Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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

Stopped flow studies of D2O kinetic solvent isotope effects on the reaction catalyzed by L-glutamate dehydrogenase reveal, in addition to several effects apparently attributable simply to pKa shifts, a 2-fold pH-independent effect on the velocity of the steady state oxidative deamination of L-glutamate by enzyme and NADP. Comparable pH-independent D2O kinetic solvent isotope effects are seen both in a transient phase of the reaction in which alpha-ketoglutarate is displaced by L-glutamate from an enzyme-NADPH-alpha-ketoglutarate (product) complex and in an analogous model reaction in which alpha-ketoglutarate is displaced by D-glutamate. These results suggest that alpha-ketoglutarate dissociation from an enzyme-NADPH-alpha-ketoglutarate complex is rate-limiting in the steady state.
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PMID:Location of deuterium oxide solvent isotope effects in the glutamate dehydrogenase reaction. 23 82

Optical characteristics of enzyme-reduced coenzyme complexes of yeast NADP-specific glutamate dehydrogenase have been investigated in the presence and absence of product (L-glutamate) and in the presence or absence of phosphate. The phosphate effect, pointed out in a previous work, is found again: inorganic phosphate (Pi) destabilizes the binary complex (E - NADPH), the dissociation constant of which is equal to 14 muM, a value much higher than that determined in Tris-HCl buffer: Kd = 0.9 muM. Concerning the role of phosphate some assumptions are drawn up with respect to a similar behaviour of Pi toward yeast glutamate dehydrogenase and ADP toward the beef liver enzyme. In the same way, L-glutamate induces a stabilization of the binary complex; this latter effect is unchanged in the presence of phosphate, yet it is less marked than in the case of beef liver glutamate dehydrogenase. Protein fluorescence, nucleotide fluorescence and circular dichroism measurements allowed the determination of three identical and independent NADPH binding sites per hexameric active unit. In analogy with beef liver enzyme, it seems that yeast glutamate dehydrogenase is a good model to study anticooperativity in ligand binding.
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PMID:Binding studies of NADPH to NADP-specific L-glutamate dehydrogenase from Saccharomyces cerevisiae. 24 Jul 22

The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values.
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PMID:The effects of methanol on the glutamate dehydrogenase reaction at 0 degrees C. 26 5

Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic.Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of alpha-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of amino acid sources for parasite protein synthesis; purification and characterization of plasmodial proteinases; and in vitro translation of parasite messenger RNA.
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PMID:Amino acid metabolism and protein synthesis in malarial parasites. 33 83

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.
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PMID:Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate. The steady state. 51 77

Stopped-flow studies of the initial burst of NADPH production accompanying the oxidative deamination of L-glutamate by L-glutamate dehydrogenase and NADP+ were performed in the presence of alpha-ketoglutarate, a product of the reaction. Both binary enzyme-alpha-ketoglutarate and ternary enzyme--NADP+-alpha-ketoglutarate complexes are inhibitory in the burst presence of the enzyme-catalyzed reaction. Order-of-addition experiments show the binary complex to form rapidly, in the 3 ms dead time of the stopped-flow instrument. There is a distinct lag, however, in the achievement of the full ternary complex inhibitory effect unless the enzyme is preincubated with both NADP+ and alpha-ketoglutarate prior to initiation of the catalytic reaction with L-glutamate. The formation of an inhibitory enzyme--NADP+-alpha-ketoglutarate complex appears to be sufficiently slow to give a delayed kinetic response when alpha-ketoglutarate is added to the reaction system.
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PMID:Transient-state kinetics of L-glutamate dehydrogenase: mechanism of alpha-ketoglutarate inhibition in the burst phase. 56 27

In a detailed study focused on the methodological problems in dehydrogenase histochemistry [e.g., fixation, diffusion of enzymes and of reduced inermediates, conversion of NADPH and NADP to NADH and NAD, respectively, penetration of tetrazolium salt and formazan substantivity, 'nothing dehydrogenase' reaction, use of exogenous CoQ10 and of flavoprotein substitute (PMS)], the distribution and activity of succinate dehydrogenase, NAD(P)H-tetrazolium reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase (H and M types), and of L-glutamate dehydrogenase (E.C.1.4.1.2 and E.C.1.4.1.3) have been investigated in the rat cerebellum. It was evident from the study that reliable results could only be obtained if all the aforementioned factors had been considered. The image of actual concentration of SDH in the neuropil of the molecular layer could only be recorded by adding CoQ10, while other structures exhibited greater balance between SDH and endogenous mitochondrial CoQ. Contrary to previous studies, a reversed localization of the activity of G-6-PDH and LDH was noticed. The elements of molecular and Purkinje layers were rich in G-6-PDH, while the granular layer was nearly depleted. The actual level of LDH could only be recorded if NADH-tetrazolium reductase was bypassed with PMS. The H and M types of LDH coexisted in the three cortical layers, the H type being prevalent and the M type attaining its highest level in synaptic glomeruli followed by the structures of the molecular layer and the Purkinje cells. High activity of GDH was noticed in Bergmann glia followed by synaptic glomeruli, while most other structures showed weak to moderate activity. The two GDH types coexisted in all structures showing activity, except for Bergmann cells, which only showed presence of the E.C. 1.4.1.3 type. Furthermore, Bergmann glia was exceptional by showing no activity of SDH and LDH, but strong activity of G-6-PDH and NADPH-tetrazolium reductase. The granular cells were exceptional by showing weak or no activity of all enzymes in question.
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PMID:Methodological aspects of the histochemical localization and activity of some cerebellar dehydrogenases. 66 87

