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

---

Query: EC:1.4.3.11 (glutamate dehydrogenase)
4,437 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Incubation of two types of glutamate dehydrogenase (GDH) isoproteins from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation was partially prevented by preincubation of the GDH isoproteins with 2-oxoglutarate or NADH. Spectrophotometric studies indicated that the inactivation of GDH isoproteins with o-phthalaldehyde resulted in isoindole derivatives characterized by typical fluorescence emission spectra with a stoichiometry of one isoindole derivative per molecule of enzyme subunit. There were no differences between the two GDH isoproteins in sensitivities to inactivation by o-phthalaldehyde indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Tryptic peptides of the isoproteins, modified with and without protection, identified a selective modification of one lysine as in the region containing the sequence L-Q-H-G-S-I-L-G-F-P-X-A-K for both GDH isoproteins. The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as an o-phthalaldehyde-labeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other mammalian GDHs. Also, trypsin was unable to cleave the labeled peptide at this site. Both amino acid sequencing and compositional analysis identified Lys-306 as the site of o-phthalaldehyde binding within the brain GDH isoproteins.
...
PMID:Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde. 1060 7

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.
...
PMID:Regulatory properties of glutamate dehydrogenase from Sulfolobus solfataricus. 1077 43

The extensive use of 13C enrichments in precursor metabolites for flux quantification does not rely on NADPH stoichiometries and can therefore be used to quantify reducing power fluxes. As an application of this concept, the NADPH fluxes were quantified in an L-lysine producer of Corynebacterium glutamicum grown into metabolic and isotopic steady state with [1-13C]glucose. In this case, where the organism's NADPH-dependent glutamate dehydrogenase consumes reducing power, the NADPH flux generated is 210% (molar flux relative to glucose uptake rate) with its major part (72% of the total) generated via the pentose phosphate pathway activity. An isogenic strain in which the glutamate dehydrogenase of C. glutamicum was replaced by the NADH-dependent glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was made and the metabolite fluxes were again estimated. The major response to this local perturbation is a drastically reduced NADPH generation of only 139%. Most of the NADPH (62% of the total) is now generated via the tricarboxylic acid cycle activity. This shows the extraordinary flexibility of the central metabolism and provides a picture of the global regulatory properties of the central metabolism. Furthermore, a detailed analysis of the fluxes and exchange fluxes within the anaplerotic reactions is given. It is hypothesized that these reactions might also serve to balance the total reducing power budget as well as the energy budget within the cell.
...
PMID:Response of the central metabolism in Corynebacterium glutamicum to the use of an NADH-dependent glutamate dehydrogenase. 1093 53

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine epsilon-aminotransferase. This enzyme catalyzed the oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required alpha-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine epsilon-amino transferase and glutamate oxidase, respectively. L-lysine epsilon-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine epsilon-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine epsilon-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine epsilon-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65-70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine epsilon-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine epsilon-aminotransferase. N-alpha-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine epsilon-aminotransferase.
...
PMID:Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N(2)- 1093 17

By reaction of adenosine 5'-monothiophosphate with benzophenone-4-maleimide, we synthesized adenosine 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (AMPS-Succ-BP) as a photoreactive ADP analogue. Bovine liver glutamate dehydrogenase is known to be allosterically activated by ADP, but the ADP site has not been located in the crystal structure of the hexameric enzyme [Peterson, P. E., and Smith, T. J. (1999) Structure 7, 769-782]. In the dark, AMPS-Succ-BP reversibly activates GDH. Irradiation of the complex of glutamate dehydrogenase and AMPS-Succ-BP at lambda >300 nm causes a time-dependent, irreversible 2-fold activation of the enzyme. The k(obs) for photoactivation shows nonlinear dependence on the concentration of AMPS-Succ-BP, with K(R) = 4.9 microM and k(max) = 0.076 min(-)(1). The k(obs) for photoreaction by 20 microM AMPS-Succ-BP is decreased 10-fold by 200 microM ADP, but is reduced less than 2-fold by NAD, NADH, GTP, or alpha-ketoglutarate. Modified enzyme is no longer activated by ADP, but is still inhibited by GTP and high concentrations of NADH. These results indicate that reaction of AMPS-Succ-BP occurs within the ADP site. The enzyme incorporates up to 0.5 mol of [(3)H]AMPS-Succ-BP/mol of enzyme subunit or 3 mol of reagent/mol of hexamer. The peptide Lys(488)-Glu(495) has been identified as the only reaction target, and the data suggest that Arg(491) is the modified amino acid. Arg(491) (in the C-terminal helix close to the GTP #2 binding domain of GDH) is thus considered to be at or near the enzyme's allosteric ADP site. On the basis of these results, the AMPS-Succ-BP was positioned within the crystal structure of glutamate dehydrogenase, where it should also mark the ADP binding site of the enzyme.
...
PMID:Adenosine 5'-0-[S-(4-succinimidyl-benzophenone)thiophosphate]: a new photoaffinity label of the allosteric ADP site of bovine liver glutamate dehydrogenase. 1132 16

Biocatalytic processes were used to prepare chiral intermediates required for the synthesis of Omapatrilat 1 by three different routes. The synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 3 to L-6-hydroxynorleucine 2 was demonstrated by reductive amination using beef liver glutamate dehydrogenase. To avoid the lengthy chemical synthesis of the ketoacid 3, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 4] with D-amino acid oxidase from porcine kidney or Trigonopsis variabilis followed by reductive amination to convert the mixture completely to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess (e.e.). The enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 5) was demonstrated using phenylalanine dehydrogenase (PDH) from T. intermedius. Phenylalanine dehydrogenase was cloned and overexpressed in Escherichia coli and Pichia pastoris. Using PDH from E. coli or P. pastoris, the enzymatic process was scale-up to prepare kg quantity of allysine ethylene acetal 5. The reaction yields of >94% and e.e. of >98% were obtained for allysine ethylene acetal 5. An enzymatic process was developed for the synthesis of [4S-(4a,7a,10ab)]1-octahydro-5-oxo-4 [[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid [BMS-199541-01]. The enzymatic oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[[(phenyl-methoxy)carbonyl] L-homocysteinyl] L-lysine)-1,1-disulphide [BMS-201391-01] to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase (LAT) from Sphingomonas paucimobilis SC 16113 was demonstrated. This enzyme was overexpressed in E. coli and a process was developed using the recombinant enzyme.
...
PMID:Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug. 1133 76

It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5'-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.
...
PMID:Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase. An essential residue in catalysis. 1138 22

It has been reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in glutamate dehydrogenase (GDH) gene that affects enzyme sensitivity to GTP-induced inhibition. To identify the GTP binding site(s) within human GDH, mutant GDHs at Tyr-266 or Lys-450 position were constructed by cassette mutagenesis. More than 90% of the initial activities were remained at the concentration of GTP up to 300 microm for the Lys-450 mutant GDHs regardless of their size, hydrophobicity, and ionization of the side chains, whereas the wild type GDH and the Tyr-266 mutant GDHs were completely inhibited by 30 microm GTP. The binding of GTP to the wild type GDH or the mutant GDHs was further examined by photoaffinity labeling with 8-[gamma-(32)P]azidoguanosine 5'-triphosphate (8-N(3)-GTP). Saturation of photoinsertion with 8-N(3)-GTP occurred apparent K(d) values near 20 microm for the wild type GDH or the Tyr-266 mutant GDH, and the photoinsertion of 8-N(3)-[gamma-(32)P]GTP was significantly decreased in the presence of 300 microm GTP. Unlike the wild type GDH or the Tyr-266 mutant GDH, less than 10% of photoinsertion was detected in the Lys-450 mutant GDH, and the photoinsertion was not affected by the presence of 300 microm GTP. The results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the GTP binding site and suggest that Lys-450, but not Tyr-266, is required for efficient binding of GTP to GDH. Interestingly, studies of the steady-state velocity showed that both the wild type GDH and the Tyr-266 mutant GDHs were inhibited by ATP at concentrations between 10 and 100 microm, whereas less than 10% of the initial activities of the Lys-450 mutant GDHs were diminished by ATP. These results indicate that Lys-450, but not Tyr-266, may be also responsible for the ATP inhibition; therefore, ATP bound to the GTP site.
...
PMID:Identification of the GTP binding site of human glutamate dehydrogenase by cassette mutagenesis and photoaffinity labeling. 1160 May 2

A chemiluminometric flow-through sensor for simultaneous determination of L-glutamate (Glu) and L-lysine (Lys) in a single sample has been developed. Immobilized uricase, immobilized peroxidase, support material, coimmobilized glutamate oxidase/peroxidase, support material, and coimmobilized lysine oxidase/peroxidase were packed sequentially in a transparent PTFE tube, and the tube was placed in front of a photomultiplier tube as a flow cell. A three-peak recording was obtained by one injection of the sample solution. The peak height of the first peak was due to the concentrations of urate and other reductants in the sample; the immobilized uricase was used to decompose urate, and the hydrogen peroxide produced was decomposed with a luminol-hydrogen peroxide reaction by immobilized peroxidase. The peak heights of the second and third peaks were free from the interferences from the reductants and were dependent only on the concentrations of Glu and Lys, respectively. Calibration graphs for Glu and Lys were linear at 40-1,000 and 50-1,200 nM, respectively. The sampling rate was 11/h without carryover. The sensor was stable for two weeks. The sensor system was applied to the simultaneous determination of Glu and Lys in serum.
...
PMID:Chemiluminometric sensor for simultaneous determination of L-glutamate and L-lysine with immobilized oxidases in a flow injection system. 1192 93

Biocatalytic processes were used to prepare chiral intermediates for pharmaceuticals. These include the following processes. Enzymatic synthesis of [4S-(4a,7a,10ab)]1-octahydro-5-oxo-4-[[(phenylmethoxy) carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01), a key chiral intermediate for synthesis of a new vasopeptidase inhibitor. Enzymatic oxidation of the epsilon-amino group of lysine in dipeptide dimer N2-[N[[(phenylmethoxy)carbonyl] L-homocysteinyl] L-lysine)1,1-disulfide (BMS-201391-01) to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase from S. paucimobilis SC16113 was demonstrated. This enzyme was overexpressed in E. coli, and a process was developed using recombinant enzyme. The aminotransferase reaction required alpha-ketoglutarate as the amine acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from S. noursei SC6007. Synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 5 to L-6-hydroxy norleucine 4 was demonstrated by reductive amination using beef liver glutamate dehydrogenase. To avoid the lengthy chemical synthesis of ketoacid 5, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine (readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin, 6) with D-amino acid oxidase from porcine kidney or T. variabilis followed by reductive amination to convert the mixture to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess. Enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 7), one of three building blocks used for synthesis of a vasopeptidase inhibitor, was demonstrated using phenylalanine dehydrogenase from T. intermedius. The reaction requires ammonia and NADH. NAD produced during the reaction was recycled to NADH by oxidation of formate to CO2 using formate dehydrogenase. Efficient synthesis of chiral intermediates required for total chemical synthesis of a beta 3 receptor agonist was demonstrated. These include: (a) microbial reduction of 4-benzyloxy-3-methanesulfonylamino-2'-bromoacetophenone 9 to corresponding (R)-alcohol 10 by S. paucimobilis SC16113, (b) enzymatic resolution of racemic alpha-methyl phenylalanine amide 11 and alpha-methyl-4-hydroxyphenylalanine amide 13 by amidase from M. neoaurum ATCC 25795 to prepare corresponding (S)-amino acids 12 and 14, and (c) asymmetric hydrolysis of methyl-(4-methoxyphenyl)-propanedioic acid ethyl diester 15 to corresponding (S)-monoester 16 by pig liver esterase. (S)[1-(acetoxyl)-4-(3-phenyl)butyl]phosphonic acid diethyl ester 21, a key chiral intermediate required for total chemical synthesis of BMS-188494 (an anticholesterol drug) was prepared by stereoselective acetylation of racemic [1-(hydroxy)-4-(3-phenyl)butyl]phosphonic acid diethyl ester 22 using G. candidum lipase. Lipase-catalyzed stereoselective acetylation of racemic 7-[N,N'-bis-(benzyloxy-carbonyl)N-(guanidinoheptanoyl)]-alpha-hydroxy-glycine 24 to corresponding S-(-)-acetate 25 was demonstrated. S-(-)-acetate 25 is a key intermediate for total chemical synthesis of (-)-15-deoxyspergualin 23, an immunosuppressive agent and antitumor antibiotic. Stereoselective microbial reduction of (1S)[3-chloro-2-oxo-1-(phenyl-methyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester 26 to corresponding chiral alcohol 27a (a key chiral intermediate for HIV protease inhibitors) was also demonstrated. Stereospecific enzymatic hydