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Disequilibrium in triose phosphate isomerase system in rat liver

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Abstract

1. The equilibrium constant at 38 degrees and I 0.25 of the triose phosphate isomerase reaction was found to be 22.0 and that of the aldolase reaction, 0.99x10(-4)m. The [dihydroxyacetone phosphate]/[glyceraldehyde phosphate] ratio was found to be 9.3 in rat liver. The causes of the apparent deviation of the triose phosphate isomerase system from equilibrium in vivo have been investigated. 2. The equilibria of the triose phosphate isomerase and aldolase reactions were studied with relatively large concentrations of crystalline enzymes and small concentrations of substrates, approximating to those found in rat liver and muscle. There was significant binding of fructose diphosphate by aldolase under these conditions. There was no evidence that binding of glyceraldehyde phosphate by either enzyme affected the equilibria. 3. The deviation from equilibrium of the triose phosphate isomerase system in rat liver can be accounted for by the low activity of the enzyme, in relation to the flux, at low physiological concentrations of glyceraldehyde phosphate (about 3mum). It has been calculated that a flux of 1.8mumoles/min./g. wet weight of liver would be expected to cause the measured degree of disequilibrium found in vivo. 4. The conclusion that the triose phosphate isomerase is not at equilibrium is in accordance with the situation postulated by Rose, Kellermeyer, Stjernholm & Wood (1962) on the basis of isotope-distribution data. 5. The triose phosphate isomerase system is closer to equilibrium in resting muscle probably because of a very low flux and a high enzyme concentration. 6. The aldolase system deviated from equilibrium in rat liver by a factor of about 10 and by a much greater factor in resting muscle. 7. The measurement of total dihydroxyacetone phosphate and glyceraldehyde phosphate content indicates the concentrations of the free metabolites in the tissue. This may not hold for fructose diphosphate, a significant proportion of which may be bound to aldolase.

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... The relative intensities of Fru-P2:DHAP: GAP were 95:21:4, where the DHAP intensity was determined from the sum of its two forms in peaks B and F. We estimate that [Fru-P2] 10 mM-first on the basis of its integrated intensity being about twice that of ATP, which under our conditions has been shown (11) to be about 5 mM, and second by its near equality to the Pi intensity, which was originally fixed at 10 mM. In this event the ratio ([DHAP][GAP])/[Fru-P2] 0.10 mM, which is in excellent agreement with the reported values of the equilibrium constant, around 0.1 mM at 380C (12)(13)(14). In contrast to these equilibrium concentrations, the ratio [DHAP]/[GAP] is about 5.2, quite different from the equilibrium ratio of about 20 previously reported (14) for the isomerization of these two compounds, which is catalyzed by triose phosphate isomerase. ...
... In this event the ratio ([DHAP][GAP])/[Fru-P2] 0.10 mM, which is in excellent agreement with the reported values of the equilibrium constant, around 0.1 mM at 380C (12)(13)(14). In contrast to these equilibrium concentrations, the ratio [DHAP]/[GAP] is about 5.2, quite different from the equilibrium ratio of about 20 previously reported (14) for the isomerization of these two compounds, which is catalyzed by triose phosphate isomerase. In the past, nonequilibrium values of the [DHAP]/[GAP] ratio had been observed in cellular extracts and it was concluded that this reflected intracellular nonequilibrium concentrations of these metabolites (14,15). ...
... In contrast to these equilibrium concentrations, the ratio [DHAP]/[GAP] is about 5.2, quite different from the equilibrium ratio of about 20 previously reported (14) for the isomerization of these two compounds, which is catalyzed by triose phosphate isomerase. In the past, nonequilibrium values of the [DHAP]/[GAP] ratio had been observed in cellular extracts and it was concluded that this reflected intracellular nonequilibrium concentrations of these metabolites (14,15). However, their low concentrations, in the micromolar range, meant that appreciable fractions might be bound in the cell. ...
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High-resolution 31P nuclear magnetic resonance spectra at 145.7 MHz are reported for intact Ehrlich ascites tumor cells and their perchloric acid extracts. In the extracts it was possible to assign resonances to fructose 1,6-bisphosphates, dihydroxyacetone phosphate, ATP, ADP, AMP, Pi, NAD+, phosphorylcholine, glycero-3-phosphorylcholine, glycero-3-phosphorylethanolamine, and glyceraldehyde 3-phosphate from their chemical shifts, pH behavior, and spin couplings. All but glyceraldehyde 3-phosphate were observed and assigned in the intact cells. It was possible to show that the hydrolysis of fructose 1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate is in equilibrium, that the dihydroxyacetone phosphate leads to glyceraldehyde 3-phosphate reaction is not, and that in the intact cell without added oxygen or glucose the reaction 2ADP in equilibrium ATP + AMP is in equilibrium. From the known pH dependence of the Pi resonance it was possible to show that during aerobic or anerobic glycolysis the difference between intracellular and extracellular pH values was less than 0.2 pH units. Upon oxygenation the ATP concentration increased while the ADP concentration fell. Introducing deoxyglucose depleted the ATP and resulted in an AMP signal and one from deoxyglucose 6-phosphate, which is transported and phosphorylated but not catabolized.
... (1). In the recent years, new standard data (especially R g 0 ) for glycolytic reactions have been accessed by equilibrium concentrations [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] combined with thermodynamic modeling 12,17,18,[22][23][24]26 . In these works, it was proven that the equilibrium concentrations of glycolytic reactions strongly depend on the medium conditions. ...
... K a (pH 7, 298.15 K) finally yields www.nature.com/scientificreports/ R g 0 of the aldolase reaction was determined in this work based on equilibrium concentrations measured at pH 7, 311.15 K, 10 mM sodium phosphate buffer and 230 mM KCl by Veech et al. 14 . To obtain K a , it was again assumed that concentration c i equals molality m i and ePC-SAFT was used to calculate K γ = 5.8, yielding K a (pH 7, 311.15 K) with parameters from Tables 2 and 3 5)) was then used to convert K a (pH 7, 311.15 K) to K a (pH 7, 298.15 K). ...
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Thermodynamic feasibility analyses help evaluating the feasibility of metabolic pathways. This is an important information used to develop new biotechnological processes and to understand metabolic processes in cells. However, literature standard data are uncertain for most biochemical reactions yielding wrong statements concerning their feasibility. In this article we present activity-based equilibrium constants for all the ten glycolytic reactions, accompanied by the standard reaction data (standard Gibbs energy of reaction and standard enthalpy of reaction). We further developed a thermodynamic activity-based approach that allows to correctly determine the feasibility of glycolysis under different chosen conditions. The results show for the first time that the feasibility of glycolysis can be explained by thermodynamics only if (1) correct standard data are used and if (2) the conditions in the cell at non-equilibrium states are accounted for in the analyses. The results here will help to determine the feasibility of other metabolisms and to understand metabolic processes in cells in the future.
... Synthesis of [U- 13 Figure 1F). In the presence of triose phosphate isomerase (TPI), this was converted to an equilibrium mixture containing >95% [U-13 C]DHAP, 41 which was separated from unreacted FBP and GAP by HVPE at pH 6.5 ( Figure 2E). The final yield of [U-13 C]DHAP after HVPE purification was 37%, mainly reflecting the inefficiency of the aldolase reaction (Table S2, Supporting Information). ...
