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PubMed1993

Purification and characterization of Clostridium difficile glutamate dehydrogenase.

Recombinant Clostridium difficile glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, EC 1.4.1.2) was purified 177-fold to electrophoretic homogeneity with a 62% recovery through a four-step procedure involving gel filtration and ion-exchange and dye affinity chromatography. The approximate molecular weights of the native enzyme by gel filtration and subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with a hexameric structure for the purified enzyme. The enzyme-catalyzed glutamate oxidation was an NAD-dependent sequential process in which NADP could not be substituted as coenzyme. Several dinucleotide analogs of NAD structurally altered in either the pyridine or the purine moiety were observed to function as coenzymes when substituted for NAD. Nicotinamide mononucleotide did not serve as a coenzyme for glutamate oxidation. Product inhibition by NADH was competitive with respect to NAD. In deadend inhibition studies, adenosine diphosphoribose was shown to be an effective coenzyme-competitive inhibitor.

Chromatography, Affinity↗

PubMed1972

Glutamate dehydrogenase from apodachlya (oomycetes).

A glutamate dehydrogenase specific for nicotinamide-adenine-dinucleotide has been purified 50-fold from Apodachlya brachynema (Leptomitales). Certain physical, chemical, and kinetic properties of this enzyme have been studied, particularly specificity for coenzymes and substrates. With glucose as the sole carbon source, the synthesis of glutamate dehydrogenase was repressed, whereas glutamate, proline, alanine, or ornithine plus aspartate as sole carbon sources induced synthesis of the enzyme. These data indicate that the function of this enzyme is primarily degradative, although there is no evidence for a nicotinamide-adenine-dinucleotide-phosphate-specific biosynthetic glutamate dehydrogenase in Apodachlya.

Journal Article↗

PubMed1997

GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae.

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP+-glutamate dehydrogenase (NADP+-GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP+-GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.

Ammonium Sulfate↗

PubMed1969

Isoenzymes of glutamate dehydrogenase in plants.

Glutamate dehydrogenase of several different plants was resolved by polyacrylamide gel electrophoresis into separate molecular forms and the isoenzymic patterns detected by the tetrazolium technique were compared. The number of isoenzymes and their electrophoretic mobilities varied among the different plants studied. The isoenzymes were found to have the same coenzyme specificity and to localize in the mitochondrial fraction of the cell in all the plants examined. Electrophoretic heterogeneity in tissue homogenates was observed in some of the plants studied. The pattern of isoenzymes of mungbean hypocotyl was followed and shown to change during germination.

Journal Article↗

PubMed1975

Formation of transient complexes in the glutamate dehydrogenase catalyzed reaction.

The reaction of glutamate dehydrogenase and glutamate (gl) with NAD+ and NADP+ has been studied with stopped-flow techniques. The enzyme was in all experiments present in excess of the coenzyme. The results indicate that the ternary complex (E-NAD(P)H-kg) is present as an intermediate in the formation of the stable complex (E-NAD(P)H-gl). The identification of the complexes is based on their absorption spectra. The binding of the coenzyme to (E-gl) is the rate-limiting step in the formation of (E-NAD(P)H-kg) while the dissociation of alpha-ketoglutarate (kg) from this complex is the rate-limiting step in the formation of (E-NAD(P)H-gl). The Km for glutamate was 20-25 mM in the first reaction and 3 mM in the formation of the stable complex. The Km values were independent of the coenzyme. The reaction rates with NAD+ were approximately 50% greater than those with NADP+. Furthermore, high glutamate concentration inhibited the formation of (E-NADH-kg) while no substrate inhibition was found with NADP+ as coenzyme. ADP enhanced while GTP reduced the rate of (E-NAD(P)H-gl) formation. The rate of formation of (E-NAD(P)H-kg) was inhibited by ADP, while it increased at high glutamate concentration when small amounts of GTP were added. The results show that the higher activity found with NAD+ compared to NADP+ under steady-state assay conditions do not necessarily involve binding of NAD+ to the ADP activating site of the enzyme. Moreover, the substrate inhibition found at high glutamate concentration under steady-state assay condition is not due to the formation of (E-NAD(P)H-gl) as this complex is formed with Km of 3 mM glutamate, and the substrate inhibition is only significant at 20-30 times this concentration.

