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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In previous transient state kinetic work from this laboratory, we proposed a new mechanism for the glutamate dehydrogenase-catalyzed oxidative deamination reaction involving an initial replacement of a proton from lysine 126 by a single bound water molecule, followed by closure of the active site cleft and expulsion of bulk water, providing a hydrophobic environment for the ensuing hydride transfer step. Here, we report the results of further transient state fluorescence, absorbance, and kinetic isotope effect studies, which demonstrate the occurrence of an unusual intermediate in the early steps of that reaction. This phenomenon is revealed by an initial fluorescence burst that occurs in the time period where the absorbance signal is still in its lag phase. Using an extension of the proton/product ratio approach we have described earlier, we show that this intermediate is a strongly fluorescent but weakly absorbing species whose absorption maximum is red-shifted beyond that of other known complexes of this enzyme. The transient state kinetic isotope effects of the fluorescence and absorbance signals are compatible only with a reaction scheme in which the formation of the fluorescent complex precedes the hydride transfer step. The optical properties of this enzyme-oxidized coenzyme-substrate intermediate strongly suggest that it is a charge-transfer complex, similar in nature to the complex responsible for the well known "Racker band" reported in 1952 for glyceraldehyde-3-phosphatase dehydrogenase (Racker, E., and Krimsky, I. (1952) Nature 169, 1043-1044). The crystal structure studies of the enzyme-coenzyme and enzyme-L-glutamate complexes of the closely analogous Clostridium symbosium glutamate dehydrogenase, reported by the Sheffield group (Stillman, T. J., Baker, P. J., Britton, K. L., and Rice, D. W. (1993) J. Mol. Biol. 234, 1131-1139), provide a basis for a physical explanation of the phenomenon. We conclude that the charge transfer phenomenon is caused by the near apposition of the unprotonated alpha-amino group of the substrate in a form of the enzyme in which a conformational change has caused the complete closing of the active site cleft.
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PMID:The demonstration of a glutamate dehydrogenase-NADP-L-glutamate charge-transfer complex and its location on the reaction pathway. 796 46

Previous studies have shown the pathogenic effects of grains cultivated in the endemic areas of Keshan disease and selenium is effective in the prevention of this disease. In this study, liver damages induced by feeding grains from an endemic area (endemic diet), and the effects of selenium and alpha-tocopherol supplement were examined. After 3 months on the endemic diet, the amounts of serum enzymes were significantly increased when compared to controls (animals receiving diet from a non-endemic area). Liver enzymes (alkaline phosphatase and choline esterase) were also found to be altered in the serum, further suggesting liver damages in animals on an endemic diet. Supplement of the endemic diet with selenium or alpha-tocopherol reversed the changes in serum enzymes. Increase in lipid peroxidation in the liver of animals on the endemic diet was observed when compared to that in control animals. Selenium and alpha-tocopherol supplements prevented the increase in lipid peroxidation in the liver by the endemic diet. Semi-quantitative histochemical analysis of glutamate dehydrogenase and succinate dehydrogenase in liver tissue showed that the livers of animals on an endemic diet were more sensitive to ischemic damages in vitro. Supplementation of the endemic diet with either selenium or alpha-tocopherol reduced the sensitivity to ischemic damages. The results suggest that increased lipid peroxidation in the liver of rats on an endemic diet may be responsible for liver damages and elevation of serum enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1994 Mar 30
PMID:Effects of selenium and alpha-tocopherol on liver damage induced by feeding grains from an endemic area of Keshan disease in rats. 796 93

The effects of urea, cations (K+,NH4,Na+,Cs+,Li+), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle were analyzed in two anuran amphibians, an estivating species, the spadefoot toad Scaphiopus couchii, and a semi-aquatic species, the leopard frog Rana pipiens. Urea, which accumulates naturally to levels of 200-300 mM during estivation in toads, had only minor effects on the Vmax, kinetic constants and pH curves of PK from either species and no effects on PFK Vmax or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the Vmax of toad PFK and of PK from both species and altered the Km values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the Vmax of PK from either species was reduced by more than 80% by the addition of 200 mM NH4Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the Km values for substrates of toad lactate dehydrogenase and strongly reduced the Vmax of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1994 Feb 09
PMID:Urea and salt effects on enzymes from estivating and non-estivating amphibians. 804 69