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Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, co-eluting components in tissue extracts can mutually interfere with ionization at the interface of the LC and MS/MS phases, potentially causing under- or over-estimation of metabolite concentrations. Spiking of samples with known amounts of stable isotope labelled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS, and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose- and triose-phosphates, UDP-glucose and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were these were validated by LC-MS/MS analyses of extracts from leaves, non-photosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and baker's yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC-MS/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With a suitable tissue sampling procedure and use of SIL-IS to correct for matrix effects, LC-MS/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms.
... In mammalian metabolism, MG is formed predominantly as a side product of glycolysis by non-enzymatic degradation of triosephosphates, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetonephosphate (DHAP) [100,101]. Usually, 0.05% to 0.1% of the glycolytic flux is converted into MG [102], resulting in concentrations of 50-150 nM in human plasma [103,104]. Since MG is produced proportional to the glycolytic flux, it is not surprising that elevated MG plasma levels can be found in diseases characterized by altered glycolytic flux (e.g., diabetes mellitus). ...
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Sepsis remains one of the leading causes of death in intensive care units. Although sepsis is caused by a viral, fungal or bacterial infection, it is the dysregulated generalized host response that ultimately leads to severe dysfunction of multiple organs and death. The concomitant profound metabolic changes are characterized by hyperglycemia, insulin resistance, and profound transformations of the intracellular energy supply in both peripheral and immune cells. A further hallmark of the early phases of sepsis is a massive formation of reactive oxygen (ROS; e.g., superoxide) as well as nitrogen (RNS; e.g., nitric oxide) species. Reactive carbonyl species (RCS) form a third crucial group of highly reactive metabolites, which until today have been not the focus of interest in sepsis. However, we previously showed in a prospective observational clinical trial that patients suffering from septic shock are characterized by significant methylglyoxal (MG)-derived carbonyl stress, with the glyoxalase system being downregulated in peripheral blood mononuclear cells. In this review, we give a detailed insight into the current state of research regarding the metabolic changes that entail an increased MG-production in septicemia. Thus, we point out the special role of the glyoxalase system in the context of sepsis.
... The value of k cat = (300 ± 30) s −1 determined for the Tbb TIM-catalyzed reaction of DHAP is also smaller than an earlier published value of (730 ± 200) s −1 (49). However, the ratio of the values of k cat /K m for the Tbb TIM-catalyzed reactions of GAP and DHAP is (8.0 × 10 6 M −1 s −1 )/(4.3 × 10 5 M −1 s −1 ) = 19 is in good agreement with the value of the equilibrium constant for the conversion of GAP to DHAP, K eq = 22, determined at 38°C (62), as required by the Haldane relationship. Table 1 also lists kinetic parameters for the isomerization reactions catalyzed by TIMs from yeast, rabbit muscle and chicken muscle. ...
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Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by (1)H NMR spectroscopy and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O'Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005), Biochemistry 44, 2610 - 2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((k(C1))(D)/(k(C2))(D) = 1.1) than for the wildtype enzyme ((k(C1))(D)/(k(C2))(D) = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-(13)C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-(13)C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle [Amyes, T. L. and Richard, J. P. (2007) Biochemistry 46, 5841-5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM.
... The difference (5.8 Ϯ 0.2) was due to the PPP activity; it cannot arise from metabolism in the CAC, transaldolase, incomplete equilibration at TPI, or any combination of the pathways. When the label was provided in lactate rather than glycerol, the difference between [1,2-13 C 2 ]/[2,3-13 C 2 ] and [5,6-13 C 2 ]/[4,5-13 C 2 ] was smaller, only 3.1 Ϯ 0.4 (Fig. 4, B and C). 13 C enrichment in plasma glucose was measured by the sum of all glucose isotopomers with excess 13 C, including singlylabeled glucose plus multiply labeled glucose such as [1,2-13 C 2 ]-, [2,3-13 C 2 ]-, [1,2,3-13 C 3 ]-, [5,6-13 C 2 ]-, [4,[5][6][7][8][9][10][11][12][13] C 2 ]-, [4,5,6-13 C 3 ]-, and [U-13 C 6 ]glucose. In fasted rats supplied with [U-13 C 3 ]glycerol, unlabeled glucose, and unlabeled lactate, the sum of excess-labeled glucose isotopomers was 14.1 Ϯ 1.6% whereas the sum in rats given [U-13 C 3 ]lactate, unlabeled glucose, and unlabeled glycerol was 11.8 Ϯ 0.4%. ...
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After exposure to [U-13C3]glycerol, the liver produces primarily [1,2,3-13C3]- and [4,5,6-13C3]glucose in equal proportions through gluconeogenesis from the level of trioses. Other 13C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex 13C-labeling patterns in exported glucose. Here, we investigated 13C labeling in plasma glucose in rats given [U-13C3]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a 13C-13C bond within the trioses contributing to glucose, the appearance of [1,2-13C2]-, [2,3-13C2]-, [5,6-13C2]-, and [4,5-13C2]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-13C2]glucose/[2,3-13C2]glucose was significantly greater than [5,6-13C2]glucose/[4,5-13C2]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by 13C distribution in blood glucose after [U-13C3]glycerol administration.
... 8-fold more reactive than DHAP in degrading to MG but as the concentration ratio of DHAP/GA3P in cells in situ is ca. 9 or similar [9], both of these triosephosphates are important sources of MG formation in physiological systems in situ [10]. MG formation is a minor fate of triosephosphates: early studies with red blood cells suggested only 0.089 % glucotriose (2 x glucose consumption) was converted to MG [11] and our subsequent studies with endothelial cells and fibroblasts suggest a similar flux. ...
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The reactive dicarbonyl metabolite methylglyoxal (MG) is the precursor of the major quantitative advanced glycation endproducts (AGEs) in physiological systems - arginine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. The glyoxalase system in the cytoplasm of cells provides the primary defence against dicarbonyl glycation by catalysing the metabolism of MG and related reactive dicarbonyls. Dicarbonyl stress is the abnormal accumulation of dicarbonyl metabolites leading to increased protein and DNA modification contributing to cell and tissue dysfunction in ageing and disease. It is produced endogenously by increased formation and/or decreased metabolism of dicarbonyl metabolites. Dicarbonyl stress contributes to ageing, disease and activity of cytotoxic chemotherapeutic agents. It contributes to ageing through age-related decline in glyoxalase 1 (Glo-1) activity. Glo-1 has a dual role in cancer as a tumour suppressor protein prior to tumour development and mediator of multi-drug resistance in cancer treatment, implicating dicarbonyl glycation of DNA in carcinogenesis and dicarbonyl-driven cytotoxicity in mechanism of action of anticancer drugs. Glo-1 is a driver of cardiovascular disease, likely through dicarbonyl stress-driven dyslipidemia and vascular cell dysfunction. Dicarbonyl stress is also a contributing mediator of obesity and vascular complications of diabetes. There are also emerging roles in neurological disorders. Glo-1 responds to dicarbonyl stress to enhance cytoprotection at the transcriptional level through stress-responsive increase of Glo-1 expression. Small molecule Glo-1 inducers are in clinical development for improved metabolic, vascular and renal health and Glo-1 inhibitors in preclinical development for multidrug resistant cancer chemotherapy.
