19F NMR quantitation of lens aldose reductase activity using 3-deoxy-3-fluoro-D-glucose

In the present study we have determined the kinetics of 3-deoxy-3-fluoro-D-glucose (3-FG) as a substrate for the aldose reductase reaction in vitro. In addition, we compared the 3-deoxy-3-fluoro-sorbitol (3-FS) production rates from 3-FG in the intact lens using 19F NMR with conventional aldose reductase determinations in extracts from the same lenses. The affinity of in vitro aldose reductase for 3-FG was approximately 20 times greater (9.3 mM) than that for glucose (188 mM). An excellent correlation between the rate of 3-FS production in the intact canine lens, determined with 19F NMR, and extracted aldose reductase activity was observed. The relatively high affinity of aldose reductase for 3-FG and the correlation of 3-FS production with enzyme activity in the intact lens suggests that 3-FS production from 3-FG detected by 19F NMR could provide an accurate noninvasive determination of aldose reductase activity in the eye lens.     

Renal hypertrophy in experimental diabetes. Effect of diabetes on the pathways of glucose metabolism: differential response in adult and immature rats.

The effect of short-term diabetes, 5 days after the administration of streptozotocin, on renal growth and the activity of alternative pathways of glucose metabolism was studied in immature (21-day-old) rats and in adult rats. The kidney weight increased by 28% in the adult diabetic rats, but by only 10% in the immature diabetic rats, relative to their age-matched control groups. The flux of glucose via the pentose phosphate pathway was increased 2-3-fold in the adult diabetic rats, but was unchanged in the immature diabetic group. Enzymes of this pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) increased by 29% and 77% respectively in adult diabetic rats; in the immature group they showed changes of +5% and +28% respectively. The rate of glucose phosphorylation increased significantly in both groups of diabetic rats; only minor changes were observed in oxidation via the tricarboxylic acid cycle. Increases of 40-50% were found in the activity of enzymes involved in UDP-glucose metabolism (phosphoglucomutase, UDPglucose pyrophosphorylase and UDPglucose dehydrogenase) and in lactate dehydrogenase in both young and adult animals. The results suggest a differential renal response to streptozotocin-diabetes according to the stage of renal growth and development, and it is proposed that the difference is related to the developmental emergence of aldose reductase. Enzymes involved in formation of ribose 5-phosphate and NADPH are strikingly increased in the adult diabetic, whereas metabolic functions dependent on a high ambient glucose concentration, e.g. synthesis of glycogen and glucuronate, are similarly affected in adult and immature diabetic groups, both showing certain aspects of 'glucose overutilization'.     

Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts

The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.     

Changes in pathways of pentose phosphate formation in relation to phosphoribosyl pyrophosphate synthesis in the developing rat kidney. Effects of glucose concentration and electron acceptors

Phosphoribosyl pyrophosphate (PPRibP), required in nucleotide synthesis, increases 2-fold in rat kidney from 1 day post partum to adult stage; there is no accompanying increase in PPRibP synthetase activity measured in vitro. Ribose 5-phosphate is a key factor in the regulation of PPRibP synthesis. The activity and regulation of 3 routes of ribose 5-phosphate formation have been measured in renal growth: (i) the flux through the oxidative pentose phosphate pathway was high in the neonatal period but increased only +50% thereafter; (ii) the non-oxidative pentose phosphate pathway, including transketolase, increased by +145%; (iii) the rate-limiting enzymes of the glucuronate-xylulose route increased +200% from 1 day to the adult stage. The importance of systems reoxidizing NADPH was shown by: (i) the stimulation of renal PPRibP formation from glucose by phenazine methosulphate; (ii) the early involvement of the oxidative pentose phosphate pathway at the stage where NADPH is used for biosynthetic routes; (iii) the increasing involvement of the glucuronate-xylulose route, which acts as a transhydrogenase producing NADP+ in addition to pentose phosphate formation and (iv) the correlation between renal PPRibP content and the activity of aldose reductase, which, by utilization of NADPH, stimulates ribose 5-phosphate formation via the oxidative pentose phosphate pathway. Evidence is adduced that the contribution of the 3 routes of ribose 5-phosphate formation in the kidney varies at different stages of development.     

