Glutamate Synthase: Properties of the Reduced Nicotinamide Adenine Dinucleotide-Dependent Enzyme from Saccharomyces cerevisiae

A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and alpha-ketoglutarate (K(m) values of 2.6 muM, 1.0 mM, and 140 muM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase.     

Glutamate synthase: a possible role in nitrogen metabolism of the developing maize endosperm

Glutamate synthase activity was demonstrated in the developing endosperm of maize (Zea mays L.). The enzyme shows specificity for glutamine and alpha-ketoglutarate as amino donor and acceptor, respectively. Both NADH and NADPH function as electron donors although lower activities were often, but not always, obtained with NADPH. The apparent K(m) values for glutamine, alpha-ketoglutarate, and NADH were 1.35 mm, 0.57 mm, and 7 mum, respectively.The pattern of activity during endosperm development revealed a well defined peak coinciding with the period of most active N accumulation. Activity in general was related to the rates of accumulation throughout development. Maximum glutamate synthase activity was the order of 56 nmoles of glutamate formed per minute per endosperm compared with a rate of N accumulation of 9.5 nmoles per minute.It is suggested that glutamate synthase plays a key role in the N nutrition of the maize endosperm providing a means whereby N transported in the form of glutamine is made available for the synthesis of other seed protein amino acids via transaminase reactions. Transaminase activity involving glutamate, the product of the glutamate synthase reaction, was also demonstrated.     

Glutamate formation by a new In vitro enzyme system consisting of purified glutamine synthetase and glutamate synthase

After the addition of ammonia to the culture medium, the concentration of glutamine in B. flavum cells increased in 20 s with a decrease in glutamate. In the subsequent 30 s, the glutamine concentration deceased again with an increase in glutamate. An enzyme system, which consisted of purified glutamine synthetase (GS) and glutamate synthase (GOGAT) with ATP- and NADPH-regenerating systems, was made up to study the functions of the GS/GOGAT pathway: concentrations of the substrates and of the enzymes were decided on according to the intracellular conditions. Changes in the concentrations of amino acids caused by the addition of ammonia to the system were very similar to those of intracellular glutamate and glutamine when ammonia was added to the bacterial culture. The time required for the complete formation of glutamate from 0.5 mM ammonia was about 4-times shorter in the GS/GOGAT system than in the system using purified glutamate dehydrogenase (GDH) and the NADPH-regenerating system. The glutamate synthase reaction in the GS/GOGAT system was inhibited by some amino acids much more markedly than in the standard assay mixture consisting of glutamine, α-ketoglutarate and NADPH. These results gave further evidence elucidating the operation of the GS/GOGAT pathway in ammonia assimilation, and suggested that a reconstructed enzyme system is useful for studying physiological mechanisms.     

Some properties of glutamine synthetase from the nitrifying bacterium Nitrosomonas europaea

Nitrosomonas europaea oxidizes ammonia to nitrite, thereby deriving energy for growth. Glutamate dehydrogenase (NADP+) (EC 1.4.1.4) is the main route for the incorporation of ammonia into glutamic acid, because glutamate synthase (NADPH)(EC 1.4.1.13) was not detected in cell-free extracts of N. europaea. Some properties of a partially purified glutamine synthetase (EC 6.3.1.2) have been determined, namely the effects of pH and metal ions, substrate requirements, Km and Ki values, based on biosynthetic and gamma-glutamyltransferase (EC 2.3.2.2) assays. The molecular weight of the enzyme preparation was approximately 440 000. The gamma-glutamyltransferase activity was markedly inhibited by alanine, lysine, glutamic acid, aspartic acid and serine and to a lesser extent by glycine, asparagine, arginine and histidine. Except for tryptophan and cystine, the gamma-glutamyltransferase activity was inhibited to a greater extent by these amino acids than was the biosynthetic activity. Different pairs of amino acids in various combinations resulted in a cumulative inhibition of enzyme activity determined by either method. Of the various nucleotides tested, the gamma-glutamlytransferase activity of the enzyme was inhibited to a greater extent by di- and triphosphate nucleotides--IDP, CDP, UDP, ITP, CTP, TTP and ATP (except GDP and GTP) than by monophosphate nucleotides except AMP. Saturating concentrations of pyruvate, oxalate, oxaloacetate and alpha-ketoglutarate depressed enzyme activity. Various combinations of amino acids with adenine nucleotides exerted cumulative inhibitory effects on the transferase activity.     

