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PubMed2004

Crystallization and preliminary X-ray diffraction analysis of the hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum.

NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum was crystallized in the apo- and holoenzyme forms. Crystals were obtained using 2-propanol and polyethylene glycol MME 550 as precipitants for the apoenzyme and holoenzyme, respectively. The apoenzyme crystals belong to the trigonal space group P3(1)21 or its enantiomorph P3(2)21. The asymmetric unit contains three subunits; the values of the Matthews coefficient (VM) and the solvent content are 2.9 A3 Da-1 and 57%, respectively. A native data set was collected to a highest resolution limit of 4.0 A on an in-house X-ray source using a rotating-anode generator (overall Rsym of 12.3% and completeness of 97%). The holoenzyme crystals belong to the orthorhombic space group P2(1)2(1)2(1); the asymmetric unit contains one hexamer, giving a VM of 2.79 A3 Da-1 and a solvent content of 55%. Native and derivative data sets were collected. The crystals diffract to a maximum resolution of 2.8 A on the KEK-NW12 beamline at the Photon Factory and gave a data set with an overall Rsym of 7.9% and a completeness of 91%. Attempts are being made to solve the structure by the SIRAS method.

Archaeal Proteins↗
PubMed1977

The interaction of phospholipid membranes and detergents with glutamate dehydrogenase.

1. The interaction of beef liver glutamate dehydrogenase with cardiolipin from both beef liver mitochondria and beef heart mitochondria, with phosphatidylcholine from both beef liver mitochondria and egg-yolk, and with beef brain phosphatidylserine was investigated by steady-state kinetic methods. 2. the phosphatidylcholine did not inhibit the enzyme under a wide range of conditions. The cardiolipins and phosphatidylserine inhibited the enzyme. The inhibition by these lipids was found to diminish with time if the lipids were prepared and the reaction was studied in either phosphate or Tris buffers, but in zwitterionic buffers these lipid brought about a rapid, reversible inhibition which remained stable with time for at least 150 min. 3. The kinetic type of the inhibition was difficult to determine because of variation between lipid sonicates. Complex mixed types of inhibition were found with cardiolipin, and with phosphatidylserine the inhibition approximated to a non-competitive interaction with Ki(app) values varying between (0.9-6.1) x 10(-6)M. 4. The extent of inhibition decreased with increasing pH and with increasing ionic strength. Basic proteins, such as cytochrome c, show a higher affinity for the anionic membranes and can dissociate the enzyme-lipid complexes. Cosonicates of the cardiolipin and phosphatidylcholine inhibited the enzyme, the extent of inhibition increasing in proportion to the amount of acidic lipid. 5. Sodium dodecylsulphate causes a time-dependent inhibition of the enzyme. The kinetics of this effect and its variation with detergent concentration were studied. 6. The relationship of these observations to the structure and function of the enzyme is discussed. It is suggested that their apparent regulation of the enzyme by oestrogens and other small molecules is due to their binding in vitro at sites on the enzyme designed for binding cardiolipin, when the enzyme is functioning in vivo. The association of the enzyme oligomer in vitro may, for similar reasons, be an artifact.

Animals↗
PubMed1988

Regulation of insulin release by factors that also modify glutamate dehydrogenase.

Leucine and monomethyl succinate initiate insulin release, and glutamine potentiates leucine-induced insulin release. Alanine enhances and malate inhibits leucine plus glutamine-induced insulin release. The insulinotropic effect of leucine is at least in part secondary to its ability to activate glutamate oxidation by glutamate dehydrogenase (Sener, A., Malaisse-Lagae, F., and Malaisse, W. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5460-5464). The effect of these other amino acids or Krebs cycle intermediates on insulin release also correlates with their effects on glutamate dehydrogenase and their ability to regulate inhibition of this enzyme by alpha-ketoglutarate. For example, glutamine enhances insulin release and islet glutamate dehydrogenase activity only in the presence of leucine. This could be because leucine, especially in the presence of alpha-ketoglutarate, increases the Km of glutamate and converts alpha-ketoglutarate from a noncompetitive to a competitive inhibitor of glutamate. Thus, in the presence of leucine, this enzyme is more responsive to high levels of glutamate and less responsive to inhibition by alpha-ketoglutarate. Malate could decrease and alanine could increase insulin release because malate increases the generation of alpha-ketoglutarate in islet mitochondria via the combined malate dehydrogenase-aspartate aminotransferase reaction, and alanine could decrease the level of alpha-ketoglutarate via the alanine transaminase reaction. Monomethyl succinate alone is as stimulatory of insulin release as leucine alone, and glutamine enhances the action of both. Succinyl coenzyme A, leucine, and GTP are all bound in the same region on glutamate dehydrogenase, where GTP is a potent inhibitor and succinyl coenzyme A and leucine are comparable activators. Thus, the insulinotropic properties of monomethyl succinate could result from it increasing the level of succinyl coenzyme A and decreasing the level of GTP via the succinate thiokinase reaction.