Detailed study of the effects of oxygen on the carbohydrate metabolism of Giardia lamblia revealed that low concentrations of oxygen (< 0.25 microM) produced profound alterations in the carbon balance of this organism. Although this concentration of oxygen could not be detected by mass spectrometry, a marked stimulation of ethanol production was observed. Associated with this was an inhibition of alanine production and oxidation of the intracellular NAD(P)H pool. Higher concentrations of oxygen inhibited ethanol production and further reduced levels of alanine. These results suggest that this stimulation is due to changes in carbon flux. Analysis of cell and medium hydrolysates after the growth of trophozoites in [U-14C]glucose suggests that G. lamblia does not synthesise detectable levels of labelled amino acids, except alanine and to a lesser extent valine, from this sugar. Trophozoites of G. lamblia have both glutamate dehydrogenase and alanine aminotransferase activity. As glutamate is taken up from the medium, it is suggested that glutamate dehydrogenase and alanine aminotransferase cooperate to convert pyruvate to alanine, with the concomitant oxidation of NAD(P)H.
Mol Biochem Parasitol 1993 Jan
PMID:The effects of oxygen on fermentation in Giardia lamblia. 809 74

Hepatic serine dehydratase activity was significantly lower in the obese Zucker rats. In both skeletal muscle and kidney adenylate deaminase showed a lower activity in the obese animals. In the small intestine the activity of glutamate dehydrogenase was increased while that of glutamine synthetase was reduced. No changes were found in the enzymatic activities of white adipose tissue while those found in brown adipose tissue were lower for glutamine synthetase. Starvation resulted in increase in liver serine dehydratase in the lean animals and in aspartate transaminase in both lean and obese. Kidney aspartate transaminase and glutamine synthetase were increased with starvation in the lean rats while kidney adenylate deaminase and small intestine glutamine synthetase and branched-chain amino acid transaminase were increased with starvation in the obese animals. In brown adipose tissue starvation caused an increase in branched-chain amino acid transaminase in the lean rats while it significantly lowered the adenylate deaminase and increased branched-chain amino acid transaminase in the obese rats.
Cell Mol Biol (Noisy-le-grand) 1993 Jun
PMID:Amino acid metabolism enzyme activities in the obese Zucker rat. 810 Nov 20

Pathophysiological concentrations of ammonia, both in vivo and in vitro, suppressed the oxidation of glutamate by rat cerebellar mitochondria. The transport of glutamate into mitochondria was either unaltered or enhanced during hyperammonemic states. Activities of mitochondrial enzymes, aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, glutaminase, and GABA-transaminase were suppressed during hyperammonemic states. Suppression of 14CO2 production with (aminooxy)acetic acid but not with glutamic acid diethyl ester indicated that transamination but not oxidative deamination of glutamate plays a major role in glutamate oxidation during normal and hyperammonemic states.
Mol Chem Neuropathol 1993 Aug
PMID:Transport and metabolism of glutamate by rat cerebellar mitochondria during ammonia toxicity. 810 3

We have used gel mobility shift assays to scan 1.7 kb of 5' non-coding sequence of the am (glutamate dehydrogenase) gene of Neurospora crassa for binding by partially fractionated Neurospora proteins. Using genetic analysis this region had been shown to play an important role in the control of glutamate dehydrogenase (GDH) expression. Gel mobility shift analysis identified three regions to which Neurospora proteins bind specifically. Two of these corresponded to the two elements previously defined by genetic analysis (URSam alpha and URSam beta). The third protein binding site appears to be unrelated to am gene expression. Competition experiments showed that the proteins that bind to the URSam alpha and URSam beta elements are different. The URSam alpha element was shown to contain two independent binding sites for the URSam alpha binding protein(s). Both fragments contain a CCAAT motif, suggesting that URSam alpha binding protein(s) may be members of one of the CCAAT-binding protein families. The effect of deletion of either the URSam alpha or URSam beta elements on catabolite induction of am expression was also determined. Both elements appear to act as constitutive enhancers of gene expression.
Mol Gen Genet 1994 Feb
PMID:Sequential gel mobility shift scanning of 5' upstream sequences of the Neurospora crassa am (GDH) gene. 812 95

