/z) and in SIM mode on the generated anions at m/z = 339 (F1,6BP) and m/z = 259 (F6P). Under the optimised final conditions, the method was validated for accuracy, specificity, precision (inter- and intradays RSD comprised between 1.0 and 6.3% over the range of concentrations used), linearity (50-400 microM), LODs (0.44 microM) and LOQs (1.47 microM), and the method was applied to F6P determination in the F1,6BPase catalysed hydrolysis of F1,6BP.

Difference in the xylitol sensitivity of acid production among Streptococcus mutans strains and the biochemical mechanism.

Xylitol inhibits the glycolysis and growth of Streptococcus mutans, but to different degrees among strains. Thus, we studied the biochemical mechanism through which the inhibition varies, using S. mutans strains ATCC 31989, NCTN 10449, and NCIB 11723, which are highly sensitive, moderately sensitive, and resistant to xylitol, respectively, under strictly anaerobic conditions such as those found in deep layers of dental plaque. Xylitol (30 mM) decreased the rate of acid production from glucose (10 mM) in ATCC 31989, NCTC 10449, and NCIB 11723 by 86, 26, and 0%, respectively. The activities of the xylitol : phosphoenolpyruvate phosphotransferase system (PEP-PTS) relative to those of glucose : PEP-PTS were 120, 16, and 3%, respectively. In ATCC 31989 and NCTC 10449, intracellular accumulation of xylitol 5-phosphate and decreases of fructose 1,6-bisphosphate and glucose 6-phosphate were observed. Furthermore, in the presence of xylitol (30 mM), glucose : PEP-PTS activities decreased by 34, 17, and 0%, respectively. These findings indicated that the higher the xylitol : PEP-PTS activity was and the more effectively xylitol decreased glucose : PEP-PTS activity, the more sensitive the strain was to xylitol. These results suggest that the following inhibitory mechanisms are active in the xylitol-sensitive mutans streptococci: direct inhibition of glycolytic enzymes by xylitol 5-phosphate derived from xylitol : PEP-PTS and, possibly, indirect inhibition through competition for the phosphoryl donor, HPr-P, between glucose and xylitol : PEP-PTSs.

Effect of acetic acid feeding on the circadian changes in glycogen and metabolites of glucose and lipid in liver and skeletal muscle of rats.

The aim of the present study is to investigate the effect of acetic acid feeding on the circadian changes in glycogen concentration in liver and skeletal muscle. Rats were provided meal once daily (09.00-13.00 hours) for 10 d. On the 11th day, they were either killed immediately or given 9 g diet containing either 0 (control) or 0.7 g/kg-diet acetic acid beginning at 09.00 hours for 4 h, as in the previous regimen. Rats in the fed group were killed at 4, 8 or 24 h after the start of feeding. At 4 h after the start of feeding, the acetic acid group had significantly greater liver and gastrocnemius muscle glycogen concentrations (P<0.05). Also, at this same point, liver xylulose-5-phosphate, a key stimulator of glycolysis, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate in skeletal muscle, which reflects phosphofructokinase-1 activity, and liver malonyl-CoA, an allosteric inhibitor of carnitine palmitoyl-transferase, were significantly lower in the acetic acid group than in the control group (P<0.05). In addition, the acetic acid group had a significantly lower serum lactate concentration and lower ratio of insulin to glucagon than the control group at the same point (P<0.05). We conclude that a diet containing acetic acid may enhance glycogen repletion but not induce supercompensation, a large increase in the glycogen level that is beneficial in improving performance, in liver and skeletal muscle by transitory inhibition of glycolysis. Further, we indicate the possibility of a transient enhancement of fatty acid oxidation in liver by acetic acid feeding.

Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques.

The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement ("metabolic engineering"). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand "in vivo" reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume.

Tungstate is an effective antidiabetic agent in streptozotocin-induced diabetic rats: a long-term study.

AIMS/HYPOTHESIS: Recent studies have shown the anti diabetic effects of oral sodium tungstate treatment in several animal models of diabetes based on short-term experiments. In this study, we examined the effectiveness of long-term tungstate treatment of streptozotocin-induced-diabetic rats. METHODS: Tungstate was administered to the drinking water of rats for eight months. RESULTS: The treatment resulted in a reduction in serum glucose concentrations in diabetic rats, but no change in glycaemia was detected in healthy rats. Alterations in the hepatic glucose metabolism due to diabetes were almost completely counteracted by tungstate treatment. The partial recovery of glucokinase activity, not found in diabetic animals, normalised glycogen and glucose 6-phosphate concentrations. Tungstate treatment also restored pyruvate kinase activity and fructose 2,6-bisphosphate concentrations. In healthy rats, tungstate treatment did not modify the majority of the hepatic parameters studied. Moreover, tungstate treatment prevented diabetes-induced morphological changes in the kidney and ocular lens and also reduced mortality. Furthermore, no hypoglycaemic episodes or undesirable side effects were observed in treated diabetic or healthy rats. In addition, there is no evidence of intolerance developing after prolonged use. CONCLUSION/INTERPRETATION: Tungstate could play a helpful part in the long-term treatment of diabetes.

