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PubMed1986

Affinity labeling of an allosteric ADP site of glutamate dehydrogenase by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate.

Bovine liver glutamate dehydrogenase reacts covalently with the adenine nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of about 1 mol of reagent/mol of enzyme subunit. The modified enzyme is not inactivated by this reaction as measured in the absence of allosteric effectors. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of NADH; both of these effects are irreversibly decreased upon reaction of the enzyme with 2-BDB-TAMP. The decrease in activation by ADP was used to determine the rate constant for reaction with 2-BDB-TAMP. The rate constant (kobs) for loss of ADP activation exhibits a nonlinear dependence on 2-BDB-TAMP concentration, suggesting a reversible binding of reagent (KR = 0.74 mM) prior to irreversible modification. At 1.2 mM 2-BDB-TAMP, kobs = 0.060 min-1 and is not affected by alpha-ketoglutarate or GTP, but is decreased to 0.020 min-1 by 5 mM NADH and to zero by 5 mM ADP. Incorporation after incubation with 1.2 mM 2-BDB-TAMP for 1 h at pH 7.1 is 1.02 mol/mol enzyme subunit in the absence but only 0.09 mol/subunit in the presence of ADP. The enzyme protected with 5 mM ADP behaves like native enzyme in its activation by ADP and in its inhibition by NADH. Native enzyme binds reversibly 2 mol of [14C]ADP/subunit, whereas modified enzyme binds only 1 mol of ADP/peptide chain. These results indicate that incorporation of 1 mol of 2-BDB-TAMP causes elimination of one of the ADP sites of the native enzyme. 2-BDB-TAMP acts as an affinity label of an ADP site of glutamate dehydrogenase and indirectly influences the NADH inhibitory site.

Adenosine Diphosphate↗
PubMed1995

Insights into thermal stability from a comparison of the glutamate dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis.

In the light of the solution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from the mesophile Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequences for the thermophilic glutamate dehydrogenases from Thermococcus litoralis and Pyrococcus furiosus against the sequence and the molecular structure of the glutamate dehydrogenase from C. symbiosum, to provide insights into the molecular basis of their thermostability. This homology-based modelling is simplified by the relatively small number of amino acid substitutions between the two thermophilic glutamate dehydrogenase sequences. The most frequent amino acid exchanges involve substitutions which increase the hydrophobicity and sidechain branching in the more thermostable enzyme; particularly common is the substitution of valine to isoleucine. Examination of the sequence differences suggests that enhanced packing within the buried core of the protein plays an important role in maintaining stability at extreme temperatures. One hot spot for the accumulation of exchanges lies close to a region of the molecule involved in its conformational flexibility and these changes may modulate the dynamics of this enzyme and thereby contribute to increased stability.

Amino Acid Sequence↗
PubMed1994

The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium.

The complete amino acid sequence of glutamate dehydrogenase from the archaebacterium Pyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium, Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992), Eur. J. Biochem. 203, 81-87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase from Pyrococcus furiosus with that of S. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.

Amino Acid Sequence↗
PubMed1996

The dynamics of local kinetic parameters of glutamate dehydrogenase in rat liver.

Kinetic parameters of glutamate dehydrogenase (GDH, EC 1.4.1.2) for glutamate were determined in periportal and pericentral zones of adult male and female rat liver lobules under normal fed conditions and after starvation for 24 h. GDH activity was measured as formazan production over time against a range of glutamate concentrations in serial cryostat sections using image analysis. Captured gray value images were transformed to absorbance images and local initial velocities (Vini) were calculated. A hyperbolic function was used to describe the relationship between substrate concentration and local Vini. Under fed conditions, Vmax values were similar in male and female rats (8 +/- 2 and 16 +/- 2 mumol min-1 cm-3 liver tissue in periportal and pericentral zones, respectively). Starvation increased Vmax, especially in pericentral zones of females (to 27 +/- 1 mumol min-1 cm-3 liver tissue). Under fed conditions, the affinity of GDH for glutamate was similar in male and female rats (2.5 +/- 0.5 mM and 3.5 +/- 0.8 mM in periportal and pericentral zones, respectively). Starvation had no effect on K(m) values in male rats, but in female rats affinity for glutamate decreased significantly in both zones (K(m) values of 4.0 +/- 0.1 mM and 8.6 +/- 0.8 mM, respectively). These local changes in the kinetic parameters of GDH indicate that conversion of glutamate to alpha-oxoglutarate cannot be predicted on the basis of GDH concentrations or zero-order activity in the different zones of liver lobules alone.

Animals↗
PubMed2003

Structural studies on ADP activation of mammalian glutamate dehydrogenase and the evolution of regulation.

Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Unlike GDH from bacteria, mammalian GDH exhibits negative cooperativity with respect to coenzyme, activation by ADP, and inhibition by GTP. Presented here are the structures of apo bovine GDH, bovine GDH complexed with ADP, and the R463A mutant form of human GDH (huGDH) that is insensitive to ADP activation. In the absence of active site ligands, the catalytic cleft is in the open conformation, and the hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals. This is consistent with the fact that ADP promotes aggregation in solution. ADP is shown to bind to the second, inhibitory, NADH site yet causes activation. The beta-phosphates of the bound ADP interact with R459 (R463 in huGDH) on the pivot helix. The structure of the ADP-resistant, R463A mutant of human GDH is identical to native GDH with the exception of the truncated side chain on the pivot helix. Together, these results strongly suggest that ADP activates by facilitating the opening of the catalytic cleft. From alignment of GDH from various sources, it is likely that the antenna evolved in the protista prior to the formation of purine regulatory sites. This suggests that there was some selective advantage of the antenna itself and that animals evolved new functions for GDH through the addition of allosteric regulation.

Adenosine Diphosphate↗
PubMed1990

Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae.

We cloned GDH2, the gene that encodes the NAD-linked glutamate dehydrogenase in the yeast Saccharomyces cerevisiae, by purifying the enzyme, making polyclonal antibodies to it, and using the antibodies to screen a lambda gt11 yeast genomic library. A yeast strain with a deletion-disruption allele of GDH2 which replaced the wild-type gene grew very poorly with glutamate as a nitrogen source, but growth improved significantly when the strain was also provided with adenine or other nitrogenous compounds whose biosynthesis requires glutamine. Our results indicate that the NAD-linked glutamate dehydrogenase catalyzes the major, but not sole, pathway for generation of ammonia from glutamate. We also isolated yeast mutants that lacked glutamate synthase activity and present evidence which shows that normally NAD-linked glutamate dehydrogenase is not involved in glutamate biosynthesis, but that if the enzyme is overexpressed, it may function reversibly in intact cells.

Alleles↗
PubMed1983

Glutamate dehydrogenase deficiency in patients with olivopontocerebellar atrophy.

Deficiency of glutamate dehydrogenase appears to be associated with a chronic progressive degenerative disorder manifesting parkinsonian extrapyramidal features, ataxia, supranuclear oculomotor dysfunction, a peripheral neuropathy and, in some cases, amyotrophy. The clinical features resemble those of the Dejerine-Thomas type of olivopontocerebellar atrophy. The data suggest autosomal dominant inheritance with low penetrance. Measurement of leukocyte glutamate dehydrogenase should be routinely performed in the evaluation of newly diagnosed or atypical cases of parkinsonism.

Aged↗
PubMed1966

Immunochemical and enzymatic studies and glutamate dehydrogenase and a related mutant protein from Neurospora crassa.

Roberts, D. B. (University of Cambridge, Cambridge, England). Immunochemical and enzymatic studies on glutamate dehydrogenase and a related mutant protein from Neurospora crassa. J. Bacteriol. 91:1888-1895. 1966.-When an investigation was made of the inhibition of Neurospora glutamate dehydrogenase by bivalent and univalent antibodies, it was shown that the enzyme inhibition is not complete even with excess antibodies. The residual activity was some three times greater with bivalent antibodies, in spite of the observation that the ratio of inhibiting antibodies to catalytic sites was 2:1 for both types of antibody. Substrates did not affect the inhibition of enzyme activity, nor did antibodies affect the K(m) for either substrate. An allosteric mechanism for the inhibition of glutamate dehydrogenase by antibodies is proposed. It was also demonstrated that the mutant protein am-3 can be activated, to show glutamate dehydrogenase activity, by a number of activators. The requirement for the activation was the presence of a carboxymethyl group. The data suggest that the nonactivated protein has two combining sites for l-glutamate: the catalytic and activating sites. The wild-type enzyme has only one of these sites. Because the activating site is distinct from the catalytic site, an allosteric mechanism was postulated for activation. Inhibition of am-3 activity by antibodies is achieved either by a mechanism similar to the inhibition of wild-type activity or by the antibodies preventing the activation of the mutant protein. The am-3 protein can be activated by antibodies. Consequently, there appeared to be a relation the phenomena of enzyme inhibition and am-3 activation by antibodies; i.e., they alter the configuration of the catalytic site. This alteration was necessary for the activation of am-3, but inhibited the activity of the wild-type enzyme.

Antibodies↗
PubMed2001

Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation.

Glutamate dehydrogenase is found in all organisms and catalyses the oxidative deamination of l-glutamate to 2-oxoglutarate. However, only animal GDH utilizes both NAD(H) or NADP(H) with comparable efficacy and exhibits a complex pattern of allosteric inhibition by a wide variety of small molecules. The major allosteric inhibitors are GTP and NADH and the two main allosteric activators are ADP and NAD(+). The structures presented here have refined and modified the previous structural model of allosteric regulation inferred from the original boGDH.NADH.GLU.GTP complex. The boGDH.NAD(+).alpha-KG complex structure clearly demonstrates that the second coenzyme-binding site lies directly under the "pivot helix" of the NAD(+) binding domain. In this complex, phosphates are observed to occupy the inhibitory GTP site and may be responsible for the previously observed structural stabilization by polyanions. The boGDH.NADPH.GLU.GTP complex shows the location of the additional phosphate on the active site coenzyme molecule and the GTP molecule bound to the GTP inhibitory site. As expected, since NADPH does not bind well to the second coenzyme site, no evidence of a bound molecule is observed at the second coenzyme site under the pivot helix. Therefore, these results suggest that the inhibitory GTP site is as previously identified. However, ADP, NAD(+), and NADH all bind under the pivot helix, but a second GTP molecule does not. Kinetic analysis of a hyperinsulinism/hyperammonemia mutant strongly suggests that ATP can inhibit the reaction by binding to the GTP site. Finally, the fact that NADH, NAD(+), and ADP all bind to the same site requires a re-analysis of the previous models for NADH inhibition.

Adenosine Diphosphate↗
PubMed1990

Isolation and characterization of the two distinct genes for human glutamate dehydrogenase.

Two genomic clones containing a part of the glutamate dehydrogenase gene were isolated from a human genomic library. The restriction map of both clones were distinctly different from one another, although the nucleotide sequences of the three exons that they contained were virtually the same in each clone. Southern blotting analysis of the genomic DNAs from several unrelated human individuals revealed that in every case the probe hybridized with at least two DNA fragments of different sizes, each characteristic to one of the two clones. These results strongly suggest that the two clones presently obtained do not result from polymorphism but are generated from two different gene loci for glutamate dehydrogenase on the human chromosome.

Amino Acid Sequence↗
PubMed1999

Specificity of coenzyme analogues and fragments in promoting or impeding the refolding of clostridial glutamate dehydrogenase.

NAD+ facilitates high-yield reactivation of clostridial glutamate dehydrogenase (GDH) after unfolding in urea. The specificity of this effect has been explored by using analogues and fragments of NAD+. The adenine portion, unlike the nicotinamide portion, is important for reactivation. Alteration in the nicotinamide portion, in acetylpyridine adenine dinucleotide, has little effect, whereas loss of the 6-NH2 substitution on the adenine ring, in 6-deamino NAD, diminishes the effectiveness of the nucleotide in promoting refolding. Also ADP-ribose, lacking nicotinamide, promotes reactivation whereas NMN-phosphoribose, lacking the adenine, does not. Of the smaller fragments, those containing an adenosine moiety, and especially those with one or more phosphate groups, impede the refolding ability of NAD+, and are able to bind to the folding intermediate though unable to facilitate refolding. These results are interpreted in terms of the known 3D structure for clostridial glutamate dehydrogenase. It is assumed that the refolding intermediate has a more or less fully formed NAD+-binding domain but a partially disordered substrate-binding domain and linking region. Binding of NAD+ or ADP-ribose appears to impose new structural constraints that result in completion of the correct folding of the second domain, allowing association of enzyme molecules to form the native hexamer.

Chromatography, Thin Layer↗
PubMed1981

Evidence of activation of the renal glutamate dehydrogenase pathway in intact acidotic dogs.

To determine if activity of the renal glutamate dehydrogenase (GD) pathway changes during chronic acidosis in intact dogs, we assessed the deamination of glutamate formed within renal cells during glutamine and alanine infusions. Infusing glutamine into chronically acidotic, normal and acutely alkalotic dogs enhanced renal ammonia production; more was formed as glutamine loading increased. In 4 acidotic dogs, the ratio of ammonia produced to glutamine extracted by the kidneys during exogenous glutamine loading was 1.93 compared with 0.99 for 5 alkalotic dogs and 1.23 for 2 control dogs. Little glutamate and alanine were released into the renal vein in acidotic dogs, whereas over 50% of the exogenous glutamine extracted in acutely alkalotic dogs could be accounted for as glutamate and alanine released into the renal vein. Renal glutamate concentrations were not elevated in acidosis compared with alkalosis despite greater deamidation. When glutamine infusions increased renal ammoniagenesis in acutely alkalotic and control dogs to levels seen in chronically acidotic dogs receiving no exogenous glutamine, approximately 4 to 6 times more glutamate was released from the kidneys. Infusing alanine into 7 chronically acidotic dogs enhanced ammoniagenesis significantly (p less than 0.01), but lesser augmentation was seen in 3 control dogs and no augmentation was seen in 6 acutely alkalotic dogs. The increases were secondary to enhanced glutamate deamination, not secondary to any changes in glutamine extraction and/or transaminase activity. We conclude that the glutamate dehydrogenase pathway is more active in intact acidotic dogs than it is in control and alkalotic dogs.

Acidosis↗
PubMedDate not supplied

Glutamate dehydrogenase from Medicago sativa L., purification and comparative kinetic studies of the organ-specific multiple forms.

NAD-specific glutamate dehydrogenase [L-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2] from Medicago sativa constitutes organ-specific patterns of isoenzymes. The isoenzyme-patterns of seeds (GDH-I) and roots (GDH-II) were purified 1520-fold and 92-fold, respectively. All isoenzymes of both patterns remain stable throughout the purification procedures. Isoenzyme a7, the only isoenzyme common to both patterns was isolated from the GDH-I pattern. The three enzyme preparations were found to be identical in pH optima, substrate specificity and general kinetic properties. A comparative kinetic analysis revealed no pronounced differences between the various kinetic constants evaluated for the three enzyme preparations. Furthermore an identical order of substrate binding and product release could be established. Both initial rate measurements and product inhibition studies are consistent with an ordered ternary-binary kinetic mechanism. The results suggest that tissue-specific enzyme multiplicity of plant glutamate dehydrogenase is not related to differences in general or kinetic properties.

Glutamate Dehydrogenase↗
PubMed2000

Novel missense mutations in the glutamate dehydrogenase gene in the congenital hyperinsulinism-hyperammonemia syndrome.

OBJECTIVES: The objectives of this study were to clarify the involvement of the glutamate dehydrogenase gene in congenital hyperinsulinemia-hyperammonemia syndrome (CHHS) and the relationships between the mutation of the gene and clinical severity. STUDY DESIGN: Five unrelated Japanese patients (3 girls and 2 boys) with CHHS were investigated. All patients had convulsions or loss of consciousness resulting from hypoglycemia at less than 1 year of age. We examined mutations of the glutamate dehydrogenase gene using genomic or reverse-transcriptase polymerase chain reactions, followed by direct sequencing. RESULTS: We identified heterozygous missense mutations in all patients. Three patients had a previously identified mutation (C-->T at nt 1506) at codon 445 in the allosteric domain. Two novel missense mutations were identified in the other patients. These mutations included a change of A-->C at nt 1059 and a change of G-->A at nt 966, within the catalytic domain of the glutamate dehydrogenase gene. The locus of the mutations was not associated with the severity of hypoglycemia. CONCLUSIONS: Our results suggest that structural aberrations of not only the allosteric domain but also the catalytic domain of the glutamate dehydrogenase protein, caused by missense mutations, can result in the development of CHHS.

Adenine↗
PubMed1991

An NAD-specific glutamate dehydrogenase from cyanobacteria. Identification and properties.

The unicellular cyanobacterium Synechocystis sp. PCC 6803 presents a hexameric NAD-specific glutamate dehydrogenase with a molecular mass of 295 kDa. The enzyme differs from the NADP-glutamate dehydrogenase found in the same strain and is coded by a different gene. NAD-glutamate dehydrogenase shows a high coenzyme specificity, catalyzes preferentially glutamate formation and presents Km values for ammonium, NADH and 2-oxoglutarate of 4.5 mM, 50 microM and 1.8 mM respectively. An animating role for the enzyme is discussed.

Ammonia↗
PubMed1981

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean.

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD(+) and NADP(+). The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH(4)Cl and 1.5 at 12.5 millimolar NH(4)Cl. K(m) values for NH(4) (+) and NAD(P)H are reduced at low salt concentrations. The low K(m) values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.

Journal Article↗
PubMed1985

Serum glutamate dehydrogenase and ornithine carbamyl transferase in Reye's syndrome.

Parallel increases in the ornithine carbamyl transferase and glutamate dehydrogenase activities were observed in the serum of 22 Reye's syndrome patients. The increase in the activity of glutamate dehydrogenase was masked by a dialyzable inhibitor. It is proposed that the measurement of serum ornithine carbamyl transferase and glutamate dehydrogenase activities may be useful as an indicator of liver mitochondrial damage in Reye's syndrome.

Child↗