Inhibition of Calcium/Calmodulin-Dependent Protein Kinase IIα Suppresses Oxidative Stress in Cerebral Ischemic Rats Through Targeting Glucose 6-Phosphate Dehydrogenase

Ischemic stroke is a leading cause of mortality and morbidity worldwide, and oxidative stress plays a significant role in the ischemia stage and reperfusion stage. Previous studies have indicated that both calcium/calmodulin-dependent protein kinase II (CaMKII) and glucose 6-phosphate dehydrogenase (G6PD) are involved in the oxidative stress. Thus, the aim of this study was to investigate the roles of CaMKIIα, an important isoform of CaMKII, and G6PD in a rat model of middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of small interfering ribonucleic acid (siRNA) for CaMKIIα was performed at 48 h pre-MCAO surgery. Immunofluorescence Staining and western blot were performed to detect the expression of p-CaMKIIα and G6PD in the cortices. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was performed to investigate the infarct volume. In addition, neurological deficit, reactive oxygen species (ROS), ratio of reduced-to-oxidized glutathione (GSH/GSSG) and ratio of reduced-to-oxidized oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP⁺) were assessed. The results indicated that both p-CaMKIIα and G6PD were widely located in the neurons and astrocytes, and their expression was gradually increased in the cortices after MCAO, which was accompanied by increased level of ROS and decreased levels of GSH/GSSG and NADPH/NADP⁺. However, after treatment with siRNA for CaMKIIα, p-CaMKIIα expression was decreased and G6PD expression was increased. Moreover, inhibition of CaMKIIα improved the neurological deficit, reduced the infarct volume, decreased the level of ROS and increased the levels of GSH/GSSG and NADPH/NADP⁺. The results suggested that CaMKIIα inhibition exerted neuroprotective effects through regulating G6PD expression, which provides a new target for prevention and treatment of stroke.

     

Characterization of global metabolic responses of glucose-6-phosphate dehydrogenase-deficient hepatoma cells to diamide-induced oxidative stress

Glucose-6-phosphate dehydrogenase (G6PD) is crucial to NADPH generation and redox homeostasis. We have recently shown that G6PD deficiency predisposes cells to oxidant-induced cell death, and it is associated with the impairment of glutathione regeneration. It remains unclear what other metabolic pathways are affected by G6PD deficiency and whether the altered metabolism disturbs cellular redox homeostasis and underlies increased susceptibility to oxidants. In this study, we examined the effects of diamide on global metabolite profiles of SK-Hep1-derived SK-i-Gi and SK-i-Sc cells, which could inducibly express short hairpin RNA (shRNA) against G6PD (Gi) and control shRNA (Sc), respectively. There was no significant difference in their metabolite profiles under uninduced conditions. Doxycycline (Dox) addition resulted in over 70% decrease in G6PD activity in SK-i-Gi cells. This was accompanied by relatively minor changes in the metabolome of SK-i-Gi cells. Upon further diamide treatment, the metabolite profiles of both SK-i-Gi and SK-i-Sc cells changed in a time-dependent manner. A number of metabolic pathways, including those involved in energy metabolism and metabolism of amino acids and glutathione, were affected. However, the changes in the metabolite profile of Dox-treated SK-i-Gi cells were distinct from those of control cells (i.e., Dox-treated SK-i-Sc, SK-i-Gi, and SK-i-Sc cells). Cellular glutathione was depleted, whereas its disulfide form increased significantly in diamide, Dox-treated SK-i-Gi cells. Metabolites related to energy metabolism, such as AMP, ADP, and acetylcarnitine, increased to a greater extent in these cells than in diamide-treated control cells. In contrast, NAD and glutathione dropped to lower levels in SK-i-Gi cells than in control cells. The NAD⁺ depletion in SK-i-Gi cells was accompanied by a significant increase in NAD kinase activity. Targeted analyses revealed that NADP⁺ and NADPH increased significantly in diamide, Dox-treated SK-i-Gi cells compared with similarly treated control cells. Our results suggest that diamide induces oxidation and depletion of glutathione in SK-i-Gi cells under conditions of G6PD shRNA induction and subsequently induces conversion of NAD⁺ to NADP⁺ through enhanced NAD kinase activity. This may represent a compensatory mechanism to restore cellular NADPH reserve in G6PD-deficient cells. It is accompanied by alteration in pathways of cellular energy metabolism, such as glycolysis and β-oxidation.     

