Purification and Properties of Glucose-6-Phosphate Dehydrogenase from Bacillus subtilis Spores

Glucose-6-phosphate dehydrogenase [d-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent K_m values of the enzyme were 5.7 × 10^–4 M for glucose-6-phosphate and 2.4 × 10^–4 M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg⁺⁺ or Ca⁺⁺. The inactive glucoses-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

Hormonal regulation of translatable mRNA of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes

The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Characterization of glucose-6-phosphate dehydrogenase variants in the Sudan--including GdKhartoum, a hyperactive slow variant.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was characterized in blood samples of 94 male subjects in Sudan having deficient and non-deficient electrophoretic variants. They comprised 44 GdB, 17 GdA, 19 GdB-, 11 GdA- and 3 nondeficient (GdKhartoum) variants. Biochemical characteristics including enzyme activity, electrophoretic mobility, Km for glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP), heat stability and pH optimum of all the common and deficient variants were consistent with the reported characteristics of these variants. The GdKhartoum variant had 90% mobility in TEB buffer and 100% in phosphate buffer, 120% activity, Km of 130 +/- 49 microns for G6P and 0.8 +/- 0.2 microns for NADP, lowered thermostability and an optimum pH of 7.6. This variant was not inhibited by 15 mM maleic acid, 10 mM iodoacetate and dehydro-iso-androsterone. All other variants were inhibited by dehydro-iso-androsterone but uninhibited by maleic acid and iodoacetate.     

Red-cell glucose-6-phosphate dehydrogenase of “brown-howler” monkeys (Alouatta fusca)

Physico-chemical properties of erythrocyte glucose-6-phosphate dehydrogenase including erythrocyte G6PD activity, Michaelis constants, K_mG6P and NADP, pH optimum, thermostability and molecular weight were investigated in “brown-howler” monkeys and then compared with the values of human G6PD B(+). The values of Michaelis constants (K_mG6P and NADP) pH optimum were the same as the values of human G6PD B(+). The human G6PD has a dimeric form in the assay conditions employed in the present study, monkey enzyme showing great similariy with human one. Otherwise, the thermostability differed from the human G6PD. The simian enzymatic activity was about four times higher than the human G6PD. A comparison of physico-chemical properties of glucose-6-phosphate dehydrogenase among primates is also presented.     

Purification and some properties of human placental glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase was purified from human placenta using DEAE-Sepharose fast flow, 2',5'-ADP Sepharose 4B column chromatography, and chromatofocusing on PBE 94 with PB 74. The enzyme was purified with 62% yield and had a specific activity of 87 units per milligram protein. The pH optimum was determined to be 7.8, using zero buffer extrapolation method. The purified placental glucose-6-phosphate dehydrogenase gave two activity bands on native PAGE: one band, constituting about 3--5% of total activity, moved slower than the remaining 95%. Among the activity bands only the faster moving band gave a band on protein staining. The slower moving band, which probably corresponded to the higher polymeric form of the G6PD with high specific activity, was not seen on native PAGE due to insufficient protein for Coomassie brilliant blue staining. The observation of one band on SDS--PAGE with an M(r) of 54 kDa and a specific activity lower than expected, suggests the presence of both forms of the G6PD, the high polymeric form at low concentration and the inactive form at high concentration, in our preparation. Measuring the activities of placental glucose-6-phosphate dehydrogenase between 20 and 50 degrees C, the activation energy, activation enthalpy, and Q(10) were calculated to be 8.16 kcal/mol, 7.55 kcal/mol, and 1.57, respectively. It was found that human placental G6PD obeys Michaelis-Menten kinetics. K(m) values were determined using the concentration ranges of 20--300 microM for G6P and 10--200 microM for NADP(+). The K(m) value for G6P was 40 microM; the K(m) value NADP(+) was found to be 20 microM. Double-reciprocal plots of 1/Vm vs 1/G6P (at constant [NADP(+)]) and of 1/Vm vs 1/NADP(+) (at constant [G6P]) intersected at the same point on the 1/V(m) axis to give V(m) = 87 U/mg protein.     

Glucose-6-phosphate dehydrogenase. Purification and partial characterization.