Some properties of glutamine synthetase and glutamate synthase from Chlorobium vibrioforme f. thiosulfatophilum

The phototrophic green sulphur bacterium Chlorobium vibrioforme f. thiosulfatophilum assimilated ammonia via glutamine synthetase and glutamate synthase when grown with ammonia up to 30 mM, but above this level glutamate dehydrogenase was the key enzyme. Glutamine synthetase purified 42-fold was found to be adenylylated. The -glutamyltransferase activity of the enzyme was markedly inhibited by alanine, glycine, serine and lysine, and these amino acids in various combinations showed cumulative inhibition. Adenine nucleotides also inhibited enzyme activity, especially ATP. Glutamate synthase purified 222-fold had a maximum absorption at 440 nm which was reduced by sodium dithionite, and the enzyme was inhibited by atebrin indicating the presence of a flavin component. The enzyme had specific requirements for NADH, -ketoglutarate and l-glutamine, the K _m values for these were 13.5, 270 and 769 M respectively. Glutamate synthase was sensitive to feedback inhibition by amino acids, adenine nucleotides and other metabolites and the combined effects of these inhibitors was cumulative.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamic dehydrogenase     

重组CHO细胞培养过程中氨对细胞代谢的影响

研究了重组CHO细胞批培养过程中,氨浓度对细胞的葡萄糖、谷氨酰胺及其它氨基酸代谢的影响。表明,细胞对葡萄糖和谷氨酰胺的得率系数随着氨浓度的增加而降低,起始氨浓度为566mmol/L的批培养过程与起始氨浓度为021mmol/L的批培养过程相比,细胞对葡萄糖和谷氨酰胺的得率系数分别下降了78%和74%,细胞对其它氨基酸的得率系数也分别下降了50%～70%。氨浓度的增加明显地改变了细胞的代谢途径,葡萄糖代谢更倾向于厌氧的乳酸生成。在谷氨酰胺的代谢过程中,谷氨酸经谷氨酸脱氢酶进一步生成α酮戊二酸的过程受到了氨的抑制,而氨对谷氨酸经谷氨酸转氨酶反应生成α酮戊二酸的过程有促进作用,但总体上谷氨酸进一步脱氨生成α酮戊二酸的反应受到了氨的限制。     

Purification and properties of glutamate synthase from Bacillus licheniformis

Glutamate synthase [L-glutamate:NADP+ oxidoreductase (transaminating); EC 1.4.1.13](GltS) was purified to homogeneity from Bacillus licheniformis A5. The native enzyme had a molecular weight of approximately 220,000 and was composed of two nonidentical subunits (molecular weights, approximately 158,000 and approximately 54,000). The enzyme was found to contain 8.1 +/- 1 iron atoms and 8.1 +/- 1 acid-labile sulfur atoms per 220,000-dalton dimer. Two flavin moieties were found per 220,000-dalton dimer, with a ratio of flavin adenine dinucleotide to flavin mononucleotide of 1.2. The UV-visible spectrum of the enzyme exhibited maxima at 263,380 and 450 nm. The GltS from B. licheniformis had a requirement for NADPH, alpha-ketoglutarate, and glutamine. Classical hyperbolic kinetics were seen for NADPH affinity, which resulted in an apparent Km value of 13 microM. Nonhyperbolic kinetics were obtained for alpha-ketoglutarate and glutamine affinities, and the reciprocal plots obtained for these substrates were biphasic. The apparent Km values obtained for glutamine were 8 and 100 microM, and the apparent Km values obtained for alpha-ketoglutarate were 6 and 50 microM. GltS activity was found to be relatively insensitive to inhibition by amino acids, keto acids, or various nucleotides. L-Methionine-DL-sulfoximine, L-methionine sulfone, and DL-methionine sulfoxide were found to be potent inhibitors of GltS activity, yielding I0.5 values of 150, 11, and 250 microM, respectively. GltSs were purified from cells grown in the presence of ammonia and nitrate as sole nitrogen sources and were compared. Both yielded identical final specific activities and identical physical (UV-visible spectra, flavin, and iron-sulfur composition) and kinetic characteristics.     

Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting.     

Purification and properties of glutamate synthase from Nocardia mediterranei.

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.     

Transport of glutamine by Streptococcus bovis and conversion of glutamine to pyroglutamic acid and ammonia

Streptococcus bovis JB1 cells energized with glucose transported glutamine at a rate of 7 nmol/mg of protein per min at a pH of 5.0 to 7.5; sodium had little effect on the transport rate. Because valinomycin-treated cells loaded with K and diluted into Na (pH 6.5) to create an artificial delta psi took up little glutamine, it appeared that transport was driven by phosphate-bond energy rather than proton motive force. The kinetics of glutamine transport by glucose-energized cells were biphasic, and it appeared that facilitated diffusion was also involved, particularly at high glutamine concentrations. Glucose-depleted cultures took up glutamine and produced ammonia, but the rate of transport per unit of glutamine (V/S) by nonenergized cells was at least 1,000-fold less than the V/S by glucose-energized cells. Glutamine was converted to pyroglutamate and ammonia by a pathway that did not involve a glutaminase reaction or glutamate production. No ammonia production from pyroglutamate was detected. S. bovis was unable to take up glutamate, but intracellular glutamate concentrations were as high as 7 mM. Glutamate was produced from ammonia via a glutamate dehydrogenase reaction. Cells contained high concentrations of 2-oxoglutarate and NADPH that inhibited glutamate deamination and favored glutamate formation. Since the carbon skeleton of glutamine was lost as pyroglutamate, glutamate formation occurred at the expense of glucose. Arginine deamination is often used as a taxonomic tool in classifying streptococci, and it had generally been assumed that other amino acids could not be fermented. To our knowledge, this is the first report of glutamine conversion to pyroglutamate and ammonia in streptococci.     