Alanine↗
PubMed1975

A product-inhibition study of bovine liver glutamate dehydrogenase.

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.

Ammonia↗
PubMed1994

Lobular patterns of expression and enzyme activities of glutamine synthase, carbamoylphosphate synthase and glutamate dehydrogenase during postnatal development of the porcine liver.

Carbamoylphosphate synthase and glutamine synthase show a complementary distribution in the liver lobule of the rat. In the human liver lobule, which is approximately 2-fold larger than that of the rat, an intermediate, 'empty' zone is present between the periportal carbamoylphosphate synthase-positive and the pericentral glutamine synthase-positive zone. To investigate whether these differences in gene expression can be attributed to the size of the liver lobule, we investigated the patterns of expression of carbamoylphosphate synthase, glutamine synthase and glutamate dehydrogenase during postnatal development of the pig, a species in which the total number of lobules does not increase after birth. We demonstrate that lobular size increases 3-fold between 1 week and 8 months after birth. In the same developmental period the number of hepatocytes on the porto-central axis increases 2-fold, resulting in a 3-fold increase in cellular volume. However, the lobular patterns of expression of carbamoylphosphate synthase, glutamate dehydrogenase and glutamine synthase do not change anymore after 1 month, i.e., when lobular diameter is comparable to that in rat liver, showing that lobular size is not a major determinant of the heterogeneous patterns of expression of these enzymes. The adult patterns of expression of glutamine synthase, glutamate dehydrogenase and, in particular carbamoylphosphate synthase in the porcine liver resemble those of man. Changes in the enzyme activities of glutamate dehydrogenase and carbamoylphosphate synthase are not related to the lobular size. However, the 70% decrease of GS activity in the 8-month-old pigs corresponds with the gradual 2-3-fold decrease in the size of the GS-positive compartment during postnatal development. During adulthood GS activity increases again to values observed 1 week after birth demonstrating a 2-fold increase in cellular glutamine synthase content. The present data show that the pig is an excellent model to study the regulation and functional implication of zonation of gene expression in the human liver.

Animals↗
PubMed1984

NADP-dependent glutamate dehydrogenase from a facultative methylotroph, Pseudomonas sp. strain AM1.

The NADP-dependent glutamate dehydrogenase (EC 1.4.1.4.) elaborated by the methylotrophic bacterium Pseudomonas sp. strain AM1 when growing on succinate and ammonium chloride was studied. The enzyme, which has a pH optimum of 9.0, was purified 140-fold and shown to have Km values of 20.2 mM, 0.76 mM, 0.033 mM, and 31.6 mM for ammonia, alpha-ketoglutarate, NADPH, and glutamate, respectively. The native molecular weight was determined by polyacrylamide gel electrophoresis to be 190,000, and electrophoresis under denaturing conditions in the presence of sodium dodecyl sulfate revealed a minimum molecular weight of 50,000. The enzyme was highly specific; NADH was unable to replace NADPH in the reaction, various alpha-keto acids could not replace alpha-ketoglutarate, and neither methylamine nor hydroxylamine could substitute for ammonia. Glutamate dehydrogenase was synthesized by the bacteria only when ammonia was its nitrogen source and was repressed if methylamine or nitrate were provided as sources of nitrogen instead of ammonia.

Ammonium Chloride↗
PubMed1991

Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

Amino Acid Sequence↗
PubMed1989

Oxidation of Neurospora crassa NADP-specific glutamate dehydrogenase by activated oxygen species.

The glutamine synthetase and the NADP-specific glutamate dehydrogenase activities of Neurospora crassa were lost in a culture without carbon source only when in the presence of air. Glutamine synthetase was previously reported to be liable to in vitro and in vivo inactivation by activated oxygen species. Here we report that NADP-specific glutamate dehydrogenase was remarkably stable in the presence of activated oxygen species but was rendered susceptible to oxidative inactivation when chelated iron was bound to the enzyme and either ascorbate or H2O2 reacted on the bound iron. This reaction gave rise to further modifications of the enzyme monomers by activated oxygen species, to partial dissociation of the oligomeric structure, and to precipitation and fragmentation of the enzyme. The in vitro oxidation reaction was affected by pH, temperature, and binding to the enzyme of NADPH. Heterogeneity in total charge was observed in the purified and immunoprecipitated enzymes, and the relative amounts of enzyme monomers with different isoelectric points changes with time of the oxidizing reaction.

Aerobiosis↗
PubMed2000

Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms: implications for biologic function.

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.

ATP-Binding Cassette Transporters↗
PubMed1989

Glutamate metabolism of leukocytes and skin fibroblasts in spinocerebellar degeneration with lowered glutamate dehydrogenase activity.

From 21 patients with spinocerebellar degeneration 5 had markedly decreased glutamate dehydrogenase (GDH) activities and high values of serum plasma glutamate level after oral glutamate loading tests. Skin fibroblasts from patients with GHD deficiency showed intracellularly higher glutamate and lower glutathione contents than those from controls and showed significantly decreased viability in L-glutamate-containing medium. These data suggest that glutamate toxicity may at least play a part in this degeneration process.

Adolescent↗
PubMed1996

Crystallization of the glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis.

The NADP(+)-dependent glutamate dehydrogenase from Thermococcus litoralis has been crystallized by the hanging-drop method of vapour diffusion using an ammonium sulfate and PEG mixture as the precipitant. The crystals belong to the monoclinic system and are in space group C2 with unit-cell dimensions a = 142.7, b = 202.0, c = 125.8 A with beta = 113.1 degrees with a hexamer in the asymmetric unit. T. Litoralis, a hyperthermophilic organism, belongs to the family of Archaea and has a maximum growth temperature of about 370 K. The glutamate dehydrogenase isolated from this organism has a half-life of 2 h at 373 K and a comparison of this structure with that of other GluDH's from hyperthermophilic organisms and from mesophiles will contribute to an understanding of the molecular mechanisms which underlie thermostability.

Journal Article↗
PubMed2004

CcpA-dependent regulation of Bacillus subtilis glutamate dehydrogenase gene expression.

The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B. subtilis cells in glucose-glutamate medium. Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium.

Bacillus subtilis↗
PubMed1980

Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron.

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.

Anions↗
PubMed1995

Crystallization of the NAD(P)-dependent glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

The NAD(P)-dependent glutamate dehydrogenase from Pyrococcus furiosus has been crystallized by the hanging-drop method of vapour diffusion using lithium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group P4(2)2(1)2 with unit-cell dimensions of a = b = 167.2, c = 172.9 A. Consideration of the values of V(m) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a trimer. P. furiosus belongs to the family of Archaea and is one of the most thermostable organisms known, having an optimal growth temperature of 376 K. The glutamate dehydrogenase isolated from this organism has a half-life of 12 h at 373 K and, therefore, the determination of the structure of this enzyme will be important in advancing our understanding of how proteins are adapted to enable them to survive at such extreme temperatures.

Journal Article↗
PubMed1987

Role of glutamate dehydrogenase in ammonia assimilation in nitrogen-fixing Bacillus macerans.

