Human erythrocyte glucose 6-phosphate dehydrogenase. Interaction with oxidized and reduced coenzyme

The complex between tetrameric glucose 6-phosphate dehydrogenase (G6PD) and four moles of structural NADP can bind four additional NADP equivalents with a K_diss of 0.85 μM. Alternatively, this complex shows a maximal binding capacity of two NADPH equivalents, with a corresponding K_diss of 0.1 μM. Therefore, a clear discrepancy has emerged from these spectrofluorimetric titrations with either the oxidized or the reduced form of the coenzyme, an “all-of-the-sites reactivity” being observed for NADP and a “half-of-the-sites reactivity” being conversely involved in NADPH binding.     

Human erythrocyte glucose 6-phosphate dehydrogenase. Evidence for competitive binding of NADP and NADPH.

The tetrameric form of human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) was investigated in respect to interaction of coenzyme at its ron-structural sites. 1:N⁶-ethenoadenine dinucleotide phosphate (ε-NADP), although displaying a lower catalytic efficiency compared to NADP, showed identical binding patterns, i.e. four moles per tetramer with a K_diss of 1.0 μM. Furthermore, spectrofluorometric titrations with NADPH in the absence and in presence of varying NADP concentrations revealed a typically competitive mechanism of binding of NADP (four moles) and NADP (two moles) at the non-structural sites of the tetramer.     

A genetic variant of human erythrocyte glucose 6-phosphate dehydrogenase

Human erythrocyte G6PD activity was measured in more than 500 subjects in Isfahan, Iran, and the percent of enzyme deficiency for males and females are reported. Some properties of the abnormal enzyme is compared with its normal counterpart. Apparent Km values of glucose 6-phosphate for the variant and normal enzymes were 37 and 101 microM, respectively. The variant enzyme was less resistant to inhibition by 40 microM NADPH (72% inhibition) than the normal enzyme (48% inhibition). The mode of inhibition for both enzymes was competitive with NADP+. ATP at 1.5 mM concentration also inhibited normal and variant enzymes at 17% and 10%, respectively. The inhibition was competitive with glucose 6-phosphate. Polyacrylamide gel electrophores showed that normal enzyme has one major and another weak active bands, while the variant enzyme under identical conditions shows only one active band corresponding to the major band of the normal enzyme. Thermostability of variant G6PD was slightly lower that normal but no significant differences observed in their energy of activation. The activity pH profile of the variant enzyme was truncate.     

Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history.

Glucose 6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. Inherited G6PD deficiency is associated with either episodic hemolytic anemia (triggered by fava beans or other agents) or life-long hemolytic anemia. We show here that an evolutionary analysis is a key to understanding the biology of a housekeeping gene. From the alignment of the amino acid (aa) sequence of 52 glucose 6-phosphate dehydrogenase (G6PD) species from 42 different organisms, we found a striking correlation between the aa replacements that cause G6PD deficiency in humans and the sequence conservation of G6PD: two-thirds of such replacements are in highly and moderately conserved (50-99%) aa; relatively few are in fully conserved aa (where they might be lethal) or in poorly conserved aa, where presumably they simply would not cause G6PD deficiency. This is consistent with the notion that all human mutants have residual enzyme activity and that null mutations are lethal at some stage of development. Comparing the distribution of mutations in a human housekeeping gene with evolutionary conservation is a useful tool for pinpointing amino acid residues important for the stability or the function of the corresponding protein. In view of the current explosive increase in full genome sequencing projects, this tool will become rapidly available for numerous other genes.     

In vitro correction of erythrocyte glucose 6-phosphate dehydrogenase (G6PD) deficiency.

The permeability characteristics of egg phosphatidylcholine (EPC) vesicles to Eu³⁺ has been examined by ³¹P-nmr, in various mixed lipid systems. It has been found that incorporation of small amounts of sodium taurocholate (NaTC) in the EPC vesicle greatly increased the vesicular permeability to Eu³⁺. Incorporation of cholesterol in the EPC vesicle significantly inhibits the ability of NaTC to induce permeability alterations in this mixed system. It has been reported that at low EPC:NaTC ratios (2:1-0.65:1), mixed micelles of the components are formed [N. A. Mazer, R. F. Kwasnick, M. C. Carey, and G. B. Benedek, 1977, in Micellization, Solubization, and Microemulsions (Mittal, K. L., ed.) Vol. 1, pp. 483-402, Plenum Press, New York]. By examination of the ³¹P-nmr linewidths of EPC, at various ratios of EPC:NaTC, it is possible to follow the decrease in size of the EPC vesicle, as it becomes incorporated into the smaller NaTC micelles. The ³¹P{¹H} nuclear Overhauser enhancement of the simple EPC vesicle is not significantly different from this same parameter measured for EPC, when it exists in mixed micellar systems with NaTC. This indicates that there must be considerable internuclear head group interactions of EPC molecules in EPC-NaTC mixed micelles. This argues for sequestering of EPC in such micelles.     

Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase.

6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.     

Regulation of coenzyme utilization by the dual nucleotide-specific glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroids.

The dual wavelength assay technique (H. R. Levy, and G. H. Daouk, 1979, J. Biol. Chem.254, 4843–4847) is used to examine the rates of the NADP- and NAD-linked reactions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase simultaneously under various conditions. Inhibition by ATP, MgATP^2?, acetyl-CoA, and palmitoyl-CoA is greatly diminished at high glucose 6-P concentration which favors the NAD-linked reaction. Increasing $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ concentration ratios inhibit the NADP-linked, but stimulate the NAD-linked reaction. The selective effects of glucose 6-P and the $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ concentration ratio, which cannot be detected by conventional assays, are explained in terms of the differing kinetic mechanisms for the NADP-linked and NAD-linked reactions previously described (C. Olive, M. E. Geroch, and H. R. Levy, 1971, J. Biol. Chem.246, 2047–2057). It is proposed that these effects constitute the mechanism whereby the nucleotide specificity of the amphibolic glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides is regulated.     

Autosomal determination of erythrocyte glucose 6-phosphate dehydrogenase in domestic chickens and ring-necked pheasants

Erythrocyte glucose 6-phosphate dehydrogenase isozymes of domestic chickens, ring-necked pheasants, and their hybrids were studied, using the starch gel zone electrophoresis technique. In domestic chickens G6PD isozymes were represented by two fast-moving bands and an indistinct third band, whereas in ring-necked pheasants a slow-moving broad band which seemed to consist of two closely apposed G6PD isozymes was observed. The F₁ hybrids showed three distinct bands combining the characteristic mobility pattern of the two parents, which seemed to indicate that both parental alleles are expressed in F₁ hybrids. Since both male and female hybrids exhibited strikingly similar isozyme patterns representing both sire and dam, it was assumed that the genes controlling the production of G6PD in chicken and pheasant red blood cells are located on the autosomes.This study was supported in part by a research grant from the National Research Council of Canada.     

Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

Kinetics of human erythrocyte glucose-6-phosphate dehydrogenase dimers

The steady-state kinetics of human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) dimers were studied by initial rate measurement. These experiments gave intersecting double-reciprocal plots suggesting a ternary complex mechanism with a Km for NADP and glucose 6-phosphate of 11 microM and 43 microM, respectively. These studies were combined with rate measurements in the presence of one product (NADPH), dead-end inhibitors, as well as alternative substrates. The inhibition by NADPH was found to be competitive with respect to both substrates. Alternate substrates experiments gave linear double-reciprocal plots over a wide range of substrate concentrations. The results suggest that the dimeric enzyme follows either a random or a Theorell-Chance mechanism.