Genetic manipulation of nitrogenase and key glutamate-forming enzymes can provide mutants that excrete fixed N2 as NH4+. A derepressed N2 fxation mutant (SK-24) has been isolated , which excretes up to 20.2 mumol of fixed N2 as NH4+ per mg of cell protein in 24 hr at room temperature. Biochemical analysis shows that this mutant, which requires glutamate for growth, releases fixed N2 as NH4+ into the environment because of (i) constitutive synthesis of nitrogenase and (ii) genetic blocks resulting in losses of glutamate synthase [L-glutamine:2-oxoglutarate aminotransferase (NADPH oxidizing), EC 2.6.1.53] and glutamate dehydrogenase [L-glutamate:NADP oxidoreductase (deaminating), EC 1.4.1.4] activities, enzymes essential for NH4+ assimilation into cell material. The parent strain (asm-1), missing only glutamate synthase activity, also actively excretes NH4+ during early phases of its growth but eventually reutilizes the NN4+. A miximum yield of 4.0 mumol of NH4+/ml per 24 hr has been noted for asm-1 only during the growth period. Biosynthesis of NH4+ PROCEEDS AT THE EXPENSE OF A Variety of fermentable sugars, such as sucrose or glucose, with a maximum energy conversion efficiency of about 5 glucose degraded per NH4+ formed. The use of microbes for production of NH4+ fertilizer is discussed.
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PMID:Microbial production of ammonium ion from nitrogen. 109 Sep 30

Wild-type glutamate dehydrogenase (EC 1.4.1.4) from Salmonella typhimurium reacts at 25 degrees C in 0.1 M phosphate buffer, pH 7, with the nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 2',5'-bisphosphate (2-BDB-TA 2',5'-DP) to give 78% inactivation. Protection against inactivation was achieved with NADPH, indicating that modification occurred in the region of the coenzyme binding site. After reaction of the enzyme with 2-BDB-TA 2',5'-DP, the dioxo moiety of the bound reagent was reduced with [3H]NaBH4. The radioactive peptide which corresponds to the sequence Leu282-Cys283-Glu284-Ile285-Lys286 was isolated by HPLC from tryptic digests of inactive modified enzyme but was absent in digests of active enzyme modified in the presence of NADPH. Mutant enzyme E284Q was 64% inactived by 2-BDB-TA 2',5'-DP and modification of the corresponding Leu282-Lys286 peptide was found, while neither mutant enzyme C283I nor C283I:E284Q was inactivated by the nucleotide analogue and no corresponding radioactive peptides were found. These results show that cysteine-283 is the target of the reagent and is located near the coenzyme binding site. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A 2',5'-DP) has also been shown to react with cysteine-283 (L. Haeffner-Gormley et al., 1991, J. Biol. Chem. 266, 5388-5394). However, the predominant form of the Leu282-Lys286 peptide after reaction with 2-BDB-TA 2',5'-DP contained only 0.17 mol tritium/mol leucine, whereas the 2-BDB-T epsilon A 2',5'-DP-modified peptide contained 1.80 mol tritium/mol leucine; these results indicate that the reaction product of 2-BDB-T epsilon A 2',5'-DP retains two reducible carbonyl groups while these are not available in the product of 2-BDB-TA 2',5'-DP. It is suggested that cysteine-283 reacts primarily at a carbonyl group of 2-BDB-TA 2',5'-DP to form a thiohemiacetal derivative, while it reacts at the methylene group of 2-BDB-T epsilon A 2',5'-DP with displacement of bromide. Both nucleotide analogues also yielded, in small amount, a crosslinked peptide containing the sequences 282-286 and 299-333, indicating proximity between these regions in the native structure.
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PMID:Reaction of the nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate at the coenzyme site of wild-type and mutant NADP(+)-specific glutamate dehydrogenases from Salmonella typhimurium. 130 91

Glutamate, glutamine, and ammonia pool size have been determined in two S. cerevisiae strains (GOGAT+ and GOGAT-) growing under ammonia excess and limitation at a dilution rate of 0.10/h. The biomass levels and glutamate dehydrogenase NADPH-dependent (NADPH-GDH) activities were also measured for both strains. The strain that lacks GOGAT activity showed lower levels of metabolites under both media and lower levels of biomass under carbon limitation (ammonia excess) compared to the GOGAT+ strain. Under nitrogen limitation, the biomass level was the same for both strains, but GOGAT- changed from rounded to ellipsoidal cells.
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PMID:Ammonia assimilation in S. cerevisiae under chemostatic growth. 132 53


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