Adenosine Diphosphate↗

PubMed1999

Reactive cysteine residue of bovine brain glutamate dehydrogenase isoproteins.

Protein chemical studies of glutamate dehydrogenase isoproteins (GDH I and GDH II) from bovine brain reveal that one cystein residue is accessible for reaction with thiol-modifying reagent. Reaction of the two types of GDH isoproteins with p-chloromercuribenzoic acid resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order kinetics with the second-order rate constant of 83 M(-1) s(-1) and 75 M(-1) s(-1) for GDH I and GDH II, respectively. The inactivation was partially prevented by preincubation of the glutamate dehydrogenase isoproteins with NADH. A combination of 10 mM 2-oxoglutarate with 2 mM NADH gave complete protection against the inactivation. There were no significant differences between the two glutamate dehydrogenase isoproteins in their sensitivities to inactivation by p-chloromercuribenzoic indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Allosteric effectors such as ADP and GTP had no effects on the inactivation of glutamate dehydrogenase isoproteins by thiol-modifying reagents. By a combination of peptide mapping analysis and labeling with [14C] p-chloromercuribenzoic acid, a reactive cystein residue was identified as Cys323 in the overall sequence. The cysteine residue was clearly identical to sequences of other GDH species known.

Amino Acid Sequence↗

PubMed1985

Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes.

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.

Binding Sites↗

PubMed1978

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate: production of an ammonia-containing intermediate in the "burst" phase.

We have studied the effects of ammonium acetate on the transient "burst" phase of the oxidation of L-glutamate by glutamate dehydrogenase. Two measurable changes are observed in the "burst" phase as ammonium acetate concentration is increased: (i) an increase in the apparent first-order rate constant, kapp, and (ii) a decrease in the amplitude of the absorbance change measured at 320 nm. The increase in kapp shows a hyperbolic dependence on ammonium acetate concentration and is independent of glutamate concentration. The results demonstrate the existence of an intermediate immediately following hydrogen transfer. The intermediate contains enzyme, reduced coenzyme, ammonia, and alpha-ketoglutarate moieties and is in equilibrium with the known complex consisting of enzyme, reduced coenzyme, and alpha-ketoglutarate. At high concentrations of ammonium acetate, the equilibrium favors the ammonia containing complex.

Ammonia↗

PubMed1975

Subcellular localization of gamma-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain. Evidence for at least two small glutamate compartments in brain.

The subcellular localizations of gamma-aminobutyrate transaminase (EC 2.6.1.19) and glutamate dehydrogenase (EC 1.4.1.2) in brain tissue of adult rats were compared with each other and with those of NAD+-isocitrate dehydrogenase (EC 1.1.41) and monoamine oxidase (EC 1.4.3.4; kynuramine as substrate). Crude mitochondrial fractions from brain tissue were centrifuged in continuous sucrose density gradients. gamma-Aminobutyrate transaminase and glutamate dehydrogenase were always found at a higher density than NAD+-isocitrate dehydrogenase and monoamine oxidase. When centrifuged for 1 h at 53 000gav., there was a slight difference between the distribution profiles of glutamate dehydrogenase and gamma-aminobutyrate transaminase. This difference was larger when the centrifugation time was only 15 min. It is concluded that there are subpopulations of brain mitochondria with differing proportions of gamma-aminobutyrate transaminase and glutamate dehydrogenase. The results are discussed in relation to evidence obtained with labelled precursors in vivo that there are at least two small glutamate compartments in adult brain.

4-Aminobutyrate Transaminase↗

PubMed1979

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate. The steady state.

Ammonia is known to inhibit the steady-state rate of oxidation of L-glutamate catalyzed by glutamate dehydrogenase. We reported previously [Brown, A., Colen, A. H., & Fisher, H. F. (1978) Biochemistry 17, 2031] kinetic evidence supporting the formation in the initial rapid phase of a complex which is composed of enzyme, reduced coenzyme, alpha-ketoglutarate, and ammonia. We show here that the effects of ammonia on the steady-state reaction can be correlated with transient-state kinetic effects related to the concentration of that ammonia-containing complex. These results indicate the existence of alternate reaction pathways which become important at high ammonia concentrations. These new pathways provide an additional route for the release of NADPH from the enzyme surface. The expanded mechanism shows that the noncompetitive product inhibition by ammonia can occur without the simultaneous presence of ammonia and L-glutamate on the enzyme. This mechanism also accommodates the observed substrate inhibition by L-glutamate.

