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At least 163 records · Page 9Linked to original sources
PubMed1985

Human brain 6-phosphogluconate dehydrogenase: purification and kinetic properties.

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.

Brain↗
PubMed1965

X-linked 6-phosphogluconate dehydrogenase in drosophila: subunit associations.

6-Phosphogluconate dehydrogenase (PGD) subunits have been dissociated and reassociated in vitro. The intermediate PGD of the heterozygous phenotype is reproduced by reassociation in vitro of subunits derived from the two types of PGD from homozygous flies. This result has a bearingon gene action in the diploid organism and on the mechanism of dosage compensation in Drosophila melanogaster.

Animals↗
PubMed1979

Combined use of strain construction and affinity chromatography in the rapid, high-yield purification of 6-phosphogluconate dehydrogenase from Escherichia coli.

A rapid, high-yield method for purification of 6-phosphogluconate dehydrogenase from Escherichia coli K-12 is described. Sonic extracts prepared from heat-induced cultures of strain RW184, doubly lysogenic for the specialized transducing bacteriophage lambdacI857St68h80dgndhis and bearing a deletion of the gene for glucose 6-phosphate dehydrogenase, contained levels of 6-phosphogluconate dehydrogenase 15- to 20-fold higher than cultures of wild-type cells. Affinity chromatography on blue dextran-Sepharose with batchwise elution with 1 mM nicotinamide adenine dinucleotide phosphate affected a further 10-fold purification. Enzyme prepared in this manner was homogeneous according to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and immunoelectrophoresis using antiserum directed against it. Fructose 1,6-diphosphate is an inhibitor of enzyme activity.

Chromatography, Affinity↗
PubMed1988

Population studies in northern Sweden. XIII. The 6-phosphogluconate dehydrogenase polymorphism.

Frequencies of the 6-phosphogluconate dehydrogenase (6-PGD) genes were studied in a series of 314 Finns from northern Finland and in a material of 4,348 conscripts and blood donors from the counties of Norrbotten and Västerbotten in northern Sweden. The Swedish individuals were distributed into 23 subpopulations according to place of birth. The frequency of the PGDC gene was comparatively high in Finns (5.3%) and showed significant variations between subpopulations (from 5.3 to 0.0%). The frequency of the PGDC gene showed a cline, with a decreasing gene frequency in the north-south direction. This geographical pattern could be explained in terms of Lappish and Finnish influence.

Gene Frequency↗
PubMed2005

Purification and kinetic properties of 6-phosphogluconate dehydrogenase from rat small intestine.

6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, DeltaH, Q10 and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4 degrees C, respectively. The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 595 +/- 213 microM for 6PG and 53.03+/-1.99 microM for NADP. 1/Vm versus 1/6PG and 1/NADP plots gave a Vm value of 8.91+/-1.92 U/mg protein. NADPH is the competitive inhibitor with a Ki of 31.91+/-1.31 microM. The relatively small Ki for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.

Animals↗
PubMed2006

Importance in catalysis of the 6-phosphate-binding site of 6-phosphogluconate in sheep liver 6-phosphogluconate dehydrogenase.

The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.

Amino Acid Sequence↗
PubMed2006

Role of the S128, H186, and N187 triad in substrate binding and decarboxylation in the sheep liver 6-phosphogluconate dehydrogenase reaction.

Crystal structures of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver indicate that S128 and N187 are within hydrogen-bonding distance of 6PG in the E:6PG binary complex and NADPH in the E:NADPH binary complex. In addition, H186 is also within hydrogen-bonding distance of NADPH in the E:NADPH binary complex, while in the E:6PG binary complex it is within hydrogen-bonding distance of S128 and close to N187. The structures suggest that this triad of residues may play a dual role during the catalytic reaction. Site-directed mutagenesis has been performed to mutate each of the three residues to alanine. All mutant enzymes exhibit a decrease in V/E(t) (the turnover number), ranging from 7- to 67-fold. An increase in the Km for 6PG (K(6PG)) was observed for S128A and H187A mutant enzymes, while for the H186A mutation, K(6PG) is decreased by a factor of 2. K(NADP) remains the same as the wild type enzyme for the S128A and H186A mutant enzyme, while it increases by 6-fold in the N187A mutant enzyme. An increased K(iNADPH) was measured for all of the mutant enzymes. The primary kinetic 13C-isotope effect is increased, while the primary deuterium kinetic isotope effect is decreased, indicating that the decarboxylation step has become more rate limiting under conditions where substrate is limiting. A quantitative analysis of the data suggests that the S128, H186, and N187 triad is multifunctional in the 6PGDH reaction and contributes as follows. The triad (1) participates in the precatalytic conformational change; (2) provides ground state binding affinity for 6PG and NADPH; and (3) affects the relative rates of reduction or decarboxylation of the 3-keto-6PG intermediate by anchoring the cofactor after hydride transfer, which is accompanied by the rotation of the nicotinamide ring around the N-glycosidic bond and displacement of C1 of 6PG, facilitating decarboxylation.

