L-glutamate Dehydrogenase Research Articles

Characterisation of a thermophilic L-glutamate dehydrogenase biosensor for amperometric determination of L-glutamate by flow injection analysis

Carbon paste wax electrodes incorporating thermophilic L-glutamate dehydrogenase, NADP and a polymeric toluidine blue O (poly-TBO) mediator have been characterised for the amperometric determination of L-glutamate at 313–318 K in a flow injection analysis (FIA) system. The biosensors exhibit good sensitivity, mechanical stability and reproducibilty, unlike carbon paste- or carbon wax-based electrodes under the same conditions. The carbon paste wax electrode responds linearly to L-glutamate up to 40 mM, the detection limit is 0.3 mM and the RSD ( n=10) for 5 mM L-glutamate was 7.6%. The response to some potential interferents has been quantified. Addition of finely ground hexaammineruthenium (III) trichloride ([Ru(NH 3) 6]Cl 3) to the carbon paste wax electrodes decreases the FIA peak width and increases the peak current. The metal complex appears to accelerate the rate of oxidation of NAD(P)H by poly-TBO.

Enzymes in Antarctic fish: Glucose-6-phosphate dehydrogenase and glutamate dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PD) and l-glutamate dehydrogenase (GDH) from Antarctic fish were isolated and characterized. G6PD was purified from the erythrocytes of red-blooded Dissostichus mawsoni and from the colorless blood of the icefish Chionodraco hamatus. Structural and functional characterization showed that the two enzymes do not differ significantly from each other. GDH was purified from the liver of the icefish Chaenocephalus aceratus. As in other fish GDHs, it showed a marked preference for NAD +. The amino acid sequence of the active-site peptide is virtually identical to that of other fish and vertebrate counterparts. Although the basic structural features of the Antarctic enzymes are similar to those of mesophilic organisms, some catalytic and thermodynamic properties make the Antarctic enzymes more suited to cold-adapted organisms.

Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications.

The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90 degrees C, contains an L-glutamate dehydrogenase (GDH). Activity of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the hyperthermophilic archaeon Pyrococcus woesei. The single-copy T. maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain. The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45,852. The gdh gene was inserted in an expression vector and expressed in E. coli as an active enzyme. The T. maritima GDH was purified to homogeneity. The NH2-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2-13 of the peptide sequence derived from the gdh gene. The purified native enzyme has a size of 265 kDa and a subunit size of 47kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75 degrees C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively. The enzyme was found to be very stable at 80 degrees C, but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (Vmax of 172 U/mg protein), but also utilizes NADPH (Vmax of 12 U/mg protein). Sequence comparisons showed that the T. maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles. Remarkably, phylogenetic analysis positions all these hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T. maritima GDH located between that of halophilic and thermophilic euryarchaeota.

Evaluation of a thermophile enzyme for a carbon paste amperometric biosensor: L-glutamate dehydrogenase

An amperometric L-glutamate sensor was prepared by the immobilization of thermophilic L-glutamate dehydrogenase (L-glutamate: NADP + oxido-reductase, deaminating and transaminating, EC 1.4.1.4.) in a carbon paste working electrode with a polyethylenimine Toluidine Blue O redox polymer mediator and lactitol/DEAE dextran stabilizing system. The response of the sensor was both pH and temperature dependent. Decreases in K m app (mM) and increases in V max (μA) were observed with increasing temperature, whilst increasing pH resulted in increases in both K m app and V max parameters. The measurements were based on the amperometric detection of the product of the enzymatic reaction, NADPH, via mediated reoxidation to NADP + at E app = 100 mV versus Ag/AgCl. The major characteristics of this method are its simplicity and reproducibility, although at higher temperatures (333 K and above) the properties of the carbon paste matrix itself limited the physical stability of the system.