Ammonia↗

PubMed1991

Purification and properties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus.

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3.) of the extreme thermophilic archaebacterium Sulfolobus solfataricus was purified to homogeneity by (NH4)2SO4 fractionation, anion-exchange chromatography and affinity chromatography on 5'-AMP-Sepharose. The purified native enzyme had a Mr of about 270,000 and was shown to be a hexamer of subunit Mr of 44,000. It was active from 30 to 95 degrees C, with a maximum activity at 85 degrees C. No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90 degrees C for 60 min, or in the presence of 4 M urea or 0.1% SDS. The enzyme was catalytically active with both NADH and NADPH as coenzyme and was specific for 2-oxoglutarate and L-glutamate as substrates. With respect to coenzyme utilization the Sulfolobus solfataricus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms than those from eubacteria.

Amino Acids↗

PubMed1979

Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa.

The regulation of the glutamate dehydrogenases was investigated in wild-type Neurospora crassa and two classes of mutants altered in the assimilation of inorganic nitrogen, as either nitrate or ammonium. In the wild-type strain, a high nutrient carbon concentration increased the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-glutamate dehydrogenase and decreased the activity of reduced nicotinamide adenine dinucleotide (NADH)-glutamate dehydrogenase. A high nutrient nitrogen concentration had the opposite effect, increasing NADH-glutamate dehydrogenase and decreasing NADPH-glutamate dehydrogenase. The nit-2 mutants, defective in many nitrogen-utilizing enzymes and transport systems, exhibited low enzyme activities after growth on a high sucrose concentration: NADPH-glutamate dehydrogenase activity was reduced 4-fold on NH(4)Cl medium, and NADH-glutamate dehydrogenase, 20-fold on urea medium. Unlike the other affected enzymes of nit-2, which are present only in basal levels, the NADH-glutamate dehydrogenase activity was found to be moderately enhanced when cells were grown on a low carbon concentration. This finding suggests that the control of this enzyme in nit-2 is hypersensitive to catabolite repression. The am mutants, which lack NADPH-glutamate dehydrogenase activity, possessed basal levels of NADH-glutamate dehydrogenase activity after growth on urea or l-aspartic acid media, like the wild-type strain, and possessed moderate levels (although three- to fourfold lower than the wild-type strain) on l-asparagine medium or l-aspartic acid medium containing NH(4)Cl. These regulatory patterns are identical to those of the nit-2 mutants. Thus, the two classes of mutants exhibit a common defect in NADH-glutamate dehydrogenase regulation. Double mutants of nit-2 and am had lower NADH-glutamate dehydrogenase activities than either parent. A carbon metabolite is proposed to be the repressor of NADH-glutamate dehydrogenase in N. crassa.

Carbon↗

PubMed2000

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17beta-Hydroxysteroid Dehydrogenase IV.

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A-conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17beta-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of alpha-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator.

Journal Article↗

PubMed1984

The binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase.

The binding of 1,N6-etheno-NAD (epsilon NAD) to bovine liver glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate has been investigated at pH 7.0, 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations. epsilon NAD binds to the protein in a simple fashion: one molecule of coenzyme per enzyme polypeptide chain in the range of enzyme concentrations investigated (from above 50 to a few micromoles of enzyme polypeptide chains/liter). The fluorescence enhancement factor, Q, of bound epsilon NAD relative to free epsilon NAD is independent of the saturation degree, as deduced from the constant value of the long fluorescence decay lifetime (about 21 ns), and is about 17, as deduced from Fmax/F0 ratio values obtained after extrapolation from double reciprocal plots of 1/delta F vs. 1/[glutamate dehydrogenase]. This value for the Q factor is also independent of enzyme concentration, as well as of the presence of either GTP or ADP. At low enzyme concentrations (below 20 mumol polypeptide chains/liter), the dissociation constant of epsilon NAD increases progressively from a plateau value of about 50 microM to about 100 microM at infinite dilution. This is interpreted as being due to a minor affinity of glutamate dehydrogenase hexamers, with respect to higher aggregation states of the enzyme, towards epsilon NAD. As expected, GTP and ADP change the affinity of glutamate dehydrogenase towards epsilon NAD in an opposite manner: GTP strongly increases it, whereas ADP strongly decreases it (Kappd around 6 microM with saturating GTP and around 300 microM with saturating ADP). Furthermore, in the case of GTP, both GTP and epsilon NAD bind to glutamate dehydrogenase with positive cooperativity, with a Hill coefficient of approx. 1.8 for both and a Kappd approximately equal to 30 microM for the binding of GTP to glutamate dehydrogenase saturated with epsilon NAD and glutarate. The value of the Q factor remains the same, even in the presence of the effectors (again from lifetime measurements), as well as the number of epsilon NAD binding sites per enzyme polypeptide chain. These results are interpreted in terms of independent active sites, in the case without effectors. With ADP the binding appears to be simple, but no careful investigation has been attempted at low enzyme concentrations because of the low saturation degree achievable, whereas with GTP the cooperativity can be explained as due to a shift towards hexamers from higher aggregation states.

