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)

The triple mutant K89L/A163G/S380A (inactive with glutamate but active with L-Nle and L-Met) and C320S (fully active with glutamate, entirely inactive with L-Nle and L-Met, and also lacking reactive cysteine) mutant of glutamate dehydrogenase (EC 1.4.1.2) of Clostridium symbiosum could be completely denatured by urea with the loss of structure and activity. The mutants denatured by urea could be reassociated to give stable hexamers with recovery of activity of approximately 67% by dilution in 0.1 M potassium phosphate buffer (pH 7.0) containing 2 mM NAD+. The native, urea-denatured, and renatured states of mutant enzymes were characterized by size exclusion chromatography on FPLC and native PAGE. Intersubunit hybrid hexamers containing five subunits of triple mutant and one subunit of C320S mutant were constructed by in vitro subunit hybridization followed by affinity chromatography. Kinetic analysis showed that a 5:1 hybrid hexamer, with only one C320S subunit able to bind NAD+ after DTNB modification, shows classical Michaelis-Menten kinetics with regard to NAD+. This contrasts with the apparent negative co-operativity shown by pure C320S hexamers and suggests that the interaction in NAD+ binding among subunits is eliminated in the hybrid. After removal of thionitrobenzoate, however, all of the subunits in the hybrid are able to bind NAD+. In this state the hybrid enzyme showed slight deviation from classical behavior with regard to NAD+, indicating reintroduction of some level of allosteric interaction. The hybrid hexamer also showed much reduced co-operativity with glutamate at pH 8.8, with a Hill coefficient of 3 for DTNB-treated hybrid (as compared to 5.2 for the pure C320S mutant) and 2.2 for the untreated hybrid. The fact that co-operativity in glutamate binding is not entirely eliminated correlates with evidence that the triple mutant subunits, though inactive toward glutamate, can nevertheless still bind this amino acid.
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PMID:Intersubunit communication in hybrid hexamers of K89L/A163G/S380A and C320S mutants of glutamate dehydrogenase from Clostridium symbiosum. 939 25

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.
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PMID:Reactive cysteine residue of bovine brain glutamate dehydrogenase isoproteins. 1010 78

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.
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PMID:Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. 1033 16

Many halogenated foreign compounds are detoxified by conversion to the corresponding cysteine S-conjugate, which is N-acetylated and excreted. However, several halogenated cysteine S-conjugates [e.g. S-(1,1,2,2-tetrafluoroethy)-L-cysteine (TFEC)] are converted to mitochondrial toxicants by cysteine S-conjugate beta-lyases. In the present work, we showed that TFEC appreciably inactivated highly purified alpha-ketoglutarate dehydrogenase complex (KGDHC) in the presence of a cysteine S-conjugate beta-lyase. Incubation of PC12 cells (which contain endogenous cysteine S-conjugate beta-lyase activity) with TFEC led to a concentration- and time-dependent loss of endogenous KGDHC activity. A 24-hr exposure to 1 mM TFEC decreased KGDHC activity in the cells by 90%. Although treatment with TFEC did not inhibit intrinsic pyruvate dehydrogenase complex (PDHC) activity, it inhibited dichloroacetate/Mg2+-mediated activation/dephosphorylation of PDHC in the PC12 cells by 90%. To determine the selectivity of enzymes targeted by TFEC, several cytosolic and mitochondrial enzymes involved in energy metabolism [malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, cytosolic and mitochondrial aspartate aminotransferases (AspAT)] were also assayed in the PC12 cells exposed to 1 mM TFEC for 24 hr. Of these enzymes, only mitochondrial AspAT, a key enzyme of the malate-aspartate shuttle, was inhibited. The present results demonstrate a selective vulnerability of mitochondrial enzymes to toxic cysteine S-conjugates. The data indicate that TFEC may be a useful cellular/mitochondrial toxicant for elucidating the consequences of the diminished mitochondrial function that accompanies numerous neurodegenerative diseases.
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PMID:Inhibition of select mitochondrial enzymes in PC12 cells exposed to S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. 1053 46

The purified glutamate dehydrogenase (GDH) from Sulfolobus solfataricus showed remarkable thermostability and retained 90-95% of the initial activity after incubation at -20 degrees C, 4 degrees C, and 25 degrees C for up to 6 months. Unlike mammalian GDHs, the activity of GDH from Sulfolobus solfataricus was not significantly affected by the presence of various allosteric effectors such as ADP, GTP, and leucine. Incubation of GDH with increasing concentration of o-phthalaldehyde resulted in a progressive decrease in enzyme activity, suggesting that the o-phthalaldehyde-modified lysine or cysteine is directly involved in catalysis. The inhibition was competitive with respect to both 2-oxoglutarate (Ki = 30 microM) and NADH (Ki = 100 microM), further supporting a possibility that the o-phthalaldehyde-modified residues may be directly involved at the catalytic site. The modification of GDH by the arginine-specific dicarbonyl reagent phenylglyoxal was also examined with the view that arginine residues might play a general role in the binding of coenzyme throughout the family of pyridine nucleotide-dependent dehydrogenases. The purified GDH was inactivated in a dose-dependent manner by phenylglyoxal. Either NADH or 2-oxoglutarate did not gave any protection against the inactivation caused by a phenylglyoxal. This result indicates that GDH saturated with NADH or 2-oxoglutarate is still open to attack by phenylglyoxal. Phenylglyoxal was an uncompetitive inhibitor (Ki = 5 microM) with respect to 2-oxoglutarate and a noncompetitive inhibitor (Ki = 6 microM) with respect to NADH. The above results suggests that the phenylglyoxal-modified arginine residues are not located at the catalytic site and the inactivation of GDH by phenylglyoxal might be due to a steric hindrance or a conformational change affected by the interaction of the enzyme with its inhibitor.
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PMID:Regulatory properties of glutamate dehydrogenase from Sulfolobus solfataricus. 1077 43

