Glucose-6-phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition

The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies, NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP+ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903-904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP+] ratio.     

Possible involvement of NADPH requirement in regulation of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase levels in rat liver

Summary Previous studies examining regulation of synthesis of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase in rat liver have focussed on the induction of these enzymes by different diets and some hormones. However, the precise mechanism regulating increases in the activities of these enzymes is unknown and the factors involved remain unidentified. Considering that many of these metabolic conditions occur simultaneously with the increase of some NADPH consuming pathway, in particular fatty acid synthesis, we suggest that the activities of Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase could be regulated through a mechanism involving changes in the NADPH requirement. Here, we have studied the effect of changes in the flux through different NADPH consuming pathways on the NADPH/NADP ratio and on Glucose-6-Phosphate and 6-Phosphogluconate levels. The results show that: i) an increase in consumption of NADPH, caused by activation of fatty acid synthesis or the detoxification system which consumes NADPH, is paralleled by an increase in levels of these enzymes; ii) when increase in consumption of NADPH is prevented, Glucose-6-Phosphate and 6-Phosphogluconate dehydrogenase levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - ME Malic Enzyme - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide - t-BHP t-Butyl hydroperoxide - BCNU 1,3,-Bis (2-chloroethyl)-1-nitrosourea - GR Glutathione Dehydrogenase - 2-ME 2-Mercaptoethanol - DTT Dithiothreitol - NADP B-Nicotinamide-Adenine Dinucleotide Phosphate - NADPH B-Nicotinamide-Adenine Dinucleotide Phosphate Reduced - EDTA Ethylenediaminetetraacetic Acid - GSH Glutathione Reduced Form - GSSG Glutathione Oxidized Form     

Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000     

Glucose-6-phosphate dehydrogenase of Anabaena sp.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three principal forms, with approximate molecular weights of 120 000 (M ₁), 240 000 (M ₂) and 345 000 (M ₃). The relative catalytic activities are: M ₁M ₂<M ₃. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M ₁ form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP⁺), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M ₁ form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M ₁ form is favored at pH values above 7; a pH of 6.5 is optimal for maintenace of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.Abbreviations G6PD glucose-6-phosphate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - RUDP ribulose-1,5-diphosphate - G6P glucose-6-phosphate - 6PG 6-phosphogluconate     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

Effects of cadmium and zinc ions on purified lamb kidney cortex glucose-6-phosphate dehydrogenase activity

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme in the pentose phosphate pathway. Cadmium is a toxic heavy metal that inhibits several enzymes. Zinc is an essential metal but overdoses of zinc have toxic effects on enzyme activities. In this study G-6-PD from lamb kidney cortex was competitively inhibited by zinc both with respect to glucose-6-phosphate (G-6-P) and NADP⁺ with Ki values of 1.066 ± 0.106 and 0.111 ± 0.007 mM respectively whereas cadmium was a non-competitive inhibitor with respect to both G-6-P and NADP⁺ Ki values of 2.028 ± 0.175 and 2.044 ± 0.289 mM respectively.     

A genetic and molecular analysis of P-induced mutations at the glucose-6-phosphate dehydrogenase locus in Drosophila melanogaster

Summary We examined P factor induced mutations of the Zw gene of Drosophila melanogaster in order to learn more about the site specificity of such mutations. Approximately 70000 chromosomes were screened using a powerful positive selection scheme. As only two mutants were discovered, Zw is a cold spot for transposable element insertion. One mutation involved a complex P element associated chromosomal rearrangement which was used to define the orientation of the gene with respect to the centromere of the X chromosome. The second mutation was either a simple, non-dysgenically induced point mutation or a very unstable insertion.     

Allosteric inhibition by phosphoenolpyruvate of glucose-6-phosphate dehydrogenase from bacteria and its taxonomic importance

Purified glucose-6-phosphate dehydrogenase from Zymomonas mobilis was examined with respect to inhibition by phosphoenolpyruvate, ADP and ATP. Its molecular weight was 260,000 and the kinetics of substrate conversion indicated a random bi bi mechanism. This enzyme and the dehydrogenases from Z. anaerobia, Azotobacter chroococcum, A. vinelandii, and “Corynebacterium” autotrophicum strain 19/-/x were found to be allosterically inhibited by phosphoenolpyruvate, while those from several coryneform bacteria and from Escherichia coli or Pseudomonas fluorescens were not.     

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.     

Regulation of specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in Penicillium chrysogenum

Abstract The specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase changed when Penicillium chrysogenum was grown on different carbon sources. In the presence of 2% lactose, the activities of these enzymes were approximately 25–35% lower than those in media containing 2% glucose or 2% fructose. We assume that an increase in cAMP concentration was responsible for the observed decreases in the enzyme activities, because a higher cAMP concentration could be detected when the mycelium was grown in a medium containing solely lactose as carbon source. The likely role played by cAMP in the regulation was also demonstrated by the addition of either cAMP or caffeine to the medium.     