Highly degenerate oligodeoxyribonucleotides (oligos) were used to PCR amplify the most conserved region of the glutamate dehydrogenase (GDH)-encoding gene from the extreme thermophilic archaeon, Sulfolobus shibatae. The amplified fragment was cloned and sequenced, and then used as a homologous probe to clone a genomic restriction fragment containing the near-complete gdhA gene. The deduced amino acid (aa) sequence shows a very high degree of similarity with the aa sequence previously determined by direct sequencing of the purified enzyme from Sulfolobus solfataricus [Maras et al., Eur. J. Biochem. 203 (1992) 81-87]. The introduction of this new sequence into our GDH phylogenetic trees [Benachenhou-Lahfa et al., J. Mol. Evol. 35 (1993) 335-346] showed that it is a new member of hexameric GDH family II, and did not modify the topology of the trees. Comparison of the primary structures of extremophilic GDH enzymes (halophilic, thermophilic and hyperthermophilic) with those of their non-halophilic and mesophilic counterparts in this family II led us to identify a few aa changes which seem to be specific either to hyperthermophilic or halophilic adaptation.
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PMID:PCR-mediated cloning and sequencing of the gene encoding glutamate dehydrogenase from the archaeon Sulfolobus shibatae: identification of putative amino-acid signatures for extremophilic adaptation. 812 34

We have solved the structure of the binary complex of the glutamate dehydrogenase from Clostridium symbiosum with glutamate to 1.9 A resolution. In this complex, the glutamate side-chain lies in a pocket on the enzyme surface and a key determinant of the enzymic specificity is an interaction of the substrate gamma-carboxyl group with the amino group of Lys89. In the apo-enzyme, Lys113 from the catalytic domain forms an important hydrogen bond to Asn373, in the NAD(+)-binding domain. On glutamate binding, the side-chain of this lysine undergoes a significant movement in order to optimize its hydrogen bonding to the alpha-carboxyl group of the substrate. Despite this shift, the interaction between Lys113 and Asn373 is maintained by a large-scale conformational change that closes the cleft between the two domains. Modelling studies indicate that in this "closed" conformation the C-4 of the nicotinamide ring and the alpha-carbon atom of the amino acid substrate are poised for efficient hydride transfer. Examination of the structure has led to a proposal for the catalytic activity of the enzyme, which involves Asp165 as a general base, and an enzyme-bound water molecule, hydrogen-bonded to an uncharged lysine residue, Lys125, as an attacking nucleophile in the reaction.
J Mol Biol 1993 Dec 20
PMID:Conformational flexibility in glutamate dehydrogenase. Role of water in substrate recognition and catalysis. 826 17

The NADP(+)-dependent hexameric glutamate dehydrogenase from Escherichia coli has been crystallized as the apo-enzyme and also in the presence of its substrates 2-oxoglutarate, glutamate or NADP+, using either pulsed equilibrium microdialysis, or the hanging drop method of vapour diffusion. Three non-isomorphous, but related, crystal forms have been obtained, all of which belong to the orthorhombic system and are most likely to be in space group P2(1)2(1)2(1). One crystal form is grown from ammonium sulphate, includes the apoenzyme and the binary complexes with 2-oxoglutarate or NADP+, and has cell dimensions a = 157.5 A, b = 212.5 A, c = 101.0 A with a hexamer in the asymmetric unit. Crystallizations using glutamate as the precipitant produced two further crystal forms, which show significant changes in the b and c cell dimensions with respect to the apo-enzyme crystals, with parameters a = 160.0 A, b = 217.5 A c = 92.4 A and a = 160.0 A, b = 223.0 A c = 92.4 A, respectively. X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 3.0 A resolution.
J Mol Biol 1993 Dec 20
PMID:Crystallization of the NADP(+)-dependent glutamate dehydrogenase from Escherichia coli. 826 29


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