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PubMed1983

Radiation inactivation of glutamate dehydrogenase hexamer: lack of energy transfer between subunits.

The effects of ionizing radiation on glutamate dehydrogenase and on fluorescein isothiocyanate--tagged glutamate dehydrogenase were analyzed by target theory. Enzymatic activity, fluorescence, and the survival of the 56,000-dalton monomer subunit were determined on frozen samples irradiated at -135 degrees C and on lyophilized samples irradiated at either -135 degrees C or +30 degrees C. The effects of temperature were the same for all three parameters. Enzymatic activity was lost after small doses of high-energy electrons, whereas fluorescence and monomer subunits survived much larger doses of radiation. Target analysis revealed that the functional unit size for enzymatic activity was the hexamer, confirming both the earlier radiation study and conventional biochemical analyses. Target sizes obtained from fluorescence and subunit structure measurements were close to that of the monomer. These results indicate that the primary ionization caused by electron bombardment results in damage to a single polypeptide strand and that there is no massive transfer of radiation energy to other units in the hexamer. The large target size observed for enzymatic activity appears to be a structural requirement for the simultaneous presence of six intact subunits rather than the result of the spread of energy from the initial site to adjacent chains with consequent damage to other subunits.

Animals↗

PubMed1979

Kinetic studies of dogfish liver glutamate dehydrogenase.

Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.

Ammonia↗

PubMed1995

A NADP-glutamate dehydrogenase mutant of the petit-negative yeast Kluyveromyces lactis uses the glutamine synthetase-glutamate synthase pathway for glutamate biosynthesis.

The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.

Amino Acid Sequence↗

PubMed1999

Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface.

The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms.

Amino Acid Substitution↗

PubMed1987

Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-glia interactions in glutamate transmission.

The principally mitochondrial enzyme glutamate dehydrogenase (GDH) exhibited low-intensity, uniform immunoreactivity in neurons and intense heterogeneous labeling of glial cells of rat brain. Simultaneous peroxidase labeling for GDH and immunoautoradiography for glial fibrillary acidic protein (GFAP) confirmed the astrocytic localization of the enzyme. Immunoreactivity in astrocytes, but not in neurons, required the presence of Triton X-100 as a solubilizing agent. Most of the intensely labeled glial processes were localized to regions previously reported as containing moderate to high densities of binding sites for the excitatory amino acids, L-glutamate or L-aspartate, and glutamatergic fibers. These included several forebrain regions, such as the superficial layers of the rostral neocortex, dorsal neostriatum, nucleus accumbens, septohippocampal nucleus, intralaminar thalamic nuclei, and external capsules. However, the central gray of the midbrain, the nuclei of the reticular formation, brain stem regions projecting to the cerebellum, and cranial nuclei of the trigeminal and vagal nerves also exhibited intense glial labeling for GDH, even though some of these regions are known to receive only weak glutamatergic projections. A second factor determining the distribution of GDH appeared to be neuronal activity, as assessed by correspondence with reported high densities of cytochrome oxidase. We conclude that GDH enriched in glial populations exists in a subcellular compartment distinct from that of neurons and may serve as one of the enzymes involved in glutamatergic transmission. Deficiencies of glial GDH and the consequent cytotoxic effects of high levels of excitatory amino acids may contribute to a number of neurodegenerative disorders.

Animals↗

PubMed1984

The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate.

In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.

Adenosine Diphosphate↗

PubMed1996

Glutamate dehydrogenase covalently binds to a reactive metabolite of acetaminophen.

The mechanism of the hepatotoxicity of the analgesic acetaminophen is believed to be mediated by covalent binding to protein; however, critical targets which effect the toxicity are unknown. It has been shown that mitochondrial respiration in vivo is inhibited in mice as early as 1 h following a hepatotoxic dose of acetaminophen, and it is postulated that covalent binding to critical mitochondrial proteins may be important. A time course of mitochondrial proteins stained with anti-acetaminophen in an immunoblot detected two major adducts of 50 and 67 kDa as early as 30 min after a hepatotoxic dose of acetaminophen in mice. To further understand the role of covalent binding to mitochondrial proteins and acetaminophen hepatotoxicity, we have purified and identified a 50 kDa mitochondrial protein which becomes covalently bound to a reactive metabolite of acetaminophen. An N-terminal sequence of the 50 kDa adduct was 100% homologous with the deduced amino acid sequence of glutamate dehydrogenase. In addition, the purified protein was immunochemically reactive with rat liver anti-glutamate dehydrogenase. Enzyme activity of glutamate dehydrogenase was significantly decreased in mice 1 h following hepatotoxic treatment with acetaminophen. These data suggest that acetaminophen hepatotoxicity may in part be mediated by covalent binding to glutamate dehydrogenase.

Acetaminophen↗

PubMed1981

Regulation of glutamate dehydrogenase in Bacillus subtilis.

The activity of the nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Bacillus subtilis was influenced by the carbon source, but not the nitrogen source, in the growth medium. The highest specific activity for this enzyme was found when B. subtilis was grown in a minimal or rich medium that contained glutamate as the carbon source. It is proposed that glutamate dehydrogenase serves a catabolic function in the metabolism of glutamate, is induced by glutamate, and is subject to catabolite repression.

Alanine Dehydrogenase↗

PubMed1991

Identification of the latex test-reactive protein of Clostridium difficile as glutamate dehydrogenase.

Computer analysis showed that the gene encoding the latex test-reactive protein of Clostridium difficile exhibited high levels of homology with glutamate dehydrogenases from various sources. Further analysis demonstrated that the recombinant protein possessed glutamate dehydrogenase activity. Our results show that the protein that reacts in commercial latex tests for C. difficile is a glutamate dehydrogenase.

Amino Acid Sequence↗

PubMed1990

Affinity labeling of bovine liver glutamate dehydrogenase with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate.

Bovine liver glutamate dehydrogenase reacts with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TA-5'-DP) and 5'-triphosphate (8-BDB-TA-5'-TP) to yield enzyme with about 1 mol of reagent incorporated/mol of enzyme subunit. The modified enzyme is catalytically active but has decreased sensitivity to inhibition by GTP, reduced extent of activation by ADP, and diminished inhibition by high concentrations of NADH. Since modified enzyme, like native glutamate dehydrogenase, reversibly binds more than 1 mol each of ADP and GTP, it is unlikely that 8-BDB-TA-5'-TP reacts directly within either the ADP or GTP regulatory sites. The rate constant for reaction of enzyme exhibits a nonlinear dependence on reagent concentration with KD = 89 microM for 8-BDB-TA-5'-TP and 240 microM for 8-BDB-TA-5'-DP. The ligands ADP and GTP alone and NADH alone produce only small decreases in the rate constant for the reaction of enzyme with 8-BDB-TA-5'-TP, but the combined addition of 5 mM NADH + 200 microM GTP reduces the reaction rate constant more than 10-fold and the reagent incorporation to about 0.1 mol/mol of enzyme subunit. These results suggest that 8-BDB-TA-5'-TP reacts as a nucleotide affinity label in the region of the GTP-dependent NADH regulatory site of bovine liver glutamate dehydrogenase.

Adenosine Diphosphate↗

PubMed2003

Characterization of the glutamate dehydrogenase gene and its regulation in a euryhaline copepod.

Glutamate dehydrogenase (GDH) plays a key role in the metabolism of free amino acids (FAA) in crustaceans and other metazoans. Glutamate synthesized by GDH via reductive amination is the amino group donor for alanine synthesis and the precursor required for proline synthesis. Since both proline and alanine are important intracellular osmolytes in many marine invertebrates, GDH has been widely implicated as playing a central role in response to hyperosmotic stress in these organisms. We have isolated the gene encoding a GDH homolog from the euryhaline copepod Tigriopus californicus and examined the regulation of GDH under salinity stress. The gene encodes a protein of 557 residues with 76% amino acid identity with Drosophila melanogaster GDH. The gene encodes an N-terminal mitochondrial signal sequence peptide. Only a single intron of 71 bp was found in the GDH gene in T. californicus when genomic sequences and cDNA sequences were compared. The levels of GDH mRNA do not increase during hyperosmotic stress in this copepod. The effects of salt and hyperosmotic stress on GDH enzyme activity were also investigated. GDH activities decrease with increasing NaCl concentrations in in vitro enzyme assays, while live animals exposed to hyperosmotic stress showed no change in GDH enzyme activities. Combined, these results indicate that GDH transcription and enzyme activity do not appear to function in the regulation of alanine and proline accumulation during hyperosmotic stress in T. californicus. The manner in which this important physiological process is regulated remains unknown.

Amino Acid Sequence↗

PubMed1989

Kinetics of serum glutamate dehydrogenase during short-term alcohol withdrawal.

The behavior of serum glutamate dehydrogenase after alcohol withdrawal was studied in 64 alcoholics admitted to a detoxification center. The enzyme activity decreased in 57 patients by a median of -35.8 percent in 24 h. Thus, the decrease of serum glutamate dehydrogenase after 24 h of ethanol abstinence can serve as a test of alcohol abuse.