Hormonal and lipogenic and gluconeogenic enzymatic responses in LA/N-corpulent rats

Genetically obese normotensive rats, LA/N-corpulent (cp), were fed ad libitum diets containing either 54% sucrose or cooked corn starch for 12 weeks. Twenty-four rats were used for the study; half were corpulent (cp/cp) and half were lean (cp/+ or +/+). Fasting levels of plasma insulin, glucose, corticosterone, glucagon and growth hormone, and activities of liver and epididymal fat pad glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), and liver and kidney glucose-6-phosphatase (G6Pase), fructose 1,6-diphosphatase (FDPase), and phosphoenolpyruvate carboxykinase (PEPCK) were measured. A significant phenotype effect was observed in insulin, corticosterone, growth hormone, and liver G6PD, ME, FDPase, and kidney PEPCK, G6Pase, FDPase, and epididymal fat pad G6PD and ME (corpulent greater than lean), and glucagon (lean greater than corpulent). Diet effect (sucrose greater than starch) was significant for plasma glucose, liver ME, and kidney G6Pase. Although not significant at the P less than 0.05 level, insulin, corticosterone, liver G6PD and FDPase and kidney FDPase tended to be higher in sucrose-fed rats. This study suggests that the corpulent rat is more lipogenic and gluconeogenic than the lean, and that the hormones responsible are effective in keeping both the lipogenic and gluconeogenic enzyme activity elevated.     

Clinical mutants of human glucose 6-phosphate dehydrogenase: Impairment of NADP+ binding affects both folding and stability

Human glucose 6-phosphate dehydrogenase (G6PD) has both the “catalytic” NADP⁺ site and a “structural” NADP⁺ site where a number of severe G6PD deficiency mutations are located. Two pairs of G6PD clinical mutants, G6PD_Wisconsin (R393G) and G6PD_Nashville (R393H), and G6PD_Fukaya (G488S) and G6PD_Campinas (G488V), in which the mutations are in the vicinity of the “structural” NADP⁺ site, showed elevated K_d values of the “structural” NADP⁺, ranging from 53 nM to 500 nM compared with 37 nM for the wild-type enzyme. These recombinant enzymes were denatured by Gdn-HCl and refolded by rapid dilution in the presence of l-Arg, NADP⁺ and DTT at 25 °C. The refolding yields of the mutants exhibited strong NADP⁺-dependence and ranged from 1.5% to 59.4% with 1000 μM NADP⁺, in all cases lower than the figure of 72% for the wild-type enzyme. These mutant enzymes also displayed decreased thermostability and high susceptibility to chymotrypsin digestion, in good agreement with their corresponding melting temperatures in CD experiments. Taken together, the results support the view that impaired binding of “structural” NADP⁺ can hinder folding as well as cause instability of these clinical mutant enzymes in the fully folded state.     

Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes

Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP⁺ in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0–2.5 mM, 2–3 h, 37 °C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500 μM. Incubation with radiolabeled MGO (0–500 µM, 0–3 h, 37 °C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP⁺ binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.     

Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP⁺-binding site. Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP⁺ and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP⁺ binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PD^def). We first solved the crystal structure for G6PD^K403Q, a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP⁺ binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PD^def), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PD^K403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PD^def and provide a foundation for future drug discovery efforts.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

On the acrylamide disc electrophoresis pattern of human G6PD

An interconversion of the electrophoretic band components of human G6PD was obtained by NADPH-producing substances in erythrocytes, leukocytes, lymphocytes, HeLa cells, fibroblast-like cells, and lymphoblastoid cells. The hypothesis is suggested that the three dimeric forms are NADP⁺-induced conformers and not heteromeric complexes of structurally different subunits. The involvement of thiol groups in the NADP⁺-binding sites is confirmed, and it appears that in their reduced state they preferentially bind NADP⁺.     