Photosynthetic carbohydrate metabolism in wheat (Triticum aestivum L.) leaves: optimization of methods for determination of fructose 2, 6-bisphosphate.

The accurate measurement of fructose 2,6-bisphosphate from plants such as wheat is fraught with difficulty. Extraction and assay methods for fructose 2,6-bisphosphate that give near 100% recovery of the metabolite, and a linear response with volume have therefore been developed for extracts prepared from wheat leaves of different ages. Amounts of fructose 2,6-bisphosphate in different regions of leaves generally showed a positive correlation with chlorophyll content. Measurements of sucrose and starch in third leaves harvested at different times of the diurnal cycle demonstrated that sucrose is the major form in which photosynthate is stored in the leaf, but starch can account for up to about 30% of the stored carbohydrate. Virtually all of the carbohydrate accumulated as starch and sucrose during the day was degraded at night. Amounts of fructose 2,6-bisphosphate were generally lower in extracts prepared from leaves harvested in the light than in the dark. Additionally, there was no change in either the amount of fructose 2, 6-bisphosphate or the ratio of sucrose to starch in samples prepared from leaves harvested at different times of the day. These results are broadly consistent with a role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis and the partitioning of carbohydrate between sucrose and starch in wheat leaves.

Analysis of sugar metabolism in an EPS producing Lactococcus lactis by 31P NMR.

Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31P NMR. Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energization by the addition of glucose. EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former. They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium. They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls. By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars. The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.

[Determination of fructose-1,6-diphosphate with aldolase-DNPH by the colorimetric method].

This paper presents a modified method of enzymatic assay for Fructose-1,6-diphosphate(FDP). FDP is split to dihydroxyacetone phosphate (DAP) and glyceraldehyde-3-phosphate (GAP) by the action of aldolase. DAP is hydrolyzed at room temperature to free triose. Under alkaline conditions, the free triose is reacted with 2,4-dinitrophenylhydrazine (DNPH), yielding a 2,4-dinitrophenylhydrazine derivative which dissolve in alkali forming a purple color mixture, with maximum absorption at 540.nm. It is proportional to the contents of FDP. Because the method depends on the colorimetric determination of triose formed from fructose-1,6-diphosphate only by aldolase, glycerophosphate dehydrogenase/triosephosphate isomerase (GDH/TIM) and reduced nicotinamide adenine dinucleotide (NADH) which usually applied in multienzymatic method, are omitted in the modified method. The method is specific, convenient and accuracy for the determination of FDP.

A potential role of the cytoskeleton of Saccharomyces cerevisiae in a functional organization of glycolytic enzymes.

Numerous individual enzymes participate in a given synthetic or degradative pathway in which the product of one reaction becomes the substrate for the subsequent enzyme. This raises the question of whether the product of one 'soluble' enzyme diffuses freely through the available cell volume, where it accidentally collides with the subsequent 'soluble' enzyme. Alternatively, enzymes acting in a given pathway may be organized in ordered structures, metabolons. Certain glycolytic enzymes have been shown to co-localize with the cytoskeleton in mammalian cells. We deleted genes coding for proteins associated with the cytoskeleton of Saccharomyces cerevisiae: TPM1 coding for tropomyosin, SAC6 for fimbrin and CIN1 for a microtubule-associated protein. Single deletions or deletions of two such genes had no effect on the specific activities of glycolytic enzymes, or on the rates of glucose consumption and ethanol production. However, the concentrations of glycolytic metabolites during a switch from a gluconeogenic mode of metabolism, growth on an ethanol medium, to glycolysis after glucose addition showed transient deviations from the normal change in metabolite concentrations, as observed in wild type cells. However, all metabolites in mutant strains reached wild-type levels within 2-4 h after the shift. Only ATP levels remained low in all but the tmp1-Delta-sac6-Delta double mutant strains. These observations can be interpreted to mean that metabolic reorganization from a gluconeogenic to a glycolytic metabolism is facilitated by an intact cytoskeleton in yeast.

Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications.

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.