Adult↗

PubMed1993

Purification and properties of extremely thermostable glutamate dehydrogenases from two hyperthermophilic archaebacteria, Pyrococcus woesei and Pyrococcus furiosus.

Glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the hyperthermophilic archaebacteria Pyrococcus woesei and P. furiosus were purified to homogeneity from crude extracts. The enzymes had similar enzymological properties: molecular mass, subunit structure, optimum pHs for the oxidative deamination and reductive amination, substrate specificity and coenzyme specificity as well as thermostability; the activity was not lost after incubation at 105 degrees C for 30 min. However, some differences were detected in resistance to urea denaturation and effects of salts on their activity and stability. The N-terminal 20 amino acid sequences of the two enzymes were identical.

Amino Acid Sequence↗

PubMed1980

Dual nucleotide specificity of bovine glutamate dehydrogenase. The role of negative co-operativity.

The thionicotinamide analogues of NAD+ and NADP+ were shown to be good alternative coenzymes for bovine glutamate dehydrogenase, with similar affinity and approx. 40% of the maximum velocity obtained with the natural coenzymes. Both thionicotinamide analogues show non-linear Lineweaver-Burk plots, which with the natural coenzymes have been attributed to negative co-operativity. Since the reduced thionicotinamide analogues have an isosbestic point at 340nm and have an absorption maximum at 400nm, it is possible to monitor reduction of natural coenzyme and thionicotinamide analogue simultaneously by dual-wavelength spectroscopy. When glutamate dehydrogenase is presented with NADP+ and thio-NADP+ simultaneously, the enzyme oligomer senses saturation of its coenzyme-binding sites irrespective of the exact nature of the coenzyme and locks the oligomer into its highly saturated form even when low saturation of the monitored coenzyme is present. These experiments substantiate the suggestion that glutamate dehydrogenase shows negative co-operativity in its catalytically active form.

Animals↗

PubMed1987

The crystal structure of glutamate dehydrogenase from Clostridium symbiosum at 0.6 nm resolution.

The structure of glutamate dehydrogenase from Clostridium symbiosum has been solved by single-crystal X-ray-diffraction studies at 0.6 nm resolution by using a combination of isomorphous replacement and molecular averaging. The electron-density map reveals that this glutamate dehydrogenase is a hexameric oligomer, arranged in 32 symmetry, of cylindrical appearance and dimensions, of length 10.8 nm and radius 4.4 nm. From an analysis of this map each subunit appears to contain some 55% alpha-helix and is organized into two distinct globular domains separated by a deep cleft. The subunits associate using the domain closest to the 32-symmetry point, making intimate contacts around the threefold and twofold interfaces. The second domain shows structural homology to the NAD-binding domain of other dehydrogenases, and difference Fourier analysis has shown that the NAD is bound in both a structurally equivalent position and a similar conformation to that observed for those related enzymes.

Animals↗

PubMed1982

Activity of adenylate deaminase and glutamate dehydrogenase in the liver: species and dietary variation.

1. Activity of glutamate dehydrogenase and adenylate deaminase were measured in the livers of carnivores (animals characterised by intake of a high dietary protein). 2. Animals studied (ferret, cat, dog, hedgehog, rat, hamster, mouse, cow, pig and rabbit) were kept on their natural diet. 3. Glutamate dehydrogenase activity showed no variation between carnivores and non-carnivores. 4. Adenylate deaminase activity was significantly higher in carnivores than in non-carnivores. 5. In carnivores, adenylate deaminase might be the rate limiting enzyme in the terminal deamination of L-amino acids. 6. Elevation of adenylate deaminase might be due to the acidogenic effect of the diet.

AMP Deaminase↗

PubMed1975

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.

Adenosine↗

PubMed1993

Age-dependent changes in some aspects of glutamate metabolism in the brain of the teleost, Channa punctatus. I. Ammonia and glutamine contents and glutamate dehydrogenase activity.

Age related changes in some aspects of glutamate metabolism were followed in the brain of a short lived species of tropical murrel, Channa punctatus. Both ammonia and glutamine contents of brain increased with advancing age of the murrel. The glutamate dehydrogenase activity of brain increased during maturation phase (year classes 0(+)-3+) followed by a decline in senescence phase (year classes 3(+)-6+). The pattern of age changes in glutamate metabolism in this species of fish shows similarity with the observations made in a majority of mammalian species, thereby suggesting a commonality in a basic metabolic mechanism during aging process in the central nervous system of vertebrates.

Aging↗