Amino Acid Substitution↗
PubMed1990

Possible involvement of NADPH requirement in regulation of glucose-6-phosphate and 6-phosphogluconate dehydrogenase levels in rat liver.

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.

Animals↗
PubMed1984

DNA sequence of the Escherichia coli gene, gnd, for 6-phosphogluconate dehydrogenase.

Expression of gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, an enzyme of the hexose monophosphate shunt, is subject to growth rate-dependent regulation and is gene dosage-dependent: the level of the enzyme increases in direct proportion to the cellular growth rate at both low and high gene copy numbers. We have determined the nucleotide sequence of gnd and flanking control regions, the 5'-end of in vivo gnd mRNA, and the start codon of the structural gene. Analysis of the sequence indicated that: (i) the gnd promoter is typical of other E. coli promoters and the structural gene is followed by a rho-independent transcription termination signal; (ii) the 56-nucleotide leader of gnd mRNA does not contain a rho-independent transcription termination signal, so growth rate-dependent regulation of 6-phosphogluconate dehydrogenase level is not carried out by an attenuation mechanism analogous to the one that controls expression of the E. coli ampC gene; (iii) the codon composition of the structural gene resembles that of other highly expressed E. coli genes and thus is not responsible for the regulation either; (iv) the structural gene is preceded at an optimal distance by a strong Shine-Dalgarno (SD) sequence, AGGAG ; (v) the leader region of the mRNA contains regions of dyad symmetry that have the potential to sequester the SD sequence and the start codon. This latter feature of the gene suggests that growth rate-dependent regulation may involve regulation of translation initiation frequency.

Amino Acid Sequence↗
PubMed1993

A potent specific inhibitor of 6-phosphogluconate dehydrogenase of Cryptococcus neoformans and of certain other fungal enzymes.

A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases from Cryptococcus neoformans and Schizophyllum commune and glucose-6-phosphate dehydrogenase from Schizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than the Cryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent.

Adenosine Monophosphate↗
PubMed1983

Amino acid sequence of ovine 6-phosphogluconate dehydrogenase.

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes.

Amino Acid Sequence↗
PubMedDate not supplied

[A comparative study of the interaction between glutathione reductase and 6-phosphogluconate dehydrogenase of rat liver with certain affinity sorbents].

A comparative study of the conditions of interaction of some group-specific ligands with rat hepatic 6-phosphogluconate dehydrogenase and glutathione reductase was accomplished. The influence of ionic strength and coenzyme concentration in buffer solution on the extent of enzymes binding with immobilized 2',5'-ADP and procion red HE-3B was studied according to the residual enzymatic activity after the interaction of enzyme and adsorbent. The optimal conditions have been found for enzymes binding and for their elution resulted in the effective purification of the enzymes mentioned above. Enzymatic preparations were free from the contaminating activity of each other. Dye-ligand chromatography step on red sepharose was introduced into the three step scheme of the purification of 6-phosphogluconate dehydrogenase. The final preparation of 6-phosphogluconate dehydrogenase obtained by gradient salt elution of the enzyme from the red sepharose column (0-0.15 M KCl) has the specific activity of 30-34 E/mg and shows electrophoretical homogeneity. The contribution of ionic strength and biospecific interaction of the nucleotide binding site of the enzymes during chromatography on these adsorbents is discussed.

Animals↗
PubMed1972

Decrease of Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenase Activities in the Xylem of Populus gelrica on Budding.

The activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases, transketolase, phosphoglucose isomerase, and fructose 6-phosphate kinase were studied in extracts of wintering poplar (Populus gelrica) xylem. The xylem of wintering poplar showed high levels of transketolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases. On recommencement of growth, the two dehydrogenase activities decreased. The three remaining enzymes appeared to be unchanged. In spring and early summer, glucose 6-phosphate dehydrogenase of the xylem was extremely low. On the other hand, 6-phosphogluconate dehydrogenase, which also became lower during the metabolic shift from winter to spring, was readily detected, and was several times higher than glucose 6-phosphate dehydrogenase throughout the year. The low dehydrogenase activities lasted into late October and then appeared to resume their original activity. A shift of metabolism at the beginning of growth was also observed by measuring the amount of sugar phosphates, soluble amino acids and amides, and proteins in the xylem. In contrast to the decrease of the two dehydrogenases and soluble proteins at the time of budding, incorporation of lysine-U-(14)C into the xylem protein ramained constant. A method to transfuse radioactive compounds into a section of stem was described.

Journal Article↗
PubMed1992

NADPH activates a decarboxylation reaction catalysed by lamb liver 6-phosphogluconate dehydrogenase.

NADP-dependent lamb liver 6-phosphogluconate dehydrogenase catalyses the oxidative decarboxylation of 2-deoxy-6-phosphogluconate, an analogue of the natural substrate. The first products of the reaction are NADPH and 3-keto-2-deoxy-6-phosphogluconate. The NADPH, released from the enzyme, binds to the coenzyme site of the same or the other subunit, activating the decarboxylation reaction in which has not a redox role, since it can be substituted by an analogue devoid of enzymatic redox power. These findings are compared to those obtained with other NADP-dependent decarboxylating dehydrogenases.

Animals↗
PubMed1988

Bass liver 6-phosphogluconate dehydrogenase: inhibition by nucleoside phosphates and by fructose 1,6 bisphosphate.

Nucleoside 5'-triphosphates, 5'-diphosphates and 5'-monophosphates are inhibitors of the 6-phosphogluconate dehydrogenase enzyme from bass liver. The 2'- and 3'-monophosphates of adenosine and guanosine are also inhibitory, the 2'-isomers being especially potent inhibitors. The catalytic activity of 6-phosphogluconate dehydrogenase has been found to be markedly inhibited by fructose 1, 6 bisphosphate. As the Km for 6-phosphogluconate, the Ki for fructose 1,6 bisphosphate and the concentration of both compounds in bass liver are all comparable, it appears that the inhibition of 6-phosphogluconate dehydrogenase by fructose 1,6 bisphosphate may be of significance in the regulation of carbohydrate metabolism in bass liver.

Animals↗
PubMed1977

Fluorescent labelling of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.

The thermophilic 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus was inhibited upon specific modification of the -SH group of cysteine residues by 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) at pH 7.0. By using 20-100-fold molar excess of NBD-CL the reaction occurs slowly at pH 7.0 as a first order process. Partial protection from inactivation was observed when the substrate 6-phosphogluconate or the coenzyme NADP was added to the reaction mixture. Complete inactivation was achieved upon modification of 1.9 of the six cysteine residues per mole of enzyme, which corresponds to nearly one residue per enzyme subunit. Circular dichroism measurements suggest that the gross structure of the protein molecule is practically unchanged upon reaction of the enzyme with NBD-Cl. Melting profile experiments revealed a single transition occurring at about 65 degrees C. Analogously, the profile of intensity of the fluorescence emission at 520 nm of the enzyme-bound S-NBD groups versus temperature indicated a midpoint of transition near 65 degrees C. Since this melting temperature corresponds closely to that observed with the native enzyme, these results would indicate that the molecular organizations of the native and modified enzyme are similar and stabilized by similar interactions within the polypeptide chain.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Implication of lysine residues in the loss of enzymatic activity in rat liver 6-phosphogluconate dehydrogenase found in aging.

During aging, there is a decrease in the activity of the 6-phosphogluconate dehydrogenase enzyme in rat liver. The "old" 6-phosphogluconate dehydrogenase enzyme is about 26% less active than the "young" enzyme. In this paper, some biochemical and chemical properties of this enzyme are studied. 2,4,6-Trinitro-benzenesulfonic acid measurements indicate that the old enzyme has 11 lysine residues less than the young enzyme. The proteolysis with trypsin produces more peptides in the young enzyme than in the old one. However, similar numbers of peptides were produced when endoproteinase Arg-C was used on both enzymes. Moreover, the treatment of the young enzyme with ascorbate for 15 min produces the loss of 8 lysine residues. These results suggest that during aging there is a modification of the lysine residue, and this could be involved in the loss of its enzymatic activity.

Aging↗