... Postal 510-3, 62250 Cuernavaca, Mor., México; Tel: +52 (777) 329-1640; E-mail: gsaab@ibt.unam.mx phates in favor of DHAP formation by 22-fold at 38°C and pH 7.5 (96% DHAP and 4% GAP) [7]. Because only GAP completes glycolysis, TIM prevents the accumulation of DHAP [8], which could deplete intracellular ATP, the trapping of inorganic phosphate, and/or the formation of toxic methylglyoxal [9]. ...
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... Substrate diffusion into catalytically competent crystals-Surprisingly, the first distinguishable ligands observed in the active site as FBP diffused into the crystals were the products of the aldolase forward reaction, namely the triose-Ps, DHAP and D-G3P. This would appear contrary to the thermodynamic equilibrium governing the aldolase reaction whose Keq for the cleavage reaction is ~ 10 -4 -10 -5 M, favoring aldol addition (24,25). However, diffusion by FBP into the crystal lattice establishes a transient concentration gradient of FBP, prior to its equilibration, providing an explanation for these observations. ...
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... Besides non-oxidative PPP intermediates, dihydroxyacetone phosphate (DHAP) was measured to provide a consistency check based on thermodynamic principles. Triose-phosphate isomerase (TPI), being the most active enzyme in glycolysis, is thought to maintain equilibrium between the pools of DHAP and GAP, in which case the ratio of their concentrations should be approximately 22:1 (K eq , TPI = 0.045 [25]). The measured ratio of DHAP vs GAP was indeed close to the TPI equilibrium constant both at steady state and throughout the pulse further supporting the validity of the results ( Table 2 and Fig. 3). ...
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... Vojinović and von Stockar: Influence of Uncertainties on TFA of Metabolism does not provide the concentration of Mg 2þ present in the experimental reaction medium. In fact, the authors state that the pH variation between 6.35 and 7.12 and the variation of the Mg 2þ concentration from 0 to 5 mM had no effect on the observed apparent equilibrium constant (Veech et al., 1969). This indicates that the acid and complex dissociation constants for the GAP and DHAP are very close in values to each other. ...
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Gluconeogenesis from lactate in hepatocytes from rainbow trout is activated by glucagon and inhibited by insulin in a dose-dependent fashion. The maximal responses to both hormones occur within their probable physiological concentration ranges. Gluconeogenic activation by glucagon is accompanied by a profound inhibition of pyruvate kinase activity. This is reflected in increases in both the S0.5 and nH for phosphoenolpyruvate and increased sensitivity to inhibition by MgATP compared to the enzyme isolated from cells not treated with hormone (control). Gluconeogenic inhibition by insulin is accompanied by activation of pyruvate kinase activity. This is observed as decreases in both the S0.5 and nH for phosphoenolpyruvate and a decreased sensitivity to inhibition by MgATP compared to enzyme isolated from control cells. These results indicate that insulin and glucagon are involved in the regulation of hepatic gluconeogenesis in trout and that their effects are mediated, at least partly, through the control of pyruvate kinase activity.
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Glycolytic intermediates and related metabolites were measured in the fat body of the American cockroach (Periplaneta americana) to locate the rate-limiting reactions that regulate glycolysis during the action of the corpus cardiacum (CC) in vitro. 1. The concentrations of glucose 1-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate were approximately doubled after 30 min treatment with CC extract, whereas that of glucose 6-phosphate increased more than four-fold. Slightly lower increases occurred after 10 and 60 min treatment. 2. Triose phosphates, 2-phosphoglyceric acid, phosphoenolpyruvate and pyruvate were unaffected by CC extract. 3. Glycerol 3-phosphate, which is 20\2-200 times more concentrated than any of the other measured metabolites in the unstimulated tissue, is increased more than two-fold by CC extract. 4. NAD, NADP, and ATP were not significantly affected by CC extract. ADP was increased significantly by the gland extract. The data support the view that glycolytic flux may be restricted at the aldolase catalyzed reaction.
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One of the challenges in strain improvement by evolutionary engineering is to subsequently determine the molecular basis of the improved properties that were enriched from the natural genetic variation during the selective conditions. This study focuses on Saccharomyces cerevisiae IMS0002 which, after metabolic and evolutionary engineering, ferments the pentose sugar arabinose. Glucose- and arabinose-limited anaerobic chemostat cultures of IMS0002 and its non-evolved ancestor were subjected to transcriptome analysis, intracellular metabolite measurements and metabolic flux analysis. Increased expression of the GAL-regulon and deletion of GAL2 in IMS0002 confirmed that the galactose transporter is essential for growth on arabinose. Elevated intracellular concentrations of pentose-phosphate-pathway intermediates and upregulation of TKL2 and YGR043c (encoding transketolase and transaldolase isoenzymes) suggested an involvement of these genes in flux-controlling reactions in arabinose fermentation. Indeed, deletion of these genes in IMS0002 caused a 21% reduction of the maximum specific growth rate on arabinose.
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Consensus design, the selection of mutations based on the most common amino acid in each position of a multiple sequence alignment, has proven to be an efficient way to engineer stabilized mutants and even to design entire proteins. However, its application has been limited to small motifs or small families of highly related proteins. Also, we have little idea of how information that specifies a protein's properties is distributed between positional effects (consensus) and interactions between positions (correlated occurrences of amino acids). Here, we designed several consensus variants of triosephosphate isomerase (TIM), a large, diverse family of complex enzymes. The first variant was only weakly active, had molten globular characteristics, and was monomeric at 25 °C despite being based on nearly all dimeric enzymes. A closely related variant from curation of the sequence database resulted in a native-like dimeric TIM with near-diffusion-controlled kinetics. Both enzymes vary substantially (30-40%) from any natural TIM, but they differ from each other in only a relatively small number of unconserved positions. We demonstrate that consensus design is sufficient to engineer a sophisticated protein that requires precise substrate positioning and coordinated loop motion. The difference in oligomeric states and native-like properties for the two consensus variants is not a result of defects in the dimerization interface but rather disparate global properties of the proteins. These results have important implications for the role of correlated amino acids, the ability of TIM to function as a monomer, and the ability of molten globular proteins to carry out complex reactions.
Article
This paper reviews the development in the 1950s of methods to determine the redox states of the free [NAD(+) ]/[NADH] in cytoplasm of yeast by Helmut Holzer and Feodore Lynen and in rat liver by Theodore Bucher and Martin Klingenberg. This work was extended in the 1960s in the laboratory of Hans Krebs, where the use of basic thermodynamic and kinetic principles allowed the extension of this approach to the determination of the free mitochondrial [NAD(+) ]/NADH] in mitochondria and the redox state of the free NADP system in cytoplasm and mitochondria. This work also outlined the linkage between the redox states in the various couples to the phosphorylation state or the free [ATP]/[ADP][P(i) ] ratio, the central energy parameter of living cells. This work has since been extended to include other energy-linked systems including the gradients of inorganic ions between extra and intracellular phases of the cell and the redox state of the co-enzyme Q couple of mitochondria. This system of linked near-equilibrium redox and phosphorylation potentials constitutes a framework of primitive metabolic control that is altered in a number of disease phenotypes. The alteration of such disease phenotypes by substrate availability is discussed, as well as the importance of a thorough grounding in basic kinetics and thermodynamics in designing new therapies to normalize the metabolic abnormalities that are the proximate cause of many common and some rare diseases states.