Adenosine Diphosphate↗

PubMed1978

Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.

Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.

Amino Acids↗

PubMed1983

Complexes between mitochondrial enzymes and either citrate synthase or glutamate dehydrogenase.

Experiments performed in polyethylene glycol and with a divalent crosslinker indicate that both mitochondrial malate dehydrogenase and aspartate aminotransferase can form hetero enzyme--enzyme complexes with either glutamate dehydrogenase or citrate synthase. In general, these as previous results indicate that complexes with the aminotransferase are favored over those with malate dehydrogenase and complexes with glutamate dehydrogenase are favored over those with citrate synthase. When the levels of enzymes are low, the only detectable complex is between the aminotransferase and glutamate dehydrogenase. Under these conditions, palmitoyl-CoA is required for complexes between the other three enzyme pairs, however, palmitoyl-CoA also enhances interactions between glutamate dehydrogenase and the aminotransferase. DPNH disrupts complexes with malate dehydrogenase and has little effect on those with the aminotransferase, while oxalacetate disrupts complexes with citrate synthase but has little effect on those with glutamate dehydrogenase. The citrate synthase-aminotransferase complex was favored in the presence of DPNH plus malate, which disrupt the other three enzyme-enzyme complexes. Glutamate dehydrogenase has a higher affinity and capacity than citrate synthase for palmitoyl-CoA. Consequently, lower levels of palmitoyl-CoA are required to enhance interactions with glutamate dehydrogenase. Furthermore, glutamate dehydrogenase can compete with citrate synthase for palmitoyl-CoA and thus can prevent palmitoyl-CoA from enhancing interactions between citrate synthase and either malate dehydrogenase or the aminotransferase.

Acyl Coenzyme A↗

PubMed1983

The precursor of rat liver mitochondrial glutamate dehydrogenase has enzymatic activity.

The cytosolic precursor for the mitochondrial glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using messenger RNA from rat liver. To check whether this precursor had enzymatic activity, a highly sensitive fluorimetric method, which can measure picogram quantities of enzyme, was used together with competitive dissociation of the precursor from an immunoprecipitate with inactive glutamate dehydrogenase. Glutamate dehydrogenase activity, corresponding to that estimated from incorporation of [35S]-methionine, was detected in the precursor. The significance of this finding is discussed.

Animals↗

PubMed1987

Adaptation of methods for glutamate dehydrogenase and alcohol dehydrogenase activities to a centrifugal analyser: assessment of their clinical use in anoxic states of the liver.

Sensitive, precise, and rapid methods for the measurement of alcohol dehydrogenase (ADH) and glutamate dehydrogenase (GDH) were developed on the Cobas Bio centrifugal analyser. The optimal pH for ADH in caucasians was 9.8. Non-linearity of ADH enzyme activity was observed when samples were diluted in saline; linearity was restored when inactivated serum was used as diluent. ADH was shown to be a sensitive index of liver anoxia due to cardiorespiratory disturbance (clinical sensitivity 90%) and generalised anoxia. GDH exhibited sensitivity equal to that of alanine aminotransferase (ALT) but was inferior to gamma-glutamyltransferase (GGT) in the detection of specific liver disease. Both ADH and GDH were sensitive indicators of alcoholic liver disease.

Alanine Transaminase↗