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)

Peanut glutamate dehydrogenase (GDH) was electrophoretically purified to homogeneity. Rotofor IEF fractionated the peanut GDH to 7 isoelectric (charge) isomers, which focused in the pH 5-8 range. Western blot analysis of the charge isomers using anti-GDH serum showed that methionine sulphoximine (MSX) treatment suppressed the b-subunit (69 KDa), but enhanced the a-subunit (45 KDa), and alpha-subunit (46 KDa) of the enzyme. The MSX-mediated suppression of the b-subunit increased the NH4+ Km value of the acidic charge isomers from 7.7 mM in the control to 50 mM in the MSX-treated peanut, and also increased the 2-ketoglutarate Km value of the basic charge isomers from 0.4 mM in the control to 7.0 mM in the MSX treatment. Therefore, the control peanut could salvage NH4+ with its GDH activity. But by increasing the NH4+ Km value, the MSX rendered the enzyme ineffective in NH4+ salvage. In the deamination direction, despite the enhancement of the a-, and alpha-subunits by MSX, the basic charge isomers of GDH had very high Km value for L-glu (50 mM), similar to that (25 mM) of the control. Thus, the GDHs of the control, and MSX treatment could not function in the deamination reaction in vivo. These results show that the treatment of peanut with MSX impaired the amination function of GDH.
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PMID:Regulation of peanut glutamate dehydrogenase by methionine sulphoximine. 937 17

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.
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PMID:Intersubunit communication in hybrid hexamers of K89L/A163G/S380A and C320S mutants of glutamate dehydrogenase from Clostridium symbiosum. 939 25

The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47,300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100 degrees C, compared to 4 h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.
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PMID:Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs. 952 Feb 68

Glutamine synthesis, the major pathway of ammonia detoxification, and the intracellular concentration of organic osmolytes in primary astrocytes and F98 glioma cells were investigated with multinuclear magnetic resonance spectroscopy. Acute exposure to ammonia (3 h incubation with NH4Cl) raised the concentration of glutamine and other amino acids, such as glutamate and aspartate, and decreased myo-inositol, hypotaurine, and taurine concentrations. The loss of these osmolytes was partially reversed by co-treatment with the glutamine synthetase inhibitor, methionine sulphoximine. Glutamate, the precursor of glutamine, is provided by stimulated anaplerotic flux via pyruvate carboxylase and glutamate dehydrogenase activity. Thus, the glutamine increase and myo-inositol decrease observed by in vivo magnetic resonance spectroscopy on patients with hepatic encephalopathy may be due to the disturbed osmoregulation in astrocytes caused by accumulation of glutamine and the subsequent loss of organic osmolytes.
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PMID:Multinuclear NMR spectroscopy studies on NH4Cl-induced metabolic alterations and detoxification processes in primary astrocytes and glioma cells. 977 80

Comparisons of the structures of glutamate dehydrogenase (GluDH) and leucine dehydrogenase (LeuDH) have suggested that two substitutions, deep within the amino acid binding pockets of these homologous enzymes, from hydrophilic residues to hydrophobic ones are critical components of their differential substrate specificity. When one of these residues, K89, which hydrogen-bonds to the gamma-carboxyl group of the substrate l-glutamate in GluDH, was altered by site-directed mutagenesis to a leucine residue, the mutant enzyme showed increased substrate activity for methionine and norleucine but negligible activity with either glutamate or leucine. In order to understand the molecular basis of this shift in specificity we have determined the crystal structure of the K89L mutant of GluDH from Clostridium symbiosum. Analysis of the structure suggests that further subtle differences in the binding pocket prevent the mutant from using a branched hydrophobic substrate but permit the straight-chain amino acids to be used as substrates. The three-dimensional crystal structure of the GluDH from C. symbiosum has been previously determined in two distinct forms in the presence and absence of its substrate glutamate. A comparison of these two structures has revealed that the enzyme can adopt different conformations by flexing about the cleft between its two domains, providing a motion which is critical for orienting the partners involved in the hydride transfer reaction. It has previously been proposed that this conformational change is triggered by substrate binding. However, analysis of the K89L mutant shows that it adopts an almost identical conformation with that of the wild-type enzyme in the presence of substrate. Comparison of the mutant structure with both the wild-type open and closed forms has enabled us to separate conformational changes associated with substrate binding and domain motion and suggests that the domain closure may well be a property of the wild-type enzyme even in the absence of substrate.
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PMID:Insights into the mechanism of domain closure and substrate specificity of glutamate dehydrogenase from Clostridium symbiosum. 987 50

It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5'-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.
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PMID:Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase. An essential residue in catalysis. 1138 22

In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards L-methionine, L-norleucine and L-norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used L-aspartate and L-leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher K(m ) values for glutamate/2-oxoglutarate at pH 7.0, but a similar k(cat)/K(m) value and lower K(m) at pH 8.0, and a nearly 22-fold lower S(0.5) (substrate concentration giving half-saturation under conditions where Michaelis-Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100-1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially L-norleucine and L-methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate k(cat) and K(m) separately, but for reductive amination the additional mutations have no significant effect on k(cat) at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in K(m). At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine approximately 2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants.
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PMID:Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site-directed mutagenesis. 1172 65

