Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.4.1.2 (glutamate dehydrogenase)
4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intersubunit ion pairs are considered to be involved for maintaining a stable structure of the glutamate dehydrogenase (GDH) from hyperthermophiles. In order to demonstrate an effect of intersubunit ion pairs on the structural stability, two kinds of mutation (T138E, Thr at position 138 was replaced by Glu; E158Q, Glu at position 158 was replaced by Gln) which add and remove ion pairs, respectively, were introduced into Pk-gdhA gene encoding GDH from Pyrococcus kodakaraensis KOD1. Addition of one ion pair (Pk-GDHA-T138E) increased the optimum temperature and thermostability. In contrast, Pk-GDH-E158Q showed lower optimum temperature and less thermostability than wild type GDH. Structure analysis of GDHs was performed by circular dichroism (CD) and indicated that all recombinant enzymes (Pk-GDH, Pk-GDH-T138E, Pk-GDH-E158Q) possess different structures from that of natural GDH. Upon heat treatment (60 degrees C, 2 h), the structures of Pk-GDH and Pk-GDH-T138E were converted to another form close to the natural structure. However, no structural conversion by heat treatment was observed in Pk-GDH-E158Q. These results indicate that intersubunit ion pairs play an important role in forming thermostable structure of Pk-GDH.
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PMID:Ion pairs involved in maintaining a thermostable structure of glutamate dehydrogenase from a hyperthermophilic archaeon. 970 28

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism in which affected children have recurrent symptomatic hypoglycemia together with asymptomatic, persistent elevations of plasma ammonium levels. We have shown that the disorder is caused by dominant mutations of the mitochondrial enzyme, glutamate dehydrogenase (GDH), that impair sensitivity to the allosteric inhibitor, GTP. In 65 HI/HA probands screened for GDH mutations, we identified 19 (29%) who had mutations in a new domain, encoded by exons 6 and 7. Six new mutations were found: Ser(217)Cys, Arg(221)Cys, Arg(265)Thr, Tyr(266)Cys, Arg(269)Cys, and Arg(269)HIS: In all five mutations tested, lymphoblast GDH showed reduced sensitivity to allosteric inhibition by GTP (IC(50), 60--250 vs. 20--50 nmol/L in normal subjects), consistent with a gain of enzyme function. Studies of ATP allosteric effects on GDH showed a triphasic response with a decrease in high affinity inhibition of enzyme activity in HI/HA lymphoblasts. All of the residues altered by exons 6 and 7 HI/HA mutations lie in the GTP-binding domain of the enzyme. These data confirm the importance of allosteric regulation of GDH as a control site for amino acid-stimulated insulin secretion and indicate that the GTP-binding site is essential for regulation of GDH activity by both GTP and ATP.
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PMID:Hyperinsulinism/hyperammonemia syndrome in children with regulatory mutations in the inhibitory guanosine triphosphate-binding domain of glutamate dehydrogenase. 1129 18

Human glutamate dehydrogenase (GDH) exists in GLUD1 (housekeeping) and in GLUD2-specified (brain-specific) isoforms, which differ markedly in their basal activity and allosteric regulation. To determine the structural basis of these functional differences, we mutagenized the GLUD1 GDH at four residues that differ from those of the GLUD2 isoenzyme. Functional analyses revealed that substitution of Ser for Arg-443 (but not substitution of Thr for Ser-331, Leu for Met-370, or Leu for Met-415) virtually abolished basal activity and totally abrogated the activation of the enzyme by l-leucine (1-10 mm) in the absence of other effectors. However, when ADP (0.025-0.1 mm) was present in the reaction mixture, l-leucine (0.3-6.0 mm) activated the mutant enzyme up to >2,000%. The R443S mutant was much less sensitive to ADP (SC(50) = 383.9 +/- 14.6 microm) than the GLUD1 GDH (SC(50) = 31.7 +/- 4.2 microm; p < 0.001); however, at 1 mm ADP the V(max) for the mutant (136.67 micromol min(-1) mg(-1)) was comparable with that of the GLUD1 GDH (152.95 micromol min(-1) mg(-1)). Varying the composition and the pH of the reaction buffer differentially affected the mutant and the wild-type GDH. Arg-443 lies in the "antenna" structure, in a helix that undergoes major conformational changes during catalysis and is involved in intersubunit communication. Its replacement by Ser is sufficient to impair both the catalytic and the allosteric function of human GDH.
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PMID:Substitution of Ser for Arg-443 in the regulatory domain of human housekeeping (GLUD1) glutamate dehydrogenase virtually abolishes basal activity and markedly alters the activation of the enzyme by ADP and L-leucine. 1232 73