The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Control of pentose phosphate-cycle activity by cellular requirement for reduced nicotinamide adenine dinucleotide phosphate

By using inhibitors and stimulators of different metabolic pathways the interdependence of the pentose phosphate cycle and lipogenesis in isolated fat-cells was studied. Rotenone, which is known to inhibit electron transport in the respiratory chain, blocked glucose breakdown at the site of pyruvate dehydrogenase. Consequently, because of the lack of acetyl-CoA, fatty acid synthesis was almost abolished. A concomitant decrease in pentose phosphate-cycle activity was observed. Phenazine methosulphate stimulated pentose phosphate-cycle activity about five- to ten-fold without a considerable effect on fatty acid synthesis. The influence of rotenone on both the pentose phosphate cycle and lipogenesis could be overcome by addition of phenazine methosulphate, indicating that rotenone has no direct effect on these pathways. The decreased rate of the pentose phosphate cycle in the presence of rotenone therefore has to be considered as a consequence of decreased fatty acid synthesis. The rate of glucose catabolism via the pentose phosphate cycle in adipocytes appears to be determined by the requirement of NADPH for lipogenesis. Treatment of cells with 6-aminonicotinamide caused an accumulation of 6-phosphogluconate, indicating an inhibition of 6-phosphogluconate dehydrogenase. The rate of glucose metabolism via the pentose phosphate cycle as well as the rate of fatty acid synthesis, however, was not affected by 6-aminonicotinamide treatment and could still be stimulated by addition of insulin. Since even in cells from starved animals, in which the pentose phosphate-cycle activity is extremely low, no accumulation of 6-phosphogluconate was observed, it is concluded that the control of this pathway is achieved by the rate of regeneration of NADP at the site of glucose 6-phosphate dehydrogenase.     

Metabolic flux analysis for a ppc mutant Escherichia coli based on 13C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements

The physiology and central metabolism of a ppc mutant Escherichia coli were investigated based on the metabolic flux distribution obtained by (13)C-labelling experiments using gas chromatography-mass spectrometry (GC-MS) and 2-dimensional nuclear magnetic resonance (2D NMR) strategies together with enzyme activity assays and intracellular metabolite concentration measurements. Compared to the wild type, its ppc mutant excreted little acetate and produced less carbon dioxide at the expense of a slower growth rate and a lower glucose uptake rate. Consequently, an improvement of the biomass yield on glucose was observed in the ppc mutant. Enzyme activity measurements revealed that isocitrate lyase activity increased by more than 3-fold in the ppc mutant. Some TCA cycle enzymes such as citrate synthase, aconitase and malate dehydrogenase were also upregulated, but enzymes of glycolysis and the pentose phosphate pathway were downregulated. The intracellular intermediates in the glycolysis and the pentose phosphate pathway, therefore, accumulated, while acetyl coenzyme A and oxaloacetate concentrations decreased in the ppc mutant. The intracellular metabolic flux analysis uncovered that deletion of ppc resulted in the appearance of the glyoxylate shunt, with 18.9% of the carbon flux being channeled via the glyoxylate shunt. However, the flux of the pentose phosphate pathway significantly decreased in the ppc mutant.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions and related enzymes of the cycle in adipose tissue