The regulation of ammonia assimilating enzymes in Lemma minor

Summary Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS Glutamine synthetase - GDH Glutamate dehydrogenase - GOGAT Glutamate synthase     

Importance of glutamate synthase in glutamate synthesis by soybean cell suspension cultures

The specific activities of glutamate synthase|EC 2.6.1.53, l-glutamine: alpha-ketoglutarate amino transferase (NADPH-oxidising)| and glutamine synthetase|EC 6.3.1.2, l-glutamate: ammonia ligase (ADP-forming)| extracted from soybean (Glycine max L.) cells grown in modified B5 medium were found to vary significantly in response to variations in the nitrogen content of the medium. The changes seen in specific activity levels could be correlated with similar patterns seen in the growth of the cells, in response to changes in the nitrogen content of the medium. By contrast, the specific activity of glutamate dehydrogenase|EC 1.4.1.2, l-glutamate: NAD(+) oxidoreductase (deaminating)|, was relatively low and invariant. Glutamate synthase was extracted from cells grown under optimal conditions, partially purified, and shown to have many properties in common with preparations of this enzyme extracted from other plant sources. Glutamate synthase was purified to homogeneity, using affinity chromatography on blue Sepharose.     

Inorganic nitrogen assimilation by the photosynthetic bacterium Rhodopseudomonas capsulata.

The photosynthetic bacterium Rhodopseudomonas capsulata lacks glutamate dehydrogenase and normally uses the glutamine synthetase/glutamate synthase sequence of reactions for assimilation of N2 and ammonia. The glutamine synthetase in cell-free extracts of the organism is completely sedimented by centrifugation at 140,000 X g for 2 h, is inhibited by L-alanine but not by adenosine 5'-monophosphate, and exhibits two apparent Km values for ammonia (ca. 13 muM and 1 mM).     

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1.

Glutamate dehydrogenase (GDH) (L-glutamate:NADP+ oxidoreductase, deaminating, EC 1.4.1.4) from the cellulolytic ruminal bacterium Ruminococcus flavefaciens has been purified and characterized. The native enzyme and subunit are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The enzyme requires 0.5 M KCl for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, alpha-ketoglutarate, and glutamate are 19, 0.41, and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The first residue in the N-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified posttranslationally. Comparison of the N-terminal sequence with those of Escherichia coli, Salmonella typhimurium, and Clostridium symbiosum revealed only 39% amino acid homologies. The GDH from R. flavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM).     

Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1.

Glutamate dehydrogenase (GDH) (L-glutamate:NADP+ oxidoreductase, deaminating, EC 1.4.1.4) from the cellulolytic ruminal bacterium Ruminococcus flavefaciens has been purified and characterized. The native enzyme and subunit are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The enzyme requires 0.5 M KCl for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, alpha-ketoglutarate, and glutamate are 19, 0.41, and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The first residue in the N-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified posttranslationally. Comparison of the N-terminal sequence with those of Escherichia coli, Salmonella typhimurium, and Clostridium symbiosum revealed only 39% amino acid homologies. The GDH from R. flavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM).     

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.     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.     

Analysis of conformationally restricted alpha-ketoglutarate analogues as substrates of dehydrogenases and aminotransferases.

Five synthetic, conformationally restricted alpha-ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransferases. The compounds were found not to be detectable substrates of glutamate dehydrogenase, L-leucine dehydrogenase, L-phenylalanine dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartate aminotransferase, alanine aminotransferase, and alpha-ketoglutarate dehydrogenase complex. However, two thermostable aminotransferases were identified that catalyze transamination between several L-amino acids (e.g., phenylalanine, glutamate) and the alpha-ketoglutarate analogues of interest. Transamination between L-glutamate (or L-phenylalanine) and the alpha-ketoglutarate analogues was found to be 0.13 to 1.08 micromol/h/mg at 45 degrees C. The products resulting from transamination between L-phenylalanine and the alpha-ketoglutarate analogues were separated by reverse-phase HPLC, and the newly formed amino acid analogues were analyzed by LC-MS in an ion selective mode. In each case, the ions obtained were consistent with the expected product and a representative example is provided. The possibility existed that although the alpha-ketoglutarate analogues are not substrates of the dehydrogenases and most of the aminotransferases investigated, they might be good inhibitors. Weak inhibition of aminotransferases and glutamate dehydrogenase was found with some of the alpha-ketoglutarate analogues. The newly available thermostable aminotransferases may have general utility in the synthesis of bulky L-amino acids from the corresponding alpha-keto acids.