The free amino acid concentrations in cotyledons and axes of soybean (Glycine max [L.] Merr. cv. Wells) seedlings were determined by automated single column analysis after germination at 10 and 23 C. After 5 days germination at 10 C, glutamate and aspartate were in high concentration in both cotyledons and axes (38 and 24% of total free amino acids recovered, respectively), whereas the concentrations of their amide derivatives, asparagine and glutamine, were low in cotyledons (4.4%) and high in axes (21%). In contrast, after 5 days germination at 23 C, asparagine and glutamine accounted for 22 and 45% of total free amino acids in cotyledons and axes respectively, and aspartate and glutamate concentrations were low. The activities of glutamine synthetase and asparagine synthetase were considerably lower in tissues from the 10 C treatment than those from the 23 C treatment.Aspartate and glutamate concentrations were nearly equal in all but one sample. Both glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were much higher in axis tissues at 23 C as compared to 10 C. Arrhenius plots of axis glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were biphasic and triphasic, respectively, with energies of activation for both increasing with low temperature. Energies of activation were identical for glutamate oxaloacetate transaminase from 10 and 23 C treatments but much higher for glutamate dehydrogenase from 23 C-treated axes. This indicates a difference in enzyme complement for glutamate dehydrogenase with the two treatments.Hydrolysis of free amino acid sample (basic fraction) aliquots showed large quantities of peptides in 23 C-treated axes at 2 days, while few or no peptides were found in the 10 C treatment. Amino acid residues most prevalent in peptides were aspartate, threonine, serine, glutamate, and glycine.
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PMID:Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Free Amino Acid Pools during Germination. 1666 May 75

Net balances of amino acids were constructed for stages of development of a leaf of white lupin (Lupinus albus L.) using data on the N economy of the leaf, its exchanges of amino acids through xylem and phloem, and net changes in its soluble and protein-bound amino acids. Asparagine, aspartate, and gamma-aminobutyrate were delivered to the leaf in excess of amounts consumed in growth and/or phloem export. Glutamine was supplied in excess until full leaf expansion (20 days) but was later synthesized in large amounts in association with mobilization of N from the leaf. Net requirements for glutamate, threonine, serine, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were met mainly or entirely by synthesis within the leaf. Amides furnished the bulk of the N for amino acid synthesis, asparagine providing from 24 to 68%. In vitro activity of asparaginase (EC 3.5.1.1) exceeded that of asparagine:pyruvate aminotransferase (EC 2.6.1.14) during early leaf expansion, when in vivo estimates of asparagine metabolism were highest. Thereafter, aminotransferase activity greatly exceeded that of asparaginase. Rates of activity of one or both asparagine-utilizing enzymes exceeded estimated rates of asparagine catabolism throughout leaf development. In vitro activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) were consistently much higher than that of glutamate dehydrogenase (EC 1.4.1.3), and activities of the former two enzymes more than accounted for estimated rates of ammonia release in photorespiration and deamidation of asparagine.
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PMID:Amino Acid transport and metabolism in relation to the nitrogen economy of a legume leaf. 1666 17

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [(15)N]H(4) (+) prior to supplying the inhibitor. Analyses of the (15)N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [(15)N]H(4) (+), are (14)N enriched and are not apparently derived from (15)N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.
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PMID:Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine. 1666 34

Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from (15)N labeling kinetics after substituting [(15)N]ammonium and [(15)N]nitrate for the (14)N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and gamma-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and gamma-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and gamma-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low (15)N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation.
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PMID:Metabolic changes associated with adaptation of plant cells to water stress. 1666 63

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.
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PMID:Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type. 1868 14

Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit alpha-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD(+)-specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.
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PMID:Regulation of glutamate metabolism by protein kinases in mycobacteria. 1901 60

Expression of the gdh2 gene encoding the alpha-subunit of mitochondrial glutamate dehydrogenase depends on redox state of the mitochondrial electron transport chain. Treatment of Arabidopsis thaliana cell suspension with antimycin A, a respiratory chain complex III inhibitor, resulted in an increase in gdh2 transcripts within 2 h. Inhibition of complex I by rotenone did not influence the transcript level, but treatment with potassium cyanide, a complex IV inhibitor, also increased the transcript content. Thus, gdh2 gene expression obviously responds to changes in the respiratory chain segment localized between complexes I and III. Lack of activation of gene expression after treatment of a cell suspension with hydrogen peroxide and the prooxidant paraquat and results of experiments with antioxidants suggest that gdh2 gene expression is not associated with increased content of reactive oxygen species generated during inhibition of the electron transport chain. Protein phosphorylation by serine/threonine protein kinases is the essential step required for signal transduction into nucleus resulting in the induction of gdh2 expression.
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PMID:Induction of Arabidopsis gdh2 gene expression during changes in redox state of the mitochondrial respiratory chain. 1923 48


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