Ammonium ion salvage by glutamate dehydrogenase during defence response in maize

When phytochemical defence response was elicited by treatment of maize with chitosan, the binomial isoenzymes of l-glutamate dehydrogenase (GDH, EC 1.4.1.2) isomerized. The amination electrode potential of the GDH charge isomers decreased by 10 mV in the chitosan-treated maize. GDH isomerization and amination are therefore spontaneous reactions. The free l-Glu and l-Gln were 3.84 and 3.68 μmol, respectively, per 10 g of the chitosan-treated maize. The amination activities of GDH showed that the 10 mV decrease in electrode potential accounted fully for the 3.84 μmol free l-Glu content of the chitosan-treated maize. Therefore the binomial GDH synthesized all the l-Glu, thereby salvaging at least 51% of the NH 4 + liberated during phytochemical defence response, while glutamine synthetase salvaged the remaining NH 4 +. The chitosan-elicited isomerization of maize GDH and the accompanying 10 mV decrease in electrode potential widened the plant-pathogen amination energy barrier, thereby minimizing the amount of NH 4 + the pathogen can absorb from the plant.

Alpha-Ketoglutarate assay based on fluorescence quenching by NADH.

A new assay procedure for the measurement of ketoglutarate concentrations is described which is based on substrate-induced quenching (SIQ) of a fluorophore. The method makes use of the photoreaction between a fluorophore (thionine) and NADH. The latter is consumed during an enzymatic reaction between ketoglutarate and L-glutamic dehydrogenase. The conversion yield of cofactor from its reduced form to oxidized forms represented as an overall change in the population of the excited state population of the fluorophore thionine. An empirical relation is described that correlates initial substrate concentration to the observed yield of the cofactor conversion via a fluorescence recovery constant,Kt. The analysis of data obtained over a range of 0-500 microM results in a constant of 2748 M-1. The applicability of the proposed method is demonstrated by performing the assay for alpha-ketoglutarate in human urine. The ketoglutarate SIQ assay was not affected by the background interference that is inherent to this complex matrix.

Flow injection methods for determination of L-glutamate using glutamate decarboxylase and glutamate dehydrogenase reactors with spectrophotometric detection

The use of glutamate decarboxylase and glutamate dehydrogenase reactors in flow injection (FI) systems for the determination of glutamate in food is described. In a gas diffusion FI system, glutamate was decarboxylated by glutamate decarboxylase immobilized on a carrier. The produced carbon dioxide was measured spectrophotometrically after gas diffusion using an acid–base indicator as acceptor. The linear range of calibration was from 2 to 20 mmol l–1 with r= 0.9978 and the RSD was 2.8%(n= 5) for 10 mmol l–1L-glutamate. Using a glutamate dehydrogenase reactor the NADH formed by the enzymic reaction was detected at 340 nm. For the normal FI approach the calibration graph was linear from 0.05–0.06 mmol l–1 with r= 0.9991 and the RSD was 2.4%(n= 5) for 0.3 mmol l–1L-glutamate. For the stop flow approach with a significant reduction in NAD+ consumption the linear range was from 0.2 to 1.0 mmol l–1L-glutamate with r= 0.9994. The RSD was 1.9%(n= 5) for 0.6 mmol l–1L-glutamate. A sample frequency of 30 h–1 using these three approaches was reached. Results from food samples using these three approaches were in good agreement with each other. Recoveries were 94.6–101.2%, 96.7–99.3% and 95.9–98.2% for L-glutamate decarboxylase, L-glutamate dehydrogenase with the normal FI approach and L-glutamate dehydrogenase with the stop flow approach, respectively. The assay results of one sample compared well with those given by the manufacturer.

Synthesis and Evaluation of Novel Substrates and Inhibitors of N-Succinyl-ll-diaminopimelate Aminotransferase (DAP-AT) from Escherichia coli