1. Measurements were made of the activities of the enzymes of the pentose phosphate pathway concerned in both the oxidative (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and the non-oxidative (ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase) reactions of this pathway, together with hexokinase and phosphoglucose isomerase, in adipose tissue in a variety of nutritional and hormonal conditions. 2. Starvation for 2 days caused a significant decrease in the activities of all the enzymes of the pentose phosphate pathway, with the exception of glucose 6-phosphate dehydrogenase, when expressed as activity/2 fat-pads; only the activities of ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase were significantly decreased on the basis of activity/mg. of protein. Re-feeding with a high-carbohydrate or high-fat diet for 3 days restored the activity of all the enzymes of the pentose phosphate pathway to the range of the control values, with the exception of transketolase, which showed a marked ;overshoot' in rats re-fed with carbohydrate. Starvation for 3 days caused a marked decrease in the activities of glucose 6-phosphate dehydrogenase and transketolase. 3. On the basis of activity/two fat-pads, alloxan-diabetes caused a marked decrease, to about half the control value, in the activities of all the enzymes concerned in the pentose phosphate pathway, transketolase showing the smallest decrease; hexokinase and phosphoglucose isomerase activities were also decreased. Treatment with insulin for 3 and 7 days raised the activities to normal or supranormal values, transketolase showing the most marked ;overshoot' effect. On the basis of activity/mg. of protein the activity of none of the enzymes was significantly decreased in alloxan-diabetes; transketolase and transaldolase activities were raised above the control values. With insulin treatment for 3 or 7 days the activities of all the enzymes were significantly increased, except that of ribulose 5-phosphate epimerase at the shorter time-interval. Glucagon treatment did not alter any of the enzyme activities expressed on either basis. 4. Thyroidectomy caused a decrease of 30-40% in the activities of enzymes of the pentose phosphate pathway, except for transketolase activity, which fell to 50% of the control value. Little change occurred in adipose-tissue weight or protein content. 5. Adrenalectomy caused a decrease of 40% in the activity of glucose 6-phosphate dehydrogenase and of 20-30% in the activities of the remaining enzymes of the pentose phosphate pathway; hexokinase activity was also decreased. Treatment with cortisone for 3 days did not significantly raise the activity from that found in adrenalectomized rats. Treatment of normal rats with high doses of cortisone had no significant effect on the activities of the enzymes of the pentose phosphate pathway in adipose tissue. 6. The changes in enzyme activities are discussed in relation to: (a) the concept of constant-proportion groups of enzymes; (b) the known changes in the flux of glucose through alternative metabolic pathways; (c) the pattern of change found in liver with similar hormonal and dietary conditions.     

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida characterization of a new mannitol-producing metabolic pathway.

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell-free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden-Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden-Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by alpha glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

Phosphonomethyl analogues of hexose phosphates.

The analogue of fructose 1,6-bisphosphate in which the phosphate group, -O-PO3H2, on C-6 is replaced by the phosphonomethyl group, -CH2-PO3H2, was made enzymically from the corresponding analogue of 3-phosphoglycerate. It was a substrate for aldolase, which was used to form it, but not for fructose 1,6-bisphosphatase. It was hydrolysed chemically to yield the corresponding analogue of fructose 6-phosphate [i.e. 6-deoxy-6-(phosphonomethyl)-D-fructose, or, more strictly, 6,7-dideoxy-7-phosphono-D-arabino-2-heptulose]. This proved to be a substrate for the sequential actions of glucose 6-phosphate isomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Thus seven out of the nine enzymes of the glycolytic and pentose phosphate pathways so far tested catalyse the reactions of the phosphonomethyl isosteres of their substrates.     

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

Mutants of the pentose phosphate pathway in Aspergillus nidulans

Mutants of the pentose phosphate pathway have been isolated in Aspergillus nidulans. These fail to grow on a variety of carbohydrates that are catabolized through the pentose phosphate pathway. They also grow poorly on nitrate and nitrite as sole nitrogen sources. The pentose phosphate pathway mutations have been assigned to two unlinked genes. Mutants with lesions in the pppB locus have reduced activities of four enzymes of the pentose phosphate pathway, of glucose-phosphate isomerase, and of mannitol-1-phosphate dehydrogenase. pppA(-) mutants have elevated activities of these same enzymes except for transaldolase, for which they have much reduced activity. Both classes of mutants accumulate sedoheptulose-7-phosphate to an extent that is increased considerably when nitrate is present in the medium. Nitrate does not cause an increase in accumulation of sedoheptulose-7-phosphate in double mutants which, in addition to the pppA1 mutation, carry a mutation that leads to the lack of nitrate reductase activity. These last results suggest that nitrate stimulates the flux through the oxidative pentose phosphate pathway, but that this stimulation depends upon the metabolism of nitrate.     

Metabolic flux analysis of postburn hepatic hypermetabolism

The hepatic response to severe injury is characterized by a marked upregulation of glucose, fatty acid, and amino acid turnover, which, if persistent, predisposes the patient to progressive organ dysfunction. To study the effect of injury on liver intermediary metabolism, metabolic flux analysis was applied to isolated perfused livers of burned and sham-burned rats. Intracellular fluxes were calculated using metabolite measurements and a stoichiometric balance model. Significant flux increases were found for multiple pathways, including mitochondrial electron transport, the TCA and urea cycles, gluconeogenesis, and pentose phosphate pathway (PPP). The burn-induced increase in gluconeogenesis did not significantly increase glucose output. Instead, glucose-6-phosphate was diverted into the PPP. These changes were paralleled by increases in glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activities. Given that G6PDH and GR are the most significant NADPH producers and consumers in the liver, respectively, and that GR is responsible for recycling the free radical scavenger glutathione, these data are consistent with the notion that hepatic metabolic changes are in part due to the induction of liver antioxidant defenses.     