In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both glutamate and NAD(+) but is catalytically inactive. The mutant proteins were denatured separately in 4 M urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mM NAD(+), 1 M fluoride and artificial chaperones (4 mM polyoxyethylene 10 lauryl ether and 1.6 mM beta-cyclodextrin). Under these conditions approximately 50% refolding was achieved for both mutant proteins separately. The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion-exchange chromatography. Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), which binds close to the NAD(+) binding site, thus abolishing coenzyme binding in the wild-type enzyme, also reacts with D165S but has no effect on C320S. Modification by DTNB was coupled with dye-ligand affinity chromatography on a Procion Red HE-3B column in order to separate the hybrid mixture into fractions of defined composition. An optimized procedure based on salt gradient elution was developed. DTNB-modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis-Menten kinetics when the NAD(+) concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that 'nonlinear' behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites. When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h = 3.0) compared with native C320S with six active subunits (h = 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely. Catalytically silent subunits can thus still contribute to cooperativity.
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PMID:Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S. 1123 Dec 68

Mitochondrial ADP-ribosylation leads to modification of two proteins of approximately 26 and 53 kDA: The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine-specific ADP-ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [14C]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP-ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD+ onto the acceptor site. ADP- ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP-ribosylation.
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PMID:Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria. 1135 Sep 29

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD(+)-agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40 mM glutamate and 1 mM NAD(+) at pH 8.8, or K89L/A163G/S380A with 70 mM norleucine and 1 mM NAD(+) at pH 8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC(50)=70 mM, h=2.4), and glutarate gave approx. 30-fold activation (EC(50)=35 mM, h=2.3). For the triple mutant, corresponding values were 80 mM and 2.2 for succinate, and 75 mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH 8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The "foreign" subunit evidently impedes inter-subunit communication to some extent.
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PMID:Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity. 1173 56

The review considers redox enzymes of Plasmodium spp., Trypanosomatida, Trichomonas, Entamoeba and Giardia, with special emphasis on their potential use as targets for drug development. Thiol-based redox systems play pivotal roles in the success and survival of these parasitic protozoa. The synthesis of cysteine, the key molecule of any thiol metabolism, has been elucidated in trypanosomatids and anaerobes. In trypanosomatids, trypanothione replaces the more common glutathione system. The enzymes of trypanothione synthesis have recently been identified. The role of trypanothione in the detoxification of reactive oxygen species is reflected in the multiplicity of trypanothione-dependent peroxidases. In Plasmodium falciparum, the crystal structures of glutathione reductase and glutamate dehydrogenase are now available; another drug target, thioredoxin reductase, has been demonstrated to be essential for the malarial parasite.
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PMID:Thiol-based redox metabolism of protozoan parasites. 1474 17

The interactions between sulphur nutrition and Cd exposure were investigated in maize (Zea mays L.) plants. Plants were grown for 12 days in nutrient solution with or without sulphate. Half of the plants of each treatment were then supplied with 100 microM Cd. Leaves were collected 0, 1, 2, 3, 4 and 5 days from the beginning of Cd application and used for chemical analysis and enzyme assays. Cd exposure produced symptoms of toxicity (leaf chlorosis, growth reduction) and induced a noticeable accumulation of non-protein SH compounds. As phytochelatins are glutamate- and cysteine-rich peptides, the effect of cadmium on some enzyme activities involved in N and S metabolism of maize leaves was studied in relation to the plant sulphur supply. In vivo Cd application to S-sufficient plants resulted in a drop of all measured enzyme activities. On the other hand, S-deficient plants showed a decrease in nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2) activity, and an increase in NAD-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) and phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) activity as a result of the Cd treatment. Furthermore, in the same plants ATP sulphurylase (ATPs; EC 2.7.7.4) and O-acetylserine sulphydrylase (OASs; EC 4.2.99.8) showed a particular pattern as both enzymes exhibited a transient maximum value of activity after 4 days from the beginning of Cd exposure. Results provide evidence that the increase of ATPs, OASs, GDH and PEPc activities, observed exclusively in S-deficient Cd-treated plants, may be part of the defence mechanism based on the production of phytochelatins.
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PMID:Role of sulphur availability on cadmium-induced changes of nitrogen and sulphur metabolism in maize (Zea mays L.) leaves. 1531 68


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