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Dietary fructose can benefit or hinder glycemic control depending on the quantity consumed and these contrasting effects are reflected by alterations in postprandial hepatic glycogen synthesis. Recently, we showed that (2)H-enrichment of glycogen positions 5 and 2 from deuterated water ((2)H(2)O) informs direct and indirect pathway contributions to glycogenesis in naturally-feeding rats. Inclusion of position 6S (2)H-enrichment data allows indirect pathway sources to be further resolved into triose-phosphate and Krebs cycle precursors. This analysis was applied to six rats that had fed on standard chow (SC), and six rats that had fed on SC plus 35% sucrose in their drinking water. After 2-weeks, hepatic glycogenesis sources during overnight feeding were determined by (2)H(2)O administration and post-mortem analysis of glycogen (2)H-enrichment at the conclusion of the dark period. Net overnight hepatic glycogenesis was similar between SC and HS rodents. Whereas direct pathway contributions were similar (403 ± 71 μmol/gdw HS versus 578 ± 76 μmol/gdw SC), triose-phosphate contributions were significantly higher for HS compared to SC (382 ± 61 μmol/gdw versus 87 ± 24 μmol/gdw, p<0.01) and Krebs cycle inputs lower for HS compared to SC (110 ± 9 μmol/gdw versus 197 ± 32 μmol/gdw, p<0.05). Analysis of plasma glucose (2)H-enrichments at the end of the feeding period also revealed a significantly higher fractional contribution of triose-phosphate to plasma glucose levels in HS versus SC. Hence, the (2)H-enrichment distributions of hepatic glycogen and glucose from (2)H(2)O informs the contribution of dietary fructose to hepatic glycogen and glucose synthesis.
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ENGLISH ABSTRACT: In Africa alone, Plasmodium, the causative agent of malaria is estimated to kill a child, under the age of five every thirty seconds140. The ability of the parasite to rapidly attain resistance, has resulted in immunity of the parasite to all, except one group of frontline drugs. The need to develop novel drugs, vaccines and prevention strategies that are accessible and affordable for third world countries is of the utmost importance to prevent needless human suffering and death. The glycolytic pathway is an attractive drug target since it is the principal source of ATP for the parasite. Many of the glycolytic enzymes have been studied and proposed as drug targets, but the importance of these enzymes for the function of the pathway as a whole has not been considered. It is known, from the frameworks of metabolic control analysis, that control of the flux and metabolite concentration can be divided among the individual steps. Differential control analysis of Plasmodium and erythrocyte glycolysis may reveal potential drug targets. These analyses require a detailed kinetic model of Plasmodium glycolysis, and the feasibility of constructing and validating such a model was the aim of this study. In this work we determined the feasibility of constructing and validating a detailed kinetic model for the Plasmodium falciparum glycolytic pathway. Whether the construction and validation of this kinetic model was feasible or not was decided on the basis of the ability to: i) culture and isolate sufficient asexual parasites for enzymatic and steady state assays , ii) obtain kinetic parameters such as Km and Vmax for each glycolytic enzyme, either from literature or experimentally, iii) measure glycolytic fluxes, iv) determine glycolytic intermediate concentrations, v) construct a kinetic model from the kinetic parameters and vi) validate it with steady state glycolytic fluxes and metabolite concentrations Each of the above criteria were successfully addressed. In summary, the kinetic parameters and glycolytic fluxes that were measured experimentally, were used to construct and partially validate a detailed kinetic model, respectively. Further validation of the model by means of steady state metabolite concentrations was shown to be possible with the development of a suitable protocol to measure the glycolytic intermediate concentrations. The model presented in this work may play an important role in drug target identification and improving the current understanding of host-parasite interactions and glycolytic regulation. AFRIKAANSE OPSOMMING: Plasmodium, die parasiet wat malaria veroorsaak, is in Afrika alleen elke dertig sekondes verantwoordelik vir die afsterwe van ’n kind jonger as vyf jaar. Die parasiet se vermoë om vinnig weerstand op te bou het daartoe gelei dat Plasmodium weerstandbiedend is teen byna alle nuwe teen-malaria middels, behalwe vir ’n enkele toonaangewende groep. Die ontwikkeling van nuwe malaria teen-middels is van uiterste belang om lyding te voorkom. ’n Goeie teiken vir teen-malaria middels is die glikolitiese padweg omdat die metaboliese padweg essensieël is vir die produksie van ATP, die energiebron van die parasiet. Desondanks die feit dat meeste van die glikolitiese ensieme al goed bestudeer en as teiken voorgestel is, is dit steeds onduidelik hoe hierdie ensieme saam funksioneer om die metaboliese weg, as geheel, tot stand te bring. Metaboliese kontrole analise het aangetoon dat die glikolitiese beheer verdeel is tussen die onderskeie glikolitiese ensieme, m.a.w. geen enkele ensiematiese stap het volledige beheer oor die fluksie van die glikolitiese padweg nie. Die afsonderlike analise en vergelyking van Plasmodium - en rooibloedselglikolise met behulp van differensiële metaboliese kontrole analise sal moontlik gebruik kan word om gasheervriendelike teikens vir nuwe middels aan te toon. So ’n analise benodig ’n omvattende kinetiese model van Plasmodium glikolise. Derhalwe was die doel van hierdie studie om vas te stel hoe uitvoerbaar dit is om ’n kinetiese model van Plasmodium glikolise te konstrueer en te valideer. Die uitvoerbaarheid van die konstruksie en validering van die kinetiese model was geasseseer op grond van die vermoë om: i) parasietkulture te kweek en genoegsame parasiete, wat in die aseksuele fase is, te isoleer sodat ensiembepalings en bestendige toestand-bepalings gedoen kan word, ii) kinetiese parameters soos Km - en Vmax-waardes vir elke glikolitiese ensiem, hetsy vanuit literatuur of eksperimentele werk, te verkry, iii) glikolitiese fluksie te meet, iv) glikolitiese intermediaatkonsentrasies te bepaal, v) ’n kinetiese model van die bepaalde kinetiese parameters op te stel en vi) die model te valideer met glikolitiese flukswaardes en metaboliet- konsentrasies wat in die bestendige toestand verkry is. Elk van die bogenoemde kriteria was met sukses in hierdie studie aangespreek. Ter opsomming, die eksperimenteel bepaalde kinetiese parameters en glikolietiese flukswaardes was gebruik om onderskeidelik ’n gedetaileerde kinetiese model te konstrueer en gedeeltelik te valideer. Daar was getoon dat verdere validering van die model deur middel van bestendige toestand metabolietkonsentrasies moontlik is met die ontwikkeling van ’n geskikte protokol om glikolitiese intermediaatkonsentrasies te meet. Die model, soos opgestel in hierdie studie, kan moontlik ’n belangrike rol speel om teikens vir nuwe malaria teen-middels te identifiseer en om gasheer-parasiet interaksies en glikolitiese regulering beter te verstaan. Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009.
Article
Metabolic diseases are an increasing health concern in the developed world. Type 2 Diabetes, (T2D) affects over 100 million people worldwide and significantly contributes to chronic diseases such as atherosclerosis and kidney failure. This condition is characterized by deregulation of glucose homeostasis through the development of insulin resistance, manifested as increased glucose production in the liver. Hepatic gluconeogenesis provides de novo formation of glucose from three carbon precursors such as glycerol, lactate, pyruvate and alanine. The upregulation of this pathway underlies the persistent hyperglycemia observed in diabetic patients. We have developed stable isotope tracer methods to reconstruct hepatic glucose production fluxes by infusion of [13C, 2H]-glycerol and mass spectrometry analysis of plasma metabolites. Using this methodology we observe physiologic changes in liver cell lines and primary hepatocyte cultures in the presence of hormones insulin/glucagon and in response to gluconeogenic precursor availability. We put forth the hypothesis that in the presence of glycerol as a gluconeogenic substrate, glucose-6-phosphatase has an important role in modulating metabolic flux through upper gluconeogenesis. Infusion of simultaneous tracer combinations in vivo including a novel [U-13C,2H5]-glycerol allow detailed net flux and reversibility reconstruction of upper gluconeogenesis to an unprecedented degree in a single experiment. We deployed the developed methods to probe glucose overproduction in the liver insulin receptor knockout (LIRKO) transgenic model of Type 2 Diabetes, and found unexpected similarities in the metabolic flux profile not observed by genomic, protein or metabolite measurements.
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Under conditions used previously for demonstrating glycolytic oscillations in muscle extracts (pH 6.65, 0.1 to 0.5 mM ATP), phosphofructokinase from rat skeletal muscle is strongly activated by micromolar concentrations of fructose diphosphate. The activation is dependent on the presence of AMP. Activation by fructose diphosphate and AMP, and inhibition by ATP, is primarily due to large changes in the apparent affinity of the enzyme for the substrate fructose 6-phosphate. These control properties can account for the generation of glycolytic oscillations. The enzyme was also studied under conditions approximating the metabolite contents of skeletal muscle in vivo (pH 7.0, 10mM ATP, 0.1 mM fructose 6-phosphate). Under these more inhibitory conditions, phosphofructokinase is strongly activated by low concentrations of fructose diphosphate, with half-maximal activation at about 10 muM. Citrate is a potent inhibitor at physiological concentrations, whereas AMP is a strong activator. Both AMP and citrate affect the maximum velocity and have little effect on affinity of the enzyme for fructose diphosphate.
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Several metabolites, including those of glycolysis, the citric acid cycle, the hexose monophosphate shunt, glutamate, aspartate, and Coenzyme A were measured in defined parietal cell-enriched freeze-dried sections of dog gastric biopsies derived from nonsecreting and secreting tissue. In addition, NH3, ribulose 5-phosphate, glycerol, and succinate were measured in perchloric acid extracts of biopsies. The onset of secretion increased the level of glycolytic intermediates including pyruvate and lactate with the most marked increase being in fructose 1,6-diphosphate levels. The level of 6-phosphogluconate and ribulose 5-phosphate also increased, in spite of a constant NADP+/NADPH ratio. The levels of all the citric acid cycle intermediates measured also rose, the most marked rise being in malate and fumarate. The levels of glycerol, acetyl-CoA, and CoA increased, but the ratio of the latter intermediates remained constant. Calculation of the ratio of the oxidized to reduced form of diphosphopyridine nucleotide indicated a fall of the ratio in the cytoplasm and a rise in the mitochondria. From these data, it is concluded that the major energy source for acid secretion is due to an increase in citric acid cycle activity and that glycolysis, and probably also fatty acid oxidation, is stimulated to provide mitochondrial substrate.
Article
Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.
Article
Three enzymes, triosephosphate isomerase (orange in picture), aldolase (cyan), and fructose 1,6-bisphosphatase (purple), which contained dockerins (red), self-assembled into a static trifunctional enzyme complex through interaction with a mini-scaffoldin protein consisting of three different cohesins (green). The synthetic enzyme complex exhibited an enhanced reaction rate compared to the noncomplexed three-enzyme mixture at the same enzyme concentration.
1.1. In the hepatopancreas and mantle of the sea mussel Mytilus galloprovincialis Lmk glycolysis shows seasonal variations.2.2. In both these tissues, glycolytic activity shows a maximum in winter and a minimum in summer.3.3. Smaller fluctuations also take place in the posterior adductor muscle.4.4. In all three tissues, Fru 2,6-P2 indicates the direction and strength of glycolysis, correlating well with other parameters such as energy charge or the phosphoenolpyruvate/pyruvate ratio.5.5. In the posterior adductor muscle, however, Fru 2,6-P2 does not correlate with the ratio from phosphofructokinase owing to the synergic activation of the enzyme by both Fru 2,6-P2 and AMP.
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Although firmly grounded in metabolic biochemistry, the study of energy metabolism has gone well beyond this discipline and become integrative and comparative as well as ecological and evolutionary in scope. At the cellular level, ATP is hydrolyzed by energy-expending processes and resynthesized by pathways in bioenergetics. A significant development in the study of bioenergetics is the realization that fluxes through pathways as well as metabolic rates in cells, tissues, organs, and whole organisms are "system properties." Therefore, studies of energy metabolism have become, increasingly, experiments in systems biology. A significant challenge continues to be the integration of phenomena over multiple levels of organization. Body mass and temperature are said to account for most of the variation in metabolic rates found in nature. A mechanistic foundation for the understanding of these patterns is outlined. It is emphasized that evolution, leading to adaptation to diverse lifestyles and environments, has resulted in a tremendous amount of deviation from popularly accepted scaling "rules." This is especially so in the deep sea which constitutes most of the biosphere. © 2012 American Physiological Society. Compr Physiol 2:2527-2540, 2012.
Chapter
EMBDEN (1) demonstrated that glucose was metabolized to lactate in an isolated perfused liver by the glycolytic pathway. With the development of more sophisticated techniques than those employed by EMBDEN and other earlier workers, it is now possible to study quantitatively the extent and nature of glycolysis in liver.
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The tissue contents of the reactants of the myokinase (EC 2.7.4.3) and the combined glyceraldehyde-3-phophate dehydrogenase (EC 1.1.1.29)-3-phosphoglycerate kinase (EC 2.7.2.3) reactions were measured in rapidly inactivated samples of human blood and rat brain, muscle, and liver. The tissue contents of the reactants of the creatine kinase (EC 2.7.3.2) reaction were measured in rat brain and muscle. In vitro the value of the expression: KG+G = [sigma3PG] . [sigmaATP] . [sigmalactate] KLDH = [sigmaHAP]/22] . [sigmaADP][sigmaPi] . [sigmaRUVATE] (1) was found to be 0.725 x 10(7) M-1 at I = 0.25, T = 38 degrees C, and free [Mg2+] = 0.15 mM and the value measured in vivo in red cell was 0.699 x 10(7) M-1. The value of the expression KMYK = ([sigma ATP] [sigma AMP]/[ADP2]) measured under the above conditions and at pH 7.2 was found to be 0.744 while the value found in red cell was 0.784 +/- 0.037. These reactions, therefore, appear to be in a state of near-equilibrium in the red cell and the measured tissue contents of ATP and ADP, which are common reactants in both reactions, approximate closely the activity of these reactants in vivo. In brain and muscle, the value of KG + G/KLDH calculated from the measured tissue contents of the reactants was a factor of 20 or more lower than that expected at equilibrium as was the measured value of the expression: KCK = [sigma ATP] [sigma creatine] divided by [sigma ADP] [sigma creatine-P] [H+] (2) Substitution of calculated free [sigma ADP] values in the expression of KG + G/KLDH gave values of 0.83 +/- 0.19 x 10(7) M-1 for brain and muscle, respectively, which agreed well with the value of 1.65 x 10(7) M-1 measured in vitro at I = 0.25, free [Mg2+] = 1 mM, T = 38 degrees C. This agreement between two highly active enzyme systems in the same compartment is taken as evidence of the existence of near-equilibrium in both these systems and suggests that free cytosolic [sigma ADP] is probably 20-fold lower than measured cell ADP content in mitochondrial-containing tissues.
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The glycolytic pathway is present in most organisms and represents a central part of the energy production mechanism in a cell. For a general understanding of glycolysis, the investigation from a thermodynamic point of view is essential and allows realising thermodynamic feasibility analyses under in vivo conditions. However, available literature standard Gibbs energies of reaction, ΔRg'0, are calculated using equilibrium-molality ratios Km', which might lead to a misinterpretation of the glycolytic pathway. It was the aim of this work to thermodynamically investigate the triosephosphate isomerase (TPI) reaction to provide new activity-based reaction data. In vitro equilibrium experiments were performed, and activity coefficients were predicted with the equation of state electrolyte PC-SAFT (ePC-SAFT). The combination of experimental concentrations and predicted activity coefficients yielded the thermodynamic equilibrium constant Ka and a new value for ΔRg'0(298.15 K, pH 7) = 7.1 ± 0.3 kJ mol‑1. The availability of the new ΔRg'0 value allowed predicting influences of the reaction medium on the reaction equilibrium of the TPI reaction. In this work, influences of the initial substrate concentration, pH and Mg2+ concentration on the reaction equilibrium were investigated and a method is presented to predict these influences. The higher the substrate concentration and the higher the temperature, the stronger the reaction equilibrium is shifted on the product side. While the pH did not have a significant influence on the reaction equilibrium, Mg2+ yielded a shift of the reaction equilibrium to the substrate side. All these effects were predicted correctly with ePC-SAFT. Based on the ePC-SAFT predictions we concluded that a charge-reduction of the product by complexation of the product with Mg2+ was responsible for the strong influence of Mg2+ on the reaction equilibrium. Finally, the standard enthalpy of reaction of ΔRh'0(pH 7) = 18 ± 7 kJ mol‑1 was determined with the equilibrium constants Ka at 298.15 K, 304.15 K and 310.15 K using the van 't Hoff equation.
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The review describes the use of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibitors to study the enzyme and to suppress its activity in various cell types. The main problem of selective GAPDH inhibition is a highly conserved nature of the enzyme active site and, especially, Cys150 environment important for the catalytic action of cysteine sulfhydryl group. Numerous attempts to find specific inhibitors of sperm GAPDH and enzymes from Trypanosoma sp. and Mycobacterium tuberculosis that would not inhibit GAPDH of somatic mammalian cells have failed, which has pushed researchers to search for new ways to solve this problem. The sections of the review are devoted to the studies of GAPDH inactivation by reactive oxygen species, glutathione, and glycating agents. The final section discusses possible effects of GAPDH inhibition and inactivation on glycolysis and related metabolic pathways (pentose phosphate pathway, uncoupling of the glycolytic oxidation and phosphorylation, etc.).
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Nucleotide coenzymes dot the map of metabolic pathways providing energy to drive the reactions of the pathway and play an important role in regulating and controlling energy metabolism through their shared potential energy, which is widely unobserved due to the paradox that the energy in the coenzyme pools cannot be determined from the concentration of the coenzyme couples. The potential energy of the nucleotide couples in the mitochondria or the cytoplasm is expressed in the enzyme reactions in which they take part. The energy in these couples, [NAD+]/[NADH], [NADP+]/[NADPH], [acetyl CoA]/[CoA], and [ATP]/[ADP]x[Pi], regulates energy metabolism. The energy contained in the couples can be altered by suppling energy equivalents in the form of ketones, such as, D‐β‐hydroxybutyrate to overcome insulin resistance, to restore antioxidants capacity, to form potential treatments for Alzheimer's and Parkinson's diseases, to enhance life span, and to increase physiological performance. © 2019 IUBMB Life, 9999(9999):1–15, 2019
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5-Deoxyribose is formed from 5'-deoxyadenosine, a toxic byproduct of radical S-adenosylmethionine (SAM) enzymes. The degradative fate of 5-deoxyribose is unknown. Here, we define a salvage pathway for 5-deoxyribose in bacteria, consisting of phosphorylation, isomerization, and aldol cleavage steps. Analysis of bacterial genomes uncovers widespread, unassigned three-gene clusters specifying a putative kinase, isomerase, and sugar phosphate aldolase. We show that the enzymes encoded by the Bacillus thuringiensis cluster, acting together in vitro, convert 5-deoxyribose successively to 5-deoxyribose 1-phosphate, 5-deoxyribulose 1-phosphate, and dihydroxyacetone phosphate plus acetaldehyde. Deleting the isomerase decreases the 5-deoxyribulose 1-phosphate pool size, and deleting either the isomerase or the aldolase increases susceptibility to 5-deoxyribose. The substrate preference of the aldolase is unique among family members, and the X-ray structure reveals an unusual manganese-dependent enzyme. This work defines a salvage pathway for 5-deoxyribose, a near-universal metabolite.
Chapter
Classical equilibrium thermodynamics (Gibbs, 1875–1878) has been used to evaluate many of the central reactions in intermediary metabolism (Holzer, et al., 1956, Huckabee, 1957, Bucher and Klingenberg, 1958, Hohorst, et al., 1959, Williamson, et al., 1967). Such near-equilibrium reactions usually have a high forward and reverse flux relative to the net flux through the enzyme and thus, due to analytical limitations, can not be distinguished from reactions which are in true equilibrium. They differ from true equilibrium reactions in that they violate the law of microscopic reversibility since there is a small net flux instead of a true equilibrium where the forward flux, v1 exactly equals the reverse flux, v−1.
Article
The incorporation of deuterium into different positions of sn-glycerol 3-phosphate in liver and into glucose in plasma of rats given [1-2H2] ethanol has been determined. The labelling of sn-glycerol 3-phosphate reached a steady state after about 1 min. The deuterium excess was about 5 atoms% at C-1, 15 atoms% at C-2, and 5 atoms% at C-3. Plasma glucose contained less than 2 atoms% deuterium. Administration of glucose or fructose did not influence the labelling of sn-glycerol 3-phosphate. The results are compared with those obtained for the glycerol moiety of phosphatidyl cholines. It is concluded that there is more than one pool of sn-glycerol 3-phosphate in the liver, and that the major part has a glycolytic origin.
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Publisher Summary This chapter describes the molecular and catalytic properties of aldose–ketose isomerases. Aldose–ketose isomerases catalyze the interconversion of isomeric aldo- and keto sugars by causing the migration of carbon-bound hydrogen between carbons 1 and 2. These enzymes may be classified in two groups according to their action on free or on phosphorylated monosaccharide substrates. Those acting on free sugars appear to be confined mainly to microorganisms, whereas some of those acting on phosphorylated substrates are common to all living organisms. Prominent among the latter are glucose-6-phosphate isomerase, triosephosphate isomerase, and ribose- 5-phosphate isomerase. The basic function of glucose-6-phosphate isomerase is catalyzing an obligatory step in glycolysis. Its potential for exerting a regulatory influence on carbohydrate metabolism, however, must be considered uncertain. Despite its strategic location near the branching point of several pathways utilizing glucose 6-phosphate, three properties would appear to make this enzyme difficult to control: (1) its equilibrium constant being close to unity, (2) its ubiquitous presence in relatively high concentration, and (3) its high catalytic efficiency.
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Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood. We analyzed the rates of superoxide/ H 2 O 2 production from different defined sites in rat skeletal muscle mitochondria oxidizing a variety of conventional substrates in the absence of added inhibitors: succinate; glycerol 3-phosphate; palmitoyl-carnitine plus carnitine; or glutamate plus malate. In all cases, the sum of the estimated rates accounted fully for the measured overall rates. There were two striking results. First, the overall rates differed by an order of magnitude between substrates. Second, the relative contribution of each site was very different with different substrates. During succinate oxidation, most of the superoxide production was from the site of quinone reduction in complex I (site I Q), with small contributions from the flavin site in complex I (site I F) and the quinol oxidation site in complex III (site III Qo). However, with glutamate plus malate as substrate, site I Q made little or no contribution, and production was shared between site I F , site III Qo and 2-oxoglutarate dehydrogenase. With palmitoylcarnitine as substrate, the flavin site in complex II (site II F) was a major contributor (together with sites I F and III Qo), and with glycerol 3-phosphate as substrate, five different sites all contributed, including glycerol 3-phosphate dehydrogenase. Thus, the relative and absolute contributions of specific sites to the production of reactive oxygen species in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely true in cells and in vivo.
Article
Internal standard based absolute quantitation of glycolytic intermediates was performed to characterize the thermodynamic states of Saccharomyces cerevisiae metabolism. A mixture of (13)C-labeled glycolytic intermediates was prepared via extraction from S. cerevisiae cells cultivated using a synthetic medium containing [U-(13)C] glucose as the sole carbon source. The (13)C-labeled metabolite mixture was used as an internal standard for the analysis of S. cerevisiae cultivated in a medium containing natural glucose. The methodology was employed for the absolute quantitation of glycolytic intermediates of BY4742, pfk1Δ, and zwf1Δ strains of S. cerevisiae. Fructose-1,6-bisphosphate was the most abundant intermediate in the BY4742 strains in the log phase of growth. Estimation of the Gibbs free energy change (ΔG) from the absolute concentration revealed that several reactions, such as those catalyzed by ribose-5-phosphate keto-isomerase and phosphoglucose isomerase, were commonly at near-equilibrium in all three strains. A significant shift in thermodynamic state was also observed for the transketolase-transaldolase reaction, for which ΔG was -6.6 ± 0.5 kJ mol(-1) in the BY4742 strain and 5.4 ± 0.3 kJ mol(-1) in the zwf1Δ strain. Copyright © 2015. Published by Elsevier B.V.
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The sites and rates of mitochondrial production of superoxide and H2O2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site IIIQo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site IF) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H2O2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site IQ) and the flavin site in complex II (site IIF) each account for about a quarter of the total measured rate of H2O2 production. Site IF, site IIIQo, and perhaps site EF in the β-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site IF dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H2O2 in vivo.
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Abstract Metabolic flux analysis (MFA), a key technology in bioinformatics, is an effective way of analyzing the entire metabolic system by measuring fluxes. Many existing MFA approaches are based on differential equations, which are complicated to be solved mathematically. So MFA requires some simple approaches to investigate metabolism further. In this article, we applied continuous-time Markov chain to MFA, called MMFA approach, and transformed the MFA problem into a set of quadratic equations by analyzing the transition probability of each carbon atom in the entire metabolic system. Unlike the other methods, MMFA analyzes the metabolic model only through the transition probability. This approach is very generic and it could be applied to any metabolic system if all the reaction mechanisms in the system are known. The results of the MMFA approach were compared with several chemical reaction equilibrium constants from early experiments by taking pentose phosphate pathway as an example.
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A chemo-enzymatic cascade for the one-pot preparation of 1-deoxy-d-xylulose 5-phosphate (DXP) and 1-deoxy-d-xylulose (DX) from stable, cheap, and easily available starting material R-glycidol is reported. The epoxide ring of R-glycidol was opened with phosphate to generate l-glycerol 3-phosphate, which was subsequently converted into the target molecules by combination of multi-enzymatic reactions in the same flask with purified overall yields of 27.6% (DXP) and 33% (DX), respectively. This approach represents the first one-pot chemo-enzymatic synthesis of these two biologically important compounds.
Article
Triose phosphate isomerase was purified ca 250-fold from pea seed extracts. The km for D-glyceraldehyde-3-P was 0.44 mM and for dihydroxyacetone-P, 0.88 mM. P-enolpyruvate, 2-P-glycerate, 3-P-glycerate and 2-P-glycolate were strongly inhibitory. Pi and arsenate also inhibited pea seed triose phosphate isomerase.
1.1. The effects of temperature on the activities and kinetic characteristics of triosephosphate isomerase from the pyloric caeca of Echinaster sp. held at 25 and 15 C were measured.2.2. The apparent Michaelis constant (Km) of starfish collected in August (30 C) was significantly less (P < 0.05) at an assay temperature of 15 than at 25 C, indicating positive thermal modulation. The Km remained relatively constant from 25 to 30 C.3.3. The specific activity of TPI of starfish collected in November (25 C) was slightly lower (P < 0.1) in individuals exposed to 25 C than in those exposed to 15 C for 28 days. The Km values did not change with exposure of individuals to 25 and 15 C for 28 days.4.4. The activities and Km indicate that TPI undergoes no or only partial acclimation to temperature.
Article
—The concentrations of most of the intermediates of glycolysis and of the tricarboxylic acid cycle were determined in the cerebral cortex of rats, frozen 10 s after the induction of a generalized seizure by electroshock. The apparent equilibrium constant for the combined glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactic dehydrogenase reactions, i.e. Kapp= [Lactate] [3-Phosphoglycerate] [ATP]/[Pyruvate] [Glyceraldehyde-3-phosphate] [ADP] [HPO24], was evaluated and found to be similar to the value reported for the in vitro system at pH 7. During an estimated 4–5-fold increase in glycolytic flux imposed by the seizure, this system remained close to equilibrium. In control cortex the components of the aldolase reaction were deviated 80-fold from equilibrium but shifted slightly toward equilibrium during the seizure. The components of the aspartate aminotransferase reaction were maintained in equilibrium in both the control and the seizure states. Of 4 reactions used to assess the cytoplasmic and mitochondrial redox states, only the lactic dehydrogenase reaction was considered reliable in the acutely changing situation of the seizure, and yielded a calculated decrease in the cytoplasmic [NAD+]/[NADH] ratio. This change, coupled with an observed decrease in the [ATP]/[ADP] [HPO24] ratio during the seizure, supports the concept that in brain, as in liver (Krebs & Veech, 1969), the phosphate potential determines the redox state of the tissue.
Article
1. The concentrations of the oxidized and reduced substrates of the lactate-, beta-hydroxybutyrate- and glutamate-dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equilibrium with free NAD(+) and NADH, and the ratio of the free dinucleotides can be calculated from the measured concentrations of the substrates and the equilibrium constants (Holzer, Schultz & Lynen, 1956; Bücher & Klingenberg, 1958). The lactate-dehydrogenase system reflects the [NAD(+)]/[NADH] ratio in the cytoplasm, the beta-hydroxybutyrate dehydrogenase that in the mitochondrial cristae and the glutamate dehydrogenase that in the mitochondrial matrix. 2. The equilibrium constants of lactate dehydrogenase (EC 1.1.1.27), beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38 degrees ; I0.25). 3. The mean [NAD(+)]/[NADH] ratio of rat-liver cytoplasm was calculated as 725 (pH7.0) in well-fed rats, 528 in starved rats and 208 in alloxan-diabetic rats. 4. The [NAD(+)]/[NADH] ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that beta-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. 5. The mean [NAD(+)]/[NADH] ratio within the liver mitochondria of well-fed rats was about 8. It fell to about 5 in starvation and rose to about 10 in alloxan-diabetes. 6. The [NAD(+)]/[NADH] ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. 7. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD(+) is protein-bound. 8. The bearing of these findings on various problems, including the following, is discussed: the number of NAD(+)-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate; the relations between the redox state of cell compartments and ketosis.
Article
1. The concentrations of the oxidized and reduced substrates of the ;malic' enzyme (EC 1.1.1.40) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in freeze-clamped rat livers. By assuming that the reactants of these dehydrogenase systems are at equilibrium in the cytoplasm the [free NADP(+)]/[free NADPH] ratio was calculated. The justification of the assumption is discussed. 2. The values of this ratio obtained under different nutritional conditions (well-fed, 48hr.-starved, fed with a low-carbohydrate diet, fed with a high-sucrose diet) were all of the same order of magnitude although characteristic changes occurred on varying the diet. The value of the ratio fell on starvation and on feeding with the low-carbohydrate diet and rose slightly on feeding with the high-sucrose diet. 3. The mean values of the ratio were calculated to be between 0.001 and 0.015, which is about 100000 times lower than the values of the cytoplasmic [free NAD(+)]/[free NADH] ratio. 4. The differences in the redox state of the two nicotinamide-adenine dinucleotide couples can be explained on a simple physicochemical basis. The differences are the result of equilibria that are determined by the equilibrium constants of a number of highly active readily reversible dehydrogenases and transaminases and the concentrations of the substrates and products of these enzymes. 5. The decisive feature is the fact that the NAD and NADP couples share substrates. This sharing provides a link between the redox states of the two couples. 6. The application of the method of calculation to data published by Kraupp, Adler-Kastner, Niessner & Plank (1967), Goldberg, Passonneau & Lowry (1966) and Kauffman, Brown, Passonneau & Lowry (1968) shows that the redox states of the NAD and NADP couples in cardiac-muscle cytoplasm and in mouse-brain cytoplasm are of the same order as those in rat liver. 7. The determination of the equilibrium constant at 38 degrees , pH7.0 and I 0.25 (required for the calculation of the [free NADP(+)]/[free NADPH] ratio), gave a value of 3.44x10(-2)m for the ;malic' enzyme (with CO(2) rather than HCO(3) (-) as the reactant) and a value of 1.98x10(-2)m(-1) for glutathione reductase.
Article
1. The rates of gluconeogenesis from most substrates tested in the perfused livers of well-fed rats were about half of those obtained in the livers of starved rats. There was no difference for glycerol. 2. A diet low in carbohydrate increased the rates of gluconeogenesis from some substrates but not from all. In general the effects of a low-carbohydrate diet on rat liver are less marked than those on rat kidney cortex. 3. Glycogen was deposited in the livers of starved rats when the perfusion medium contained about 10mm-glucose. The shedding of glucose from the glycogen stores by the well-fed liver was greatly diminished by 10mm-glucose and stopped by 13.3mm-glucose. Livers of well-fed rats that were depleted of their glycogen stores by treatment with phlorrhizin and glucagon synthesized glycogen from glucose. 4. When two gluconeogenic substrates were added to the perfusion medium additive effects occurred only when glycerol was one of the substrates. Lactate and glycerol gave more than additive effects owing to an increased rate of glucose formation from glycerol. 5. Pyruvate also accelerated the conversion of glycerol into glucose, and the accelerating effect of lactate can be attributed to a rapid formation of pyruvate from lactate. 6. Butyrate and oleate at 2mm, which alone are not gluconeogenic, increased the rate of gluconeogenesis from lactate. 7. The acceleration of gluconeogenesis from lactate by glucagon was also found when gluconeogenesis from lactate was stimulated by butyrate and oleate. This finding is not compatible with the view that the primary action of glucagon in promoting gluconeogenesis is an acceleration of lipolysis. 8. The rate of gluconeogenesis from pyruvate at 10mm was only 70% of that at 5mm. This ;inhibition' was abolished by oleate or glucagon.
Article
The molecular weight and amino acid composition of triose phosphate isomerase have been determined. The molecular weight (43000) is lower and the molecular activity (500000) higher than those of most other glycolytic enzymes. Reaction with iodoacetate (studied with radioactive reagent) takes place in two phases: in the first phase, at pH6.3, cysteine and methionine groups react and enzymic activity is unimpaired; in the second phase, histidine reacts and enzymic activity is lost. Photo-oxidation leads to inactivation, with loss of cysteine, of histidine and of tryptophan, but little loss of tyrosine. The mechanism postulated for the action of the enzyme demands the intervention of a group functioning as a base, and the results obtained are consistent with histidine's being the basic group in the active centre.
Article
Evidence has been given in previous communications (Hohorst et al. 1959, 1961) that it is possible to evaluate the steady state of some red/ox-reactions in the C-compartment of rat liver cells from the overall content (level) of metabolites in the tissue.In this paper we present a further study on equilibria in living material with regard to some two-partner reactions of the Embden-Meyerhof-Krebs-pathway in striated muscle.
Article
The direction of flux imposes restrictions on the concentrations of intermediates. The concentrations of the linking intermediates in multi-enzyme systems fall within a specified range.The distance from equilibrium (in enzymic reactions) is defined in terms of the “steady-state” ratio, ϱ; the factors affecting the value of ϱ are discussed.
  • S Minakami
  • H Yoshikawa
Minakami, S. & Yoshikawa, H. (1966). J. Biochem., Tokyo, 59, 140.
  • I A Rose
  • R Kellermeyer
  • R Stjernholm
  • H G Wood
Rose, I. A., Kellermeyer, R., Stjernholm, R. & Wood, H. G. (1962). J. biol. Chem. 237, 3325.
In The Energy Level and Metabolic Control in Mitochondria
  • H A Krebs
  • R L Veech
Krebs, H. A. & Veech, R. L. (1969). In The Energy Level and Metabolic Control in Mitochondria, p. 329. Ed. by Papa, S., Tager, J. M., Quagliariello, E. & Slater, E. C. Bari: Adriatica Editrice.