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

---

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

Neurotransmitters are essential for communication between neurons and hence are vital in the overall integrative functioning of the nervous system. Previous work on acetylcholine metabolism in the fruit fly, Drosophila melanogaster, has also raised the possibility that transmitter metabolism may play a prominent role in either the achievement or maintenance of the normal structure of the central nervous system in this species. Unfortunately, acetylcholine is rather poorly characterized as a neurotransmitter in Drosophila; consequently, we have begun an analysis of the role of glutamate (probably the best characterized transmitter in this organism) in the formation and/or maintenance of nervous system structure. We present here the results of a series of preliminary analyses. To suggest where glutamatergic function may be localized, an examination of the spatial distribution of high affinity [3H]-glutamate binding sites are presented. We present the results of an analysis of the spatial and temporal distribution of enzymatic activities thought to be important in the regulation of transmitter-glutamate pools (i.e., glutamate oxaloacetic transaminase, glutaminase, and glutamate dehydrogenase). To begin to examine whether mutations in any of these functions are capable of affecting glutamatergic activity, we present the results of an initial genetic analysis of one enzymatic function, glutamate oxaloacetic transaminase (GOT), chosen because of its differential distribution within the adult central nervous system and musculature.
...
PMID:A genetic analysis of glutamatergic function in Drosophila. 310 67

Antibodies against the purified bovine brain glutamate binding protein (GBP) were raised in rabbits. Both nonderivatized and dinitrobenzene-derivatized GBP produced strong immunological responses in rabbits. Using the enzyme-linked immunosorbent assay (ELISA), we have quantified the antibody production and determined the specificity of the antibodies. Bovine brain GBP and the analogous protein from rat brain interacted most strongly with the antibodies. A bacterial glutamate-aspartate binding protein, as well as the enzymes glutamate dehydrogenase (EC 1.4.1.3), glutamine synthetase (EC 6.3.1.2), and gamma-glutamyl transpeptidase (EC 2.3.2.2), showed little or no cross-reactivity with the anti-GBP antibodies. A crude bacterial glutamate decarboxylase (EC 4.1.1.15) preparation gave a small to moderate cross-reaction with the anti-GBP antibodies. The sensitivity of the ELISA assay and the specificity of the antibodies were such that GBP levels as low as 3-10 ng could be detected.
...
PMID:Antibodies against the bovine brain glutamate binding protein. 631 9

Glutamate dehydrogenases from many sources display nonclassical kinetic behavior suggestive of allosteric interaction among the six subunits of the hexamer. A three-dimensional structure now potentially offers a framework for explaining the basis of such behavior in clostridial glutamate dehydrogenase, and this paper offers evidence of extreme, all-or-none cooperativity in the binding of glutamate by this enzyme. A site-directed mutant of clostridial glutamate dehydrogenase in which Ala163 in the glutamate binding site is replaced by glycine displays a markedly sigmoid dependence of reaction rate on glutamate concentration (S0.5 = 200 mM), with a Hill coefficient of 3.4 when assayed at pH 10.5 with 1 mM NAD+. Under the same conditions the wild-type enzyme gave no measurable rate with glutamate concentrations in the range normally used for kinetics (0-100 mM) but gave a steep rise in reaction rate from 600 to 1200 mM glutamate. At pH 9.0, where the wild-type enzyme has previously been shown to be "inactive" in a standard assay, a study extending to much higher glutamate concentrations again revealed a sigmoid dependence, with a Hill coefficient of 5.4 and an S0.5 at 150 mM glutamate. With the mutant A163G the apparent cooperativity was less, with a Hill coefficient of 2.3, and the affinity for glutamate was higher, with S0.5 of 7 mM. Both proteins gave normal hyperbolic dependence on glutamate concentration at pH 7 and pH 8. At pH 9 and with saturating glutamate, both enzymes showed a hyperbolic dependence of the rate on NAD+ concentration. The NAD+ concentration, however, affected the observed degree of cooperativity with varied glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Positive cooperativity with Hill coefficients of up to 6 in the glutamate concentration dependence of steady-state reaction rates measured with clostridial glutamate dehydrogenase and the mutant A163G at high pH. 754 69

We have solved the structure of the binary complex of the glutamate dehydrogenase from Clostridium symbiosum with glutamate to 1.9 A resolution. In this complex, the glutamate side-chain lies in a pocket on the enzyme surface and a key determinant of the enzymic specificity is an interaction of the substrate gamma-carboxyl group with the amino group of Lys89. In the apo-enzyme, Lys113 from the catalytic domain forms an important hydrogen bond to Asn373, in the NAD(+)-binding domain. On glutamate binding, the side-chain of this lysine undergoes a significant movement in order to optimize its hydrogen bonding to the alpha-carboxyl group of the substrate. Despite this shift, the interaction between Lys113 and Asn373 is maintained by a large-scale conformational change that closes the cleft between the two domains. Modelling studies indicate that in this "closed" conformation the C-4 of the nicotinamide ring and the alpha-carbon atom of the amino acid substrate are poised for efficient hydride transfer. Examination of the structure has led to a proposal for the catalytic activity of the enzyme, which involves Asp165 as a general base, and an enzyme-bound water molecule, hydrogen-bonded to an uncharged lysine residue, Lys125, as an attacking nucleophile in the reaction.
...
PMID:Conformational flexibility in glutamate dehydrogenase. Role of water in substrate recognition and catalysis. 826 17