N-Succinyl-ll-diaminopimelate aminotransferase (DAP-AT) (EC 2.6.1.17), a key enzyme in the bacterial pathway to l-lysine, was purified to near homogeneity (1500-fold) in five steps from wild type Escherichia coli ATCC 9637. This pyridoxal phosphate (PLP) dependent enzyme has a molecular weight of 39.9 kDa, appears to form an active homodimer, and uses l-glutamate as the amino group donor for its substrate, N-succinyl-α-amino-ε-ketopimelic acid (1a) (Km = 0.18 ± 0.04 mM, kcat = 86 ± 5 s-1). Progress of the reaction is monitored by spectrophotometric observation of decrease in NADPH concentration at 340 nm in a coupled enzyme assay with l-glutamate dehydrogenase (EC 1.4.1.4). Stereochemically pure 1a was synthesized as its trilithium salt by ene reaction of methyl glyoxylate with methyl N-succinyl-l-allylglycinate (4a) followed by hydrogenation of the double bond, Dess−Martin oxidation of the alcohol, and careful lithium hydroxide hydrolysis. Similar approaches allowed synthesis of a series of substrate analogues 1b−g having different N-acyl substituents, as well as derivatives missing the carboxyl group or the amide functionality (13 and 17, respectively). Compounds lacking the keto functionality (18a, 18c, and 19) were also prepared. Assay of DAP-AT shows that the enzyme has quite strict requirements for substrate recognition, but it will accept compounds with an aromatic ring in place of the terminal succinyl carboxyl group in 1a (e.g. N-Cbz-α-amino-ε-ketopimelic acid (1c)). Reaction of substrates 1a,c with hydrazine hydrate followed by NaCNBH3 reduction gives 2-(N-(succinylamino))- (20a) and 2-(N-Cbz-amino)-6-hydrazinoheptane-1,7-dioic acids (20c), respectively. These are the most potent slow-binding inhibitors of any DAP-metabolyzing enzyme reported so far (Ki* for DAP-AT: 20a is 22 ± 4 nM; 20c is 54 ± 9 nM).

Coupling of the electroenzymatic reduction of NAD + with a synthesis reaction

Hydrogenase from Alcaligenes eutrophus H16 (E.C. 1.12.1.2) catalyzes the electrochemical reduction of the pyridine cofactor NAD +. Previous studies emphasized the kinetics and thermodynamics of the reaction. We applied hydrogenase to regenerate NADH for the transformation of α-ketoglutarate into l-glutamate catalyzed by an l-glutamate dehydrogenase. The system was first studied in an electrochemical thin-layer cell. A turnover number (TN) of 450 h −1 was obtained. The influence of the ratio of l-glutamate dehydrogenase and hydrogenase activities is discussed. The separate analyses of the glutamate dehydrogenase catalysis on the one hand and the hydrogenase electrocatalysis on the other led to the hypothesis that the behavior of the coupled process was controlled by the NADH concentration level. We exploited the qualitative conclusions drawn from the analytical study to test the process on a preparative scale-batch reactor. A TN of 207 h −1 was reached. The poor stability of the hydrogenase was the major hindrance to the development of the reactor, but a possible solution was discussed by means of the stabilizing effect of the electrochemical conditions.

Involvement of NAD(P)+‐glutamate dehydrogenase isoenzymes in carbon and nitrogen metabolism in Chlamydomonas reinhardtü

The involvement of three NAD(P)+‐L‐glutamate dehydrogenase (GDH; EC 1.4.1.3) isoenzymes, named GDH1, GDH2 and GDH3, in the carbon and nitrogen metabolism of the green alga Chlamydomonas reinhardtü 6145c has been investigated under different environmental and stress conditions. GDH1 activity decreased, but GDH2 and GDH3 activities increased with the age of cultures. When the extracellular ammonium concentration was high, only GDH1 activity increased with growth whereas GDH2 and GDH3 remained unchanged. In the presence of L‐methionine‐D,L‐sulfoximine (MSX), an inhibitor of L‐glutamine synthetase (GS), a significant increase of GDH1 and a slight decrease in GDH3 activity was observed, whereas GDH2 did not change. A significant increase in the intracellular 2‐oxoglutarate was also found upon addition of azaserine, an inhibitor of L‐glutamate synthase (GOGAT) activity. However, no significant changes in GDH isoenzyme activities were observed after addition of azaserine or azaserine plus MSX, except an induction of GDH3 in the latter case. Moreover, in the presence of ethoxyzolamide (ETZ), an inhibitor of carbonic anhydrase activity, an induction of total GDH activity, mainly due to an increase in GDH1 and to a minor extent in GDH2, was observed in cells under low CO2 (0.03%). In the dark, cells showed an increase in GDH1 activity, but when acetate was present GDH1 activity was repressed. All these results taken together suggest a relationship between GDH1 and nitrogen assimilation, whereas GDH2 and GDH3 seem to be involved in the production of 2‐oxoglutarate to fuel the tricarboxylic acid (TCA) cycle.