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrificans grown on glucose/nitrate

Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ l-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD⁺-and NADP⁺-linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg²⁺ requirement. Kinetics for both NAD(P)⁺ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD⁺-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP⁺-linked activity. Neither of the two activities are inhibited by 100 M NADH but both are inhibited by NADPH. The NAD⁺-linked activity is far more sensitive to inhibition by NADPH than the NADP⁺-linked activity.     

Isolation and characterization of light- and dithiothreitol-modulatable glucose-6-phosphate dehydrogenase from pea chloroplasts

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) has been purified to electrophoretic homogeneity from pea chloroplasts. The enzyme, which has a Stokes radius of 52 Å, is a tetramer made up of four 56000 Da monomers. The pH optimum is around 8.2. The enzyme is absolutely specific for NADP. The apparent K_m(NADP) is 2.4 ± 0.1 μM. NADPH inhibition of the enzyme is competitive with respect to NADP (mean K_i, 18 ± 5 μM) and is mixed (K_p >K_m, V_max >V_p) with respect to glucose 6-phosphate (mean crossover point, 0.5 ± 0.1 mM). The apparent K_m(glucose 6-phosphate) is 0.37 ± 0.01 mM. The purified enzyme is inactivated in the light in the presence of dilute stroma and washed thylakoids, and by dithiothreitol. Enzyme which has been partially inactivated by treatment with dithiothreitol can be further inactivated in the light in the presence of dilute stroma and washed thylakoids and reactivated in the dark, but only to the extent of the reverse of light inactivation. Dithiothreitol-inactivated enzyme is not reactivated further by addition of crude stroma or oxidized thioredoxin. Dithiothreitol-dependent inactivation of the enzyme follows pseudo-first-order kinetics and shows rate saturation. The enzyme which has been partially inactivated by treatment with dithiothreitol does not differ from the untreated control with respect to thermal and tryptic inactivation. However, enzyme which has been partially light inactivated shows different thermal and tryptic inactivation patterns as compared to the dark control. These observations suggest that the changes in the enzyme brought about by light modulation are not necessarily identical with those brought about by dithiothreitol inactivation.     

Glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase of wild oat seeds

The presence of the initial enzymes of the pentose phosphate pathway, namely glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase, has been demonstrated in dormant seed of wild oat. Before a partial characterization of these enzymes was made, an inherent NADP-reducing activity and an enzyme deactivating component, both present in the crude extract, were removed by ammonium sulphate precipitation and subsequent desalting. Both enzymes were then shown to be NADP-specific. Typical Michaelis-Menten kinetics were shown by each enzyme towards NADP and their respective substrates. Soluble cytoplasmic dehydrogenase enzymes were present in both embryo and endosperm extracts.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: revised kinetic mechanism and kinetics of ATP inhibition

The kinetic mechanisms of the NAD- and NADP-linked reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides were examined using product inhibition, dead-end inhibition and alternate substrate experiments. The results are consistent with a steady-state random mechanism for the NAD-linked and an ordered, sequential mechanism with NADP+ binding first for the NADP-linked reaction. Thus, the enzyme can bind NADP+, NAD+, and glucose 6-phosphate, but the enzyme-glucose 6-phosphate complex can react only with NAD+, not with NADP+. This affects the rate equation for the NADP-linked reaction by introducing a term for a dead-end enzyme-glucose 6-phosphate complex. The kinetic mechanisms represent revisions of those proposed previously (C. Olive, M.E. Geroch, and H.R. Levy, 1971, J. Biol. Chem. 246, 2047-2057) and provide a kinetic basis for the regulation of coenzyme utilization of the enzyme by glucose 6-phosphate concentration (H.R. Levy, and G.H. Daouk, 1979, J. Biol. Chem. 254, 4843-4847) and NADPH/NADP+ concentration ratios (H.R. Levy, G.H. Daouk, and M.A. Katopes, 1979, Arch, Biochem. Biophys. 198, 406-413). The kinetic mechanisms were found to be the same at pH 6.2 and pH 7.8. The kinetics of ATP inhibition of the NAD- and NADP-linked reactions were examined at pH 6.2 and pH 7.8. The results are interpreted in terms of ATP addition to binary enzyme-coenzyme and enzyme-glucose 6-phosphate complexes.     