Pathways of ammonia assimilation into glutamic acid in Bacillus macerans were investigated by measurements of the specific activities of glutamate dehydrogenase (GDH), glutamine synthetase, and glutamate synthase. In ammonia-rich medium, GDH was the predominant pathway of ammonia assimilation. In nitrogen-fixing cells in which the intracellular NH4+ concentration was 1.4 +/- 0.5 mM, the activity of GDH with a Km of 2.2 mM for NH4+ was found to be severalfold higher than that of glutamate synthase. The result suggests that GDH plays a significant role in the assimilation of NH4+ in N2-fixing B. macerans.

Ammonia↗
PubMed2003

Characterization of the gene encoding glutamate dehydrogenase ( gdhA) from the ruminal bacterium Ruminococcus flavefaciens FD-1.

The gene encoding glutamate dehydrogenase ( gdhA) in the ruminal bacterium Ruminococcus flavefaciens FD-1 was cloned. A degenerate primer based on the N-terminal amino acid sequence of the purified protein was used in conjunction with genome walking to obtain the complete ORF of 1,365 bp, capable of encoding a polypeptide of 455 amino acid residues. The translated ORF contained the amino acid motifs characteristic of the subfamily GDH S_50(I) small glutamate dehydrogenases, including the catalytic site, and matched the originally deduced N-terminal amino acid sequence. BLAST search yielded high scores with other GdhA sequences from a variety of organisms, the closest match being with the GdhA sequence of Corynebacterium glutamicum (63% amino acid identity). Classification of the GdhA enzyme from R. flavefaciens FD-1 as a GDH S_50(I) subfamily member was further supported by phylogenetic analysis. The transcript size determined by Northern blot analysis was in good agreement with the putative regulatory region of the gene and confirmed its monocistronic structure. R. flavefaciens GdhA activity appears to be regulated primarily at the level of transcription. Brief exposure to 20 mM NH(4)Cl prior to extraction did not alter the level of activity. Transcriptional regulation, studied with quantitative real-time RT-PCR, demonstrated a three-fold increase of the gdhA transcript concentration in ammonia-limited cells in comparison with an excess of ammonia in the medium. This is in agreement with the enzyme activity data obtained under ammonia- and carbon-limited growth conditions.

Amino Acid Sequence↗
PubMed1997

Intersubunit communication in hybrid hexamers of K89L/A163G/S380A and C320S mutants of glutamate dehydrogenase from Clostridium symbiosum.

The triple mutant K89L/A163G/S380A (inactive with glutamate but active with L-Nle and L-Met) and C320S (fully active with glutamate, entirely inactive with L-Nle and L-Met, and also lacking reactive cysteine) mutant of glutamate dehydrogenase (EC 1.4.1.2) of Clostridium symbiosum could be completely denatured by urea with the loss of structure and activity. The mutants denatured by urea could be reassociated to give stable hexamers with recovery of activity of approximately 67% by dilution in 0.1 M potassium phosphate buffer (pH 7.0) containing 2 mM NAD+. The native, urea-denatured, and renatured states of mutant enzymes were characterized by size exclusion chromatography on FPLC and native PAGE. Intersubunit hybrid hexamers containing five subunits of triple mutant and one subunit of C320S mutant were constructed by in vitro subunit hybridization followed by affinity chromatography. Kinetic analysis showed that a 5:1 hybrid hexamer, with only one C320S subunit able to bind NAD+ after DTNB modification, shows classical Michaelis-Menten kinetics with regard to NAD+. This contrasts with the apparent negative co-operativity shown by pure C320S hexamers and suggests that the interaction in NAD+ binding among subunits is eliminated in the hybrid. After removal of thionitrobenzoate, however, all of the subunits in the hybrid are able to bind NAD+. In this state the hybrid enzyme showed slight deviation from classical behavior with regard to NAD+, indicating reintroduction of some level of allosteric interaction. The hybrid hexamer also showed much reduced co-operativity with glutamate at pH 8.8, with a Hill coefficient of 3 for DTNB-treated hybrid (as compared to 5.2 for the pure C320S mutant) and 2.2 for the untreated hybrid. The fact that co-operativity in glutamate binding is not entirely eliminated correlates with evidence that the triple mutant subunits, though inactive toward glutamate, can nevertheless still bind this amino acid.

Alanine↗