Glucose-6-phosphate dehydrogenase has been purified 1000-fold from pig liver. This enzyme exists as an active dimer of molecular weight 133,000 and an inactive monomer of molecular weight 67,500. The pH of maximum activity is 8.5 and the ionic strength maximum is 0.1 to 0.5 M. Glucose-6-phosphate dehydrogenase is highly specific for NADP+ and glucose 6-phosphate. Apparent Km values of 3.6 muM and 5.4 muM were obtained for glucose 6-phosphate and NADP+. This enzyme is located almost entirely within the soluble portion of the cellular cytoplasm.     

Peroxisomal localization of glucose-6-phosphate dehydrogenase and pyrophosphate-stimulated dihydroxyacetone-phosphate acyltransferase in mouse kidney.

1. The subcellular localization of dihydroxyacetone-phosphate acyltransferase (DHAPAT) (assayed in the presence of pyrophosphate) and glucose-6-phosphate dehydrogenase (NADP+-dependent) activity in mouse kidney was investigated by density-gradient centrifugation. 2. DHAPAT has a predominantly peroxisomal distribution, and the activity in purified peroxisomes is stimulated by various organic and inorganic phosphate-containing compounds. The pH optimum is acid. 3. Approx. 10% of the cellular NADP+-dependent glucose-6-phosphate dehydrogenase activity is associated with peroxisomal fractions and may provide a source of NADPH for the peroxisomal reduction of acyl-dihydroxyacetone phosphate formed by DHAPAT activity.     

Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction

Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.     

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.     

Kinetics and control of bovine adrenal glucose-6-phosphate dehydrogenase.

Michaelis-Menten kinetics are observed in studies of highly purified bovine adrenal glucose-6-phosphate dehydrogenase at pH8.0 in 0.1 M bicine. The Km for NADP+ is 3.8 muM and for glucose-6-phosphate, 61 muM. At pH 6.9 Km for NADP+ increases to 6.5 muM. The enzyme is inhibited by NADPH both at pH 6.8 and at 8.0 with a Kip of 2.36 muM at pH 8.0. Inhibition is competitive with respect to both substrates implying that addition of substrates is random ordered. The data are also interpreted in terms of "reducing charge", the mole fraction of coenzyme in the reduced form. This appears to be the major mechanism for regulation of the pentose shunt. D-glucose, oxidized by the enzyme at a very slow rate, is also a competitive inhibitor for the natural substrate with a Ki of 0.29 M. Phosphate is a competitive inhibitor for glucose-6-phosphate oxidation but both phosphate and sulfate accelerate glucose oxidation suggesting a common binding site for the two anions and the phosphate of the natural substrate. While binding of ACTH to our enzyme preparations has been observed, we have not been able, in spite of repeated attempts, to demonstrate augmentation of the activity of the enzyme by the addition of ACTH.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Stability of glucose-6-phosphate dehydrogenase in human cells cultivated in vitro]

It was shown that the thermal stability of glucose-6-phosphate dehydrogenase in human diploid cells is much higher than in human heteroploid cell lines HeLa and T-9. The purified enzymes from human diploid cells and from HeLa and T-9 cells possess similar thermal stabilities. Mixing of T-9 extracts with the purified enzyme preparations revealed that the non-stability factors of the dehydrogenase are present in the T-9 extracts. An addition of NADP- and NADPH-containing buffers and crystalline NADP to the heteroploid cell extracts stabilizes the enzyme. The thermal stability of the enzyme from "in vitro" cultivated human cells depends on the concentration of the coenzyme. It was also demonstrated that glucose-6-phosphate dehydrogenase stability in HeLa and T-9 extracts is the same at low concentrations of the coenzyme and after addition of crystalline NADP. However, at NADP concentration of 10(-3) M the enzyme stability in HeLa and T-9 extracts is different. It is assumed that the destabilizing factors are the enzymes possessing the nucleotidases activity, which is different in various cell lines.     