The specificity of oxidase and kinase preparations from Pseudomonas fluorescens towards deoxyfluoromonosaccharides.

With partially purified enzyme preparations from cell-free extracts of Pseudomonas fluorescens, 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid are substrates for glucose oxidase (EC 1.1.3.4.) and gluconate dehydrogenase (EC 1.1.99.3), with K-m values 18.2 mM and 11.8 mM, respectively. The same enzymes that oxidize glucose and gluconic acid probably oxidize 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid. The latter fluorinated carbohydrates and the presumed formation of 3-deoxy-3-fluoro-2-keto-D-gluconic acid, which has been isolated as a calcium salt and characterizied, are not substrates for gluconokinase (EC 2.7.1.12). Both 3-deoxy-3-fluoro-D-glucose and 3-deoxy-3-fluoro-D-gluconic acid act as competitive inhibitors of this enzyme preparation for gluconate, with K-i values 47.5 mM and 14.8 mM, respectively.     

Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants

The compositions of intracellular pentose phosphate pathway enzymes have been examined in mutants of Pachysolen tannophilus NRRL Y-2460 which possessed enhanced D-xylose fermentation rates. The levels of oxidoreductive enzymes involved in converting D-xylose to D-xylulose via xylitol were 1.5–14.7-fold higher in mutants than in the parent. These enzymes were still under inductive control by D-xylose in the mutants. The D-xylose reductase activity (EC 1.1.1.21) which catalyses the conversion of D-xylose to xylitol was supported with either NADPH or NADH as coenzyme in all the mutant strains. Other enzyme specific activities that generally increased were: xylitol dehydrogenase (EC 1.1.1.9), 1.2–1.6-fold; glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 1.9–2.6-fold; D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9), 1.2–2.6-fold; and alcohol dehydrogenase (EC 1.1.1.1), 1.5–2.7-fold. The increase of enzymatic activities, 5.3–10.3-fold, occurring in D-xylulokinase (EC 2.7.1.17), suggested a pivotal role for this enzyme in utilization of D-xylose by these mutants. The best ethanol-producing mutant showed the highest ratio of NADH- to NADPH-linked D-xylose reductase activity and high levels of all other pentose phosphate pathway enzymes assayed.     

Regulation of Glucose Metabolism in Thiobacillus intermedius

Glucose-yeast extract or glucose-casein hydrolysate-grown Thiobacillus intermedius cells, which use glucose for energy generation, possess high specific activities of the Entner-Doudoroff pathway and related enzymes, 6-phosphogluconate dehydrase, 2-keto-3-deoxy-6-phosphogluconate aldolase, glucokinase, and glucose-6-phosphate dehydrogenase, but low activities of enzymes unique to the pentose shunt and Embden-Meyerhof pathways. Although the synthesis of the latter enzymes remains largely unaffected by the growth environment, that of the former is stimulated by glucose. Radiorespirometric measurements demonstrate an early and parallel respiration of glucose carbon atoms one and four in glucose-casein hydrolysate broth. It is concluded that the Entner-Doudoroff pathway performs an energetic role in glucose metabolism by T. intermedius with the pentose shunt and Embden-Meyerhof pathways functioning mainly in biosynthesis. The presence of thiosulfate in the growth medium inhibits the synthesis of the Entner-Doudoroff pathway and related enzymes. In addition, both thiosulfate and glucose inhibit the synthesis of the Krebs cycle enzymes, nicotinamide adenine dinucleotide phosphate-linked isocitrate and alpha-ketoglutarate dehydrogenases. Thus, repression of enzymes is of significance in the adaptation of T. intermedius to its nutritional environment. The activity of glucose-6-phosphate dehydrogenase of T. intermedius is inhibited by adenosine triphosphate. Such a control could afford the organism a mechanism to regulate the flow of glucose into major energetic and biosynthetic routes.