In patients with severe alcoholic liver disease (i.e., cirrhosis), a deficiency of S-adenosylmethionine (SAMe) develops as a result of decreased SAMe synthetase activity. Whether a sizeable SAMe depletion occurs already at earlier stages of alcoholic liver disease has been the subject of debate. To address this issue, rats were fed alcohol (or isocaloric carbohydrate) in Lieber-DeCarli liquid diets containing adequate amounts of protein, vitamins, and lipotropic factors, including methionine. Alcohol feeding resulted in hepatic steatosis (without fibrosis) and unchanged SAMe synthetase activity, yet SAMe concentration was already greatly decreased. This most likely resulted from oxidative stress associated with the metabolism of alcohol and the induction of cytochrome P4502E1 (CYP2E1), which generates free radicals. Indeed, the decrease in hepatic SAMe correlated with parameters of oxidative stress, such as increased 4-hydroxynonenal (measured by gas chromatography-mass spectrometry) and diminished glutathione (GSH). Decreased GSH, occurring as a result of excessive GSH consumption caused by the oxidative stress, probably generated by enhanced utilization of SAMe, a precursor of GSH, thereby explaining the depletion of SAMe. In view of the known differences between rodents and primates in the metabolism of lipotropes, my colleagues and I have also studied the interaction between alcohol and SAMe in baboons and found again that, at early stages preceding the development of cirrhosis, there was already a significant lowering of hepatic SAMe concentration, associated with a striking oxidative stress documented by decreased levels and accelerated turnover of GSH. This was associated with increased lipid peroxidation and damage to cellular membranes, including those of the mitochondria, assessed by electron microscopy. Oral administration of SAMe resulted in its hepatic repletion with a corresponding attenuation of the ethanol-induced oxidative stress and liver injury, with significantly less GSH depletion, less increases in plasma aspartate aminotransferase (AST) levels, less leakage of mitochondrial glutamic dehydrogenase into the plasma, and fewer megamitochondria. In conclusion, (1) both in rodents and in non-human primates, significant SAMe depletion occurs already at early stages of alcoholic liver disease, despite the consumption of adequate diets; (2) the decreased hepatic SAMe concentration and the associated liver lesions, including mitochondrial injury, can be corrected with SAMe supplementation; and (3) accordingly, therapeutic administration of SAMe should be the subject of a comprehensive clinical trial to assess its capacity to attenuate early stages of alcoholic liver injury in human beings.
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PMID:S-Adenosyl-L-methionine and alcoholic liver disease in animal models: implications for early intervention in human beings. 1216 46

Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and cassette mutagenesis. Photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD(+) binding site within human GDH encoded by the synthetic human GDH gene and expressed in Escherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+) binding site. Amino acid sequencing and compositional analysis identified Glu(279) as the site of photoinsertion into human GDH, suggesting that Glu(279) is located at or near the NAD(+) binding site. The importance of the Glu(279) residue in the binding of NAD(+) was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The mutagenesis at Glu(279) has no effects on the expression or stability of the different mutants. The K(m) values for NAD(+) were 10-14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V(max) values were similar for wild-type and mutant GDHs. The efficiency (k(cat)/K(m)) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K(m) values for NAD(+). In contrast to the K(m) values for NAD(+), wild-type and mutant GDHs show similar K(m) values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu(279) is not directly involved in allosteric regulation. The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu(279) plays an important role for efficient binding of NAD(+) to human GDH.
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PMID:Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase. 1219 7

To investigate nitrogen assimilation and translocation in Zea mays L. colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thax. sensu Gerd.), we measured key enzyme activities, 15N incorporation into free amino acids, and 15N translocation from roots to shoots. Glutamine synthetase and nitrate reductase activities were increased in both roots and shoots compared with control plants, and glutamate dehydrogenase activity increased in roots only. In the presence of [15N]ammonium, glutamine amide was the most heavily labeled product. More label was incorporated into amino acids in VAM plants. The kinetics of 15N labeling and effects of methionine sulfoximine on distribution of 15N-labeled products were entirely consistent with the operation of the glutamate synthase cycle. No evidence was found for ammonium assimilation via glutamate dehydrogenase. 15N translocation from roots to shoots through the xylem was higher in VAM plants compared with control plants. These results establish that, in maize, VAM fungi increase ammonium assimilation, glutamine production, and xylem nitrogen translocation. Unlike some ectomycorrhizal fungi, VAM fungi do not appear to alter the pathway of ammonium assimilation in roots of their hosts.
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PMID:Ammonia Assimilation in Zea mays L. Infected with a Vesicular-Arbuscular Mycorrhizal Fungus Glomus fasciculatum. 1223 37


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