Immobilization of glutamate dehydrogenase on glass derivatives. A method for the assay of glutamates in real samples with simplex optimized automated FIA-system

An enzymatic method for the determination of glutamic acid in food samples and pharmaceuticals is described. l-Glutamate dehydrogenase (GLDH) from beef liver was immobilized on isothiocyanate modified Controlled Pore Glass for the construction of a packed bed reactor. The NADH produced from the enzymic reaction was monitored fluorimetrically. The working curve is linear up to 200 μmol/l glutamate for an injection volume of 58 μl. The detection limit of the method is 0.3 μmol/l. The composite modified simplex was employed for the selection of the proper experimental conditions using an in-house flow injection manifold. Many interfering species and several amino acids were tested to verify the specificity of the enzyme reactor. The system works selectively for glutamic acid. The method is ideally suited to the assay of glutamic acid in a large number of samples because of its simplicity, stability and low cost. Forty-five samples per hour can be analyzed with a relative standard deviation better than 2%. The reactor is stable for a period of more than four months under specified storage conditions. The accuracy of the proposed method is tested by comparison of the results with those obtained by the official methods and the manufacturer's specifications for the analyzed samples. Good correlation was attained. Recovery experiments showed results between 97 and 104%.

Glutamate Oxidase-Catalyzed Oxidation of β-N-Oxalyl-L-α,β-N-Diaminopropionic Acid (ß-ODAP), a Neurotoxin in the Seeds ofLathyrus Sativus

Abstract Seven enzymes were tested in a screening of catalytic activity towards the neurotoxic L-amino acid, s-ODAP. L-glutamate oxidase, GIOD, was found to be the only one that showed activity and its kinetic properties were examined by measuring the rate of formation of hydrogen peroxide which was monitored spectrophoto-metrically at 512 nm, using Trinder's chromogenic reagent. The Michaelis constant, KM, for the toxin is 0.24 mM at pH 7, comparable with the reported value for glutamate (0.21 mM). The activity of the enzyme was, however, considerably lower (0.78%) than that of the main substrate. Ammonia formation in the reaction was confirmed by the consumption of nicotinamide adenine dinucleotide (NADH) during reduction of α-kctoglutarate in presence of L-glutamate dehydrogenase (GIDH) and catalase.

Amperometric determination of urea using an NADH-dependent coupled enzyme

Enzyme electrodes for the amperometric measurement of urea were prepared by co-immobilizing l-glutamate dehydrogenase and urease onto an Immobilon-AV affinity membrane with attachment to a glassy carbon electrode. Reduced nicotinamide adenine dinucleotide (NADH) was used as the electroactive species. The electrochemical oxidation of NADH was monitored at +1.0 V vs. Ag/AgCl. The enzyme immobilized electrode was linear over the range of 2.0 × 10 −5 to 2 × 10 −4 M. The response time of the electrode was 3 min and the optimum pH of enzyme immobilized membrane was pH 7.4–7.6 (Dulbecco's buffer solution). It was stable for at least two weeks and 50 assays. There were no interferences from other physiological material, except for high levels of ascorbic acid.

Comparison of methods to assay ammonia in bacterial suspensions

In the determination of the concentration of ammonia in the filtrates of Pseudomonas syringae cell suspensions, similar results were obtained using the indophenol method, a gassensing membrane electrode, and the L-glutamic dehydrogenase assay. The intracellular ammonia concentration was determined using the electrode and estimated to be 17 mM, corresponding to approximately 1 4 of potassium concentration. The results suggest that ammonium is one of the major intracellular cations in P. syringae cells.

An Ultrafiltration Method for the Removal of Interfering Agents and Its Application to the Determination of Free Ammonia in Solutions of Oxystarch by the Berthelot Reaction Method

Oxystarch was chosen as a model compound for studying biological ammonia-sequestering systems. Ammonia was determined by use of an ion-selective electrode, by L-glutamate dehydrogenase (L-GDH), and by two different Berthelot procedures, in the presence and absence of oxystarch. In the presence of total oxystarch the Berthelot method, particularly when low concentration reagents were used, detected significantly less (P < 0.10) free ammonia than either L-GDH or ion-selective electrode methods. A 0.5-kDa molecular weight cutoff sample ultrafiltration step was added prior to analysis by L-GDH and Berthelot procedures. To facilitate complete removal of oxystarch by the ultrafiltration step, oxystarch was dialyzed before use, yielding a high-molecular-weight fraction (>1 kDa). Removal of high-molecular-weight oxystarch species and bound ammonia by ultrafiltration of samples prior to assay completely negated discrepancies between ammonia levels measured by L-GDH and both Berthelot methods. The correlation of the levels of measured ammonia, as determined by L-GDH and Berthelot methods, in mixtures with high-molecular-weight oxystarch was significantly improved by the addition of the sample ultrafiltration step. Improved correlation of results from such fundamentally different methods demonstrates the removal of interfering agents as well as the nonperturbatory nature of the improved procedure. The addition of such an ultrafiltration step may be applied to the determination of ammonia by the otherwise interference-prone Berthelot assay in mixtures with any interfering macromolecules, without the inconvenience or potential variabilities associated with distillation or diffusion procedures.

A rapid colorimetric assay for γ-glutamyl hydrolase (conjugase)

A simple procedure for the measurement of γ-glutamyl hydrolase (conjugase) activity is described. Glutamic acid released from pteroylpenta-γ-glutamate by hog kidney and chicken pancreas conjugases was quantitated using the dye 4,4′-bis(dimethylamino)benzophenone hydrazone. The procedure involves hydrolysis of the folylpoly-γ-glutamate substrate by conjugase, conversion of glutamate to α-ketoglutarate by l-glutamate dehydrogenase and colorimetric measurement of the BDBH derivative of α-ketoglutarate. The release of as little as one nmol of glutamic acid from the substrate can be measured by this procedure, which is well suited for the assay of a variety of conjugase preparations. In addition, the method should provide a general assay for the enzymatic hydrolysis of various folate and antifolate polyglutamates.

Mitochondrial enzymes related to glutamate and GABA metabolism in the hippocampus of young and aged rats: a quantitative histochemical study.

Quantitative histochemistry (scanning microphotometry) was used to determine the activities of the mitochondrial enzymes NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), L-glutamate dehydrogenase (EC 1.4.1.3) and GABA transaminase (EC 2.6.1.19) in various layers of the hippocampus (middle one third) of young (3-4 months old) and memory-impaired aged rats (28-30 months old). For comparison, determinations of cytochrome c oxidase (EC 1.9.3.1) as a marker for mitochondria and energy metabolism were also performed. The study showed that there was a layered reaction pattern in the hippocampus and that the cellular distribution and the levels of enzyme activity were different. However, the activities of the different enzymes (excepting GABA transaminase and cytochrome c oxidase) were significantly correlated in the hippocampus in both age groups. Age-dependent changes were only observed for NAD-linked isocitrate dehydrogenase and GABA transaminase (significant increases of activities in some layers of the hippocampus, preferentially in the terminal field of the perforant path). From the present study it is concluded that, 1. the enzymatic complement of mitochondria in neurons and glia depends upon layer specific metabolic processes of the hippocampus (also with respect to glutamatergic and GABAergic terminal fields) indicating a layer specific interaction of the enzymes studied to produce or catabolize glutamate and GABA, and 2. the age dependent changes of the studied enzymes are very restricted.

Purification and properties of three NAD(P) + isozymes of l-glutamate dehydrogenase of Chlamydomonas reinhardtii

Three isozymes of glutamate dehydrogenase (GDH) of Chlamydomonas reinhardtii, induced under different trophic and stress conditions, have been purified about 800-1000-fold to electrophoretic homogeneity. They are hexamers of Mr 266,000-269,000 as deduced from gel filtration and sedimentation coefficient data. GDH1 consisted of six identical subunits of 44 kDa each, whereas both GDH2 and GDH3 consisted of six similar-sized monomers (4 of 44 kDa and 2 of 46 kDa). Optimum pH for the three activities with each pyridine nucleotide was identical (8.5 with NADH; 7.7 with NADPH; and 9.0 with NAD+). The isozymes exhibited similar high optimum temperature values (60-62 degrees C) and isoelectric points (7.9-8.1). Activity was enhanced in vitro by Ca2+ ions and strongly inhibited by pyridoxal 5'-phosphate, KCN, o-phenanthroline and EDTA, and to a lesser extent by pHMB and methylacetimidate. In the aminating reaction the three isozymes were inhibited in a concentration-dependent process by both NADH and NADPH, with apparent Km values for NH4+ ranging from 13-53 mM; 0.36-1.85 mM for 2-oxoglutarate and 0.07-0.78 mM for NADH and NADPH. In the deaminating reaction apparent Km values ranged from 0.64-3.52 mM for L-glutamate and 0.20-0.32 for NAD+. In addition, the three isozymes exhibited a non-hyperbolic kinetics for NAD+ with negative cooperativity (n = 0.8).

Proposed Enzymes of Auxin Biosynthesis and Their Regulation II. Tryptophan Dehydrogenase Activity in Plants.

In pea, maize and tomato plants a hitherto undescribed L-tryptophan dehydrogenase activity (TDH) has been detected. This enzyme catalyzes the reversible formation of indolepyruvic acid (IPyA) from L-tryptophan (L-trp). TDH and L-glutamate dehydrogenase (GDH), related enzymes in their mode of action, could be separated by gel chromatography. Enzymatic activity of TDH was sustained by both pyridine coenzymes NAD/NADP. With pea TDH the coenzyme NAD displays, at optimum pH 8.5 and at room temperature, only about 40-70 % of the activity of NADP. The amination of IPyA is catalysed more actively than the deamination of L-trp. L-trp/IPyA, L-glu/ketoglutarate, L-ala/pyruvate reacted as dehydrogenase substrates; L-phe/ phenylpyruvate, D-trp and D-phe did not react with pea enzyme extracts. A considerable similarity between the active centres of TDH and GDH has been found using inhibitors: absence of heavy metals, presence of a carbonyl group, indispensibility of bivalent ions for the enzyme activity. Pea TDH and GDH were distinctly inhibited by sodium azide. For the activity of TDH the presence of SH groups is less important than for GDH. The TDH activity in the investigated plants was lower than the GDH activity. The possible role of TDH in the regulation of the IPyA pool is discussed.Doc. RNDr. PhMr. M. Kutacek died on 28 November, 1989. The final form for print was prepared by dr. Ivana Machdckovd of the same Institute, who will also answer the reprint requests. Received June 6, 1990; accepted October 10, 1990

Amperometric enzyme electrodes for the determination of l-glutamate

Electrodes for amperometric measurement of l-glutamate were prepared by immobilization of l-glutamate oxidase on an Immobilon-AV Affinity membrane and attachment to an oxygen/hydrogen peroxide sensor. The response of the hydrogen peroxide sensor was linear over the concentration range 5.0 × 10 −8−5.0 × 10 −4 M l-glutamate, with a limit of detection of 35n M. Attachment of a size—exclusion membrane (cut-off for molecular weight > 100) or of a hydrophobic oxygen membrane eliminated electro-oxidizable interferences, but the response was attenuated by a factor of 2–3. The response may be amplified 10-fold by co-immobilizing l-glutamate dehydrogenase with the l-glutamate oxidase. The electrode initially lost 25% of its activity but was then stable for more than 320 days and at least 200 assays. The electrode was successfully used to assay glutamate in a protein tablet and in several food products. A flow-injection system was assembled for the continuous assay of l-glutamate.