Elevation of glucose 6-phosphate dehydrogenase activity increases xylitol production in recombinant Saccharomyces cerevisiae

To increase the NAD(P)H-dependent xylitol production in recombinant Saccharomyces cerevisiae harboring the xylose reductase gene from Pichia stipitis, the activity of glucose 6-phosphate dehydrogenase (G6PDH) encoded by the ZWF1 gene was amplified to increase the metabolic flux toward the pentose phosphate pathway and NADPH regeneration. Compared with the control strain, the specific G6PDH activity was enhanced approximately 6.0-fold by overexpression of the ZWF1 gene. Amplification in the G6PDH activity clearly improved the NAD(P)H-dependent xylitol production in the recombinant S. cerevisiae strain. With the aid of an elevated G6PDH level, maximum xylitol concentration of 86 g/l was achieved with productivity of 2.0 g/l h in the glucose-limited fed-batch cultivation, corresponding to 25% improvement in volumetric xylitol productivity compared with the recombinant S. cerevisiae strain containing the xylose reductase gene only.     

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.     

Preeclampsia inactivates glucose-6-phosphate dehydrogenase and impairs the redox status of erythrocytes and fetal endothelial cells

Inactivation of glucose-6-phosphate dehydrogenase (G6PD) may contribute to vascular dysfunction in preeclampsia, and oxidative stress has been implicated in the pathogenesis of this disease. We have compared the susceptibility of erythrocytes and human umbilical vein endothelial cells (HUVEC) to oxidative stress in women with normotensive or preeclamptic pregnancies. The redox status of erythrocytes was also correlated with neutrophil-mediated superoxide (O₂⁻) production in women recruited to the “Vitamins in Preeclampsia” (VIP) trial. Erythrocytes and HUVEC from women with preeclampsia demonstrated impaired redox regulation and diminished response to glucose, detectable at 14–20 weeks gestation prior to onset of the clinical disease. Hexokinase and G6PD activities were decreased in erythrocytes and G6PD activity was decreased in HUVEC from preeclamptic pregnancies. Phorbol-ester-stimulated O₂⁻ was enhanced in preeclamptic neutrophils. Impaired redox regulation in erythrocytes and HUVEC in preeclampsia may be due to diminished hexokinase and G6PD activities resulting from increased release of reactive oxygen species from activated neutrophils. Our findings provide the first evidence that decreased G6PD activity in preeclampsia is associated with impaired redox regulation in erythrocytes and fetal endothelial cells. The deficiency in G6PD in preeclampsia potentially accounts for the lack of protection against oxidative stress afforded by antioxidant vitamin C/E supplementation in the VIP trial.     

Investigation of the effects of some sulfonamide derivatives on the activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase from human erythrocytes

In this study, the in vitro effects of some sulfonamide derivatives, which are carbonic anhydrase inhibitors, on the enzymes activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase were investigated. For this purpose, these three enzymes were purified from human erythrocytes. Purification procedure composed of four steps; preparation of the hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and gel filtration chromatography on Sephadex G-200. 5-(3α-Hydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3α,12α-Dihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3α,7α,12α-Trihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3), 5-(3α,Acetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (4), 5-(3α,7α,12α-Triacetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (5), 5-(3,7,12-Trioxo-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (6), acetazolamide, and dorzolamide were tested in this experiment. Compounds 3, 5, and dorzolamide showed inhibitory effects on the activity of 6-phosphogluconate dehydrogenase, and I₅₀ values and K_i constants were calculated as 0.0601 mM, 0.00253 mM, and 1.41 mM and 0.0878 ± 0.0274 mM, 0.0042 ± 0.0009 mM, and 3.1446 ± 0.2081 mM, respectively. Glutathione reductase was also inhibited by 1 and 2. I₅₀ values and K_i constants were 0.0471 mM and 0.0723 ± 0.0388 mM for 1 and 0.0045 mM and 0.0061 ± 0.0014 mM, for 2. If these sulfonamide derivatives are proposed as drugs, some of which are being used in glaucoma treatment such as acetazolamide and dorzolamide, these results should be taken into consideration concerning via these enzymes.     

Apoptotic effects and glucose-6-phosphate dehydrogenase responses in liver and gill tissues of rainbow trout treated with chlorpyrifos

We investigated apoptotic effects and changes in glucose-6-phosphate dehydrogenase (G6PD) enzyme activity in liver and gill tissues of fish exposed to chlorpyrifos. Three different chlorpyrifos doses (2.25, 4.5 and 6.75 μg/L) were administrated to rainbow trout at different time intervals (24, 48, 72 and 96 h). Acute exposure to chlorpyrifos showed time dependent decrease in G6PD enzyme activity at all concentrations (p < 0.05). Immunohistochemical results showed that chlorpyrifos caused mucous cell loss in gill tissue and apoptosis via caspase-3 activation in fish. The present study suggested that chlorpyrifos inhibits G6PD enzyme and causes mucous cell loss in gill and apoptosis in gill and liver tissues.     

Partial purification and kinetic properties of cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices

Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κ_m values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κ_i value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κ_i value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.