The glutamate dehydrogenase gene from the hyperthermophilic archaeon Pyrococcus furiosus has been functionally expressed in Escherichia coli under the control of the lambda PL promoter. The P. furiosus glutamate dehydrogenase amounted to 20% of the total E. coli cell protein, and the vast majority consisted of hexamers. Following activation by heat treatment, an enzyme could be purified from E. coli that was indistinguishable from the glutamate dehydrogenase purified from P. furiosus. Hybrid genes, that consisted of the coding regions for the homologous glutamate dehydrogenases from P. furiosus and the mesophilic bacterium Clostridium difficile, were constructed and successfully expressed in E. coli. One of the resulting hybrid proteins, containing the glutamate binding domain of the C. difficile enzyme and the cofactor binding domain of the P. furiosus enzyme, did not show a detectable activity. In contrast, the complementary hybrid containing the P. furiosus glutamate and the C. difficile cofactor binding domain was a catalytically active hexamer that showed a reduced substrate affinity but maintained efficient cofactor binding with the specificity found in the Clostridium symbiosum enzyme. Compared with the C. difficile glutamate dehydrogenase, the archaeal-bacterial hybrid is slightly more thermoactive, less thermostable but much more stable towards guanidinium chloride-induced inactivation and denaturation.
...
PMID:Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability. 886 41

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

The NAD-dependent glutamate dehydrogenase (GluDH) gene from the hyperthermophilic archaeon Pyrobaculum islandicum was cloned and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1266 bp encoding a protein of 421 amino acids with a molecular weight of 46,905. In the alignment of the amino acid sequence with those of mesophilic Clostridium symbiosum NAD-dependent GluDH and hyperthermophilic NADP-dependent enzymes from Thermococcus profundus and Pyrococcus furiosus, substitutions in the residues involved in dinucleotide binding were observed. On the other hand, the residues involved in glutamate binding were well conserved. This is the first description of the primary structure of NAD-dependent GluDH in hyperthermophilic archaea.
...
PMID:The NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum: cloning, sequencing, and expression of the enzyme gene(1). 1052 54

The guiding principle of the IAS Medal Lecture and of the research it covered was that searching mathematical analysis, depending on good measurements, must underpin sound biochemical conclusions. This was illustrated through various experiences with the amino acid dehydrogenases. Topics covered in the present article include: (i) the place of kinetic measurement in assessing the metabolic role of GDH (glutamate dehydrogenase); (ii) the discovery of complex regulatory behaviour in mammalian GDH, involving negative co-operativity in coenzyme binding; (iii) an X-ray structure solution for a bacterial GDH providing insight into catalysis; (iv) almost total positive co-operativity in glutamate binding to clostridial GDH; (v) unexpected outcomes with mutations at the catalytic aspartate site in GDH; (vi) reactive cysteine as a counting tool in the construction of hybrid oligomers to probe the basis of allosteric interaction; (vii) tryptophan-to-phenylalanine mutations in analysis of allosteric conformational change; (viii) site-directed mutagenesis to alter substrate specificity in GDH and PheDH (phenylalanine dehydrogenase); and (ix) varying strengths of binding of the 'wrong' enantiomer in engineered mutant enzymes and implications for resolution of racemates.
...
PMID:Making biochemistry count: life among the amino acid dehydrogenases. 2142 13

Eight weeks of latent iron deficiency in weaned female rats of Sprague Dawley strain maintained on experimental low-iron diet (18-20 mg/Kg) did not significantly change the gross body weight and tissue weights of brain and liver. Packed cell volume (PCV) and hemoglobin concentration remained unaltered. However, non-heme iron content in liver and brain decreased significantly (P<0.001). The activities of glutamate dehydrogenase, glutamic acid decarboxylase, and GABA-transaminase (GABA-T) in brain decreased by 15%, 11.4% and 25.7% respectively. However, this decrease was not statistically significant. Binding of(3)H Muscimol at pH 7.5 and 1 mg protein/assay increased by 143% (P<0.001) in synaptic vesicular membranes from iron-deficient rats as compared to the controls.(3)H glutamate binding to the synaptic vesicles was also carried out under similar condition. However, the L-glutamate binding was reduced by 63% in the vesicular membranes of iron deficient animals. These studies in dicate that iron plays an important functional role in both excitatory and inhibitory neurotransmitter receptors.
...
PMID:Effect of latent iron deficiency on gaba and glutamate neuroreceptors in rat brain. 2310 45

Eight weeks of latent iron deficiency in weaned female rats of Sprague Dawley strain maintained on experimental low-iron diet (18-20 mg/kg) did not significantly change the gross body, weight and tissue weights of brain and liver. Packed cell volume (PCV) and hemoglobin concentration remained unaltered. However, non-heme iron content in liver and brain decreased significantly (p<0.001). The activities of glutamate dehydrogenase, glutamic acid decarboxylase, and GABA-transaminase (GABA-T) in brain decreased by 15%, 11.4% and 25.7% respectively. However, this decrease was not statistically significant. Binding of(3)H Muscimol at pH 7.5 and 1 mg protein/assay increased by 143% (p<0.001) in synaptic vesicular membranes from iron-deficient rats as compared to the controls.(3)H glutamate binding to the synaptic vesicles was also carried out under similar condition. However, the L-glutamate binding was reduced by 63% in the vesicular membranes of iron deficient animals. These studies indicate that iron plays important functional role in both excitatory and inhibitory neurotransmitter receptors.
...
PMID:Effect of latent iron deficiency on GABA and glutamate neuroreceptors in rat brain. 2310 83


1 2 Next >>

---