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.     

Gradual Drought Under Field Conditions Influences the Glutathione Metabolism,Redox Balance and Energy Supply in Spring Wheat

Glutathione (GSH) metabolism, redox balance and energy supply in spring wheat (Triticum aestivum L.) during gradual drought stress under field conditions were investigated. Although levels of total and reduced GSH were decreased, the ratio of GSH/GSSG (glutathione disulfide) was markedly increased by drought. Levels of GSH biosynthetic precursors, cysteine (Cys) and -glutamylcysteine (-GC), and the activities of their biosynthetic enzymes, -glutamylcysteine synthetase (-GCS) and glutathione synthetase (GSHS) were also significantly increased in stressed plants. Glutathione reductase (GR) activity, which is responsible for the conversion of GSSG to GSH, was also increased under this field stress. However, two other important enzymes in GSH metabolism, glutathione peroxidase (GP) and glutathione S-transferase (GST), showed decreased activity in the droughted plants. These results suggest that the higher ratio of GSH/GSSG, the rate of GSH biosynthesis and the capacity of its redox cycling rather than GSH accumulation might be essential for drought resistance of plants. Activities of the two key Calvin-cycle enzymes possessing exposed sulfhydryl groups, NADP⁺-dependent glyceraldehydes-3-phosphate dehydrogenase (G3PD) and fructose-1,6-bisphosphatase (FBPase) were not affected by drought stress, whereas, activity of the key enzyme in the pentose-phosphate pathway (PPP), 6-phosphogluconate dehydrogenase (6-PGD), increased in the droughted plants. The ratios of NADPH/NADP⁺, NADH/NAD⁺ and ATP/ADP increased in the droughted plants, indicating that an up-regulation of the reduced redox state and the energy supply in the plant cells might be an important physiological strategy for plants responding to drought stress. A simple correlation between the high ratio of GSH/GSSG, the rate of GSH biosynthesis and the redox cycle and the high reduction states of redox status in the plant cells was also observed under field drought.     

Der Einfluß der Kulturbedingungen auf den Nicotinamid-Adenin-Dinucleotid(phosphat)-Gehalt in Zellen von Rhodospirillum rubrum

Zusammenfassung In Zellen von R. rubrum war das Verhältnis von oxydiertem zu reduziertem NAD(P) vom Sauerstoffpartialdruck im Medium, der Lichtintensität und der Nährbodenzusammensetzung abhängig. In ruhenden Kulturen unter aeroben Bedingungen im Licht oder im Dunkeln und anaerob bei hoher Lichtintensität, wenn der ATP-Pool in den Zellen groß ist, beobachtete man einen relativ hohen Wert für das Verhältnis von NAD(P)⁺/NAD(P)H. Unter Kulturbedingungen, bei denen der ATP-Gewinn der Zellen gering ist (anaerob Schwachlicht oder anaerob Dunkel), sank das Verhältnis von NAD(P)⁺/NAD(P)H ab. Die niedrigsten Werte für das Verhältnis von NAD(P)⁺/NAD(P)H wurden dementsprechend in anaerober Dunkelkultur, die höchsten in aerober Lichtkultur gefunden.Anaerob im Dunkeln war der NAD(P)H-Spiegel auch vom Substrat abhängig: mit Fructose oder ohne Substrat beobachtete man einen sehr großen NAD(P)H-Pool in den Zellen; nach Zugabe von Acetat, Succinat, Pyruvat oder Malat sank der Spiegel der reduzierten Coenzyme ab.In wachsenden Kulturen (außer anaerob im Dunkeln) nahm die relative Konzentration von NAD⁺ und der NADP⁺-Pool im Vergleich zu ruhenden Zellen stark zu (3-5fach).Änderungen im Verhältnis von NAD⁺/NADH und von NADP⁺/NADPH waren aber nicht unter allen Kulturbedingungen direkt korreliert.Es wird diskutiert, wieweit das Adenylatsystem und das NAD(P)-System einen regulativen Einfluß auf die Bacteriochlorophyll-Synthese und die Morphogenese bei Athiorhodaceae haben.

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The influence of culture conditions on the NAD(P) content of Rhodospirillum rubrum cells

Summary In cells of R. rubrum the ratio of oxidized to reduced NAD(P) depended on the oxygen pressure in the medium, the light intensity, and the composition of the medium. The ratio of NAD(P)⁺/NAD(P)H was high under conditions when the ATP-pool in the cell is large, viz. in resting cultures either kept aerobically in the light or in the dark or kept anaerobically in strong light. The quotient NAD(P)⁺/NAD(P)H decreased under conditions of reduced ATP-synthesis in the cells (anaerobic in dimlight or in the dark). Consequently, the lowest NAD(P)⁺/NAD(P)H value was observed in anaerobic dark cultures, the highest in aerobic light cultures.Under anaerobic conditions in the dark, the NAD(P)H level depended also on the substrate: with fructose or without any substrate, a large NAD(P)H pool was observed; the level of reduced coenzymes decreased upon addition of acetate, succinate, pyruvate, or malate.In growing cultures (except under anaerobic conditions in the dark) the relative concentration of NAD⁺ and the NADP⁺ pool showed a considerable increase (3 to 5 fold), as compared with resting cells. However, the changes in the proportions of NAD⁺/NADH and NADP⁺/NADPH were not directly correlated under all culture conditions.The regulative influence of the adenylate and the NAD(P) systems on the synthesis of bacteriochlorophyll and morphogenesis in Athiorhodaceae is discussed.

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Abkürzungen BChl Bacteriochlorophyll a - NAD(P) NAD-Nucleotide=reduziertes und oxydiertes Nicotinamid-Adenin-Dinucleotid und Nicotinamid-Adenin-Dinucleotidphosphat Herrn Prof. Dr. H. Engel zum 70. Geburtstag gewidmet.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

G6PD Vientiane: A new glucose-6-phosphate dehydrogenase variant with increased stability

Summary A new G6PD variant, called G6PD Vientiane, has been discovered in a patient from Laos.The characteristics of this variant are: mild enzyme deficiency (about 50% of the normal activity) in the granulocytes and the red cells, with normal G6PD-related antigen concentration; increased stability; normal Km glucose 6-phosphate and NADP⁺; increased inhibition constant by NADPH; decreased inhibition by ATP; slightly increased utilization of the substrate analogue; abnormal pH curve, with maximum activity at pH 9.5; slightly reduced starch gel electrophoretic migration. The implications of the molecular stability of a deficient mutant variant are discussed.     

The substrate of the glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa provides structural stability

In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP⁺. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the K_d values determined for the PaG6PDH-G6P complex (78.0 ± 7.9 μM) and the K_0.5 values determined for G6P (57.9 ± 2.5 and 104.5 ± 9.3 μM in the NADP⁺- and NAD⁺-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD⁺ and NADP⁺ from those that use NADP⁺ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.     

Isoenzymes of glucose-6-phosphate dehydrogenase from tobacco cells

Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP⁺, with K_m's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a K_m of 0.31 mM. The NADP⁺ double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent K_m and V_max values with increasing NADP⁺, denoting negative cooperativity. The two K_m's for high and low NADP⁺ concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP⁺ binding sites.     

Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation,Kinetics and Evolutionary Studies

Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD⁺ and NADP⁺ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP⁺ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2''-phosphate of NADP⁺, but the same residues formed no observable interactions in the case of NAD⁺. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the k_cat/K_M value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP⁺, only R50 makes a contribution (about -1 kcal/mol) to NAD⁺ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP⁺ from NAD⁺. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP⁺-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD⁺ in the case of the NADP⁺-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.     

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors

The steady state kinetics of glyceraldehyde 3-phosphate:NADP⁺ oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K_m values of 2 to 5 micromolar for NADP⁺ and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP⁺ as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This `G3P-inhibited state' is alleviated by saturating amounts of NADP⁺ or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP⁺ (K_i 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP⁺ and G3P (K_i 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP⁺ binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K_i 7 micromolar) and Pi (K_i 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP⁺) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.