High-performance liquid chromatography of proteins on deformed nonporous agarose beads. Affinity chromatography of dehydrogenases based on cibacron blue-derivatized agarose

Nonporous agarose beads, prepared by shrinkage and cross-linking in organic solvents, were derivatized with Cibacron Blue F3G-A. A compressed bed of these beads was used for purification of dehydrogenases (glucose-6-phosphate dehydrogenase, lactate dehydrogenase and alcohol dehydrogenase). The chromatographic conditions for the purification of glucose-6-phosphate dehydrogenase were optimized by varying the pH of the buffer; the concentrations of eluting agents, i.e. NADP (specific elution) and sodium chloride (nonspecific elution); flow rate; residence time of the protein on the column bed; and protein load. Specific elution with NADP (2 mM in 0.025 M Tris-HCl, pH 8.0) gave the highest recovery (140%) and highest purification factor (200-fold) of the enzyme. The ability of the compressed bed of nonporous agarose beads to tolerate high flow rates was essential, since the recovery of the enzyme activity increased with an increase in flow rate.     

High-Performance Liquid Chromatography of Proteins on Deformed Nonporous Agarose Beads. Affinity Chromatography of Dehydrogenases Based on Cibacron Blue-Derivatized Agarose

Nonporous agarose beads, prepared by shrinkage and cross-linking in organic solvents, were derivatized with Cibacron Blue F3G-A. A compressed bed of these beads was used for purification of dehydrogenases (glucose-6-phosphate dehydrogenase, lactate dehydrogenase and alcohol dehydrogenase). The chromatographic conditions for the purification of glucose-6-phosphate dehydrogenase were optimized by varying the pH of the buffer; the concentrations of eluting agents, i.e. NADP (specific elution) and sodium chloride (nonspecific elution); flow rate; residence time of the protein on the column bed; and protein load. Specific elution with NADP (2 mM in 0.025 M Tris-HCl, pH 8.0) gave the highest recovery (140%) and highest purification factor (200-fold) of the enzyme. The ability of the compressed bed of nonporous agarose beads to tolerate high flow rates was essential, since the recovery of the enzyme activity increased with an increase in flow rate.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

Cloning,expression, and characterization of the gsdA gene encoding thermophilic glucose-6-phosphate dehydrogenase from Aquifex aeolicus

The gsdA gene of the extreme thermophilic bacterium Aquifex aeolicus, encoding glucose-6-phosphate dehydrogenase (G6PDH), was cloned into a high-expression vector and overexpressed as a fusion protein in Escherichia coli. Here we report the characterization of this recombinant thermostable G6PDH. G6PDH was purified to homogeneity by heat precipitation followed by immobilized metal affinity chromatography on a nickel-chelate column. The data obtained indicate that the enzyme is a homodimer with a subunit molecular weight of 55 kDa. G6PDH followed Michaelis-Menten kinetics with a K(M) of 63 micro M for glucose-6-phosphate at 70 degrees C with NADP as the cofactor. The enzyme exhibited dual coenzyme specificity, although it showed a preference in terms of k(cat)/ K(M) of 20.4-fold for NADP over NAD at 40 degrees C and 5.7-fold at 70 degrees C. The enzyme showed optimum catalytic activity at 90 degrees C. Modeling of the dimer interface suggested the presence of cysteine residues that may form disulfide bonds between the two subunits, thereby preserving the oligomeric integrity of the enzyme. Interestingly, addition of dithiothreitol or mercaptoethanol did not affect the activity of the enzyme. With a half-life of 24 h at 90 degrees C and 12 h at 100 degrees C, this is the most thermostable G6PDH described.     

Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

Kinetic properties of normal human erythrocyte glucose-6-phosphate dehydrogenase dimers.

The steady-state kinetics of normal human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) dimers were studied as a function of pH and temperature. Inhibition studies using glucosamine 6-phosphate, NADPH and p-hydroxymercuribenzoate (P-OHMB) were also carried out at pH 8.0. The existence of two binding sites on the enzyme with a transition from low to high affinity for NADP+ when NADP+ concentration is increased is indicated by the nonlinear Lineweaver-Burk plots and sigmoid kinetic patterns. NADPH inhibition was found to be competitive with respect to NADP+ and non-competitive with respect to glucose-6-phosphate. Logarithmic plot of Vmax against pH and inactivation by P-OHMB indicate the participation in the reaction mechanism of imidazolium group of histidine and sulhydryl groups. The initial velocity and product inhibition data gave results which are consistent with the dimeric enzyme following an ordered sequential mechanism. A possible random mechanism is ruled out by the inhibition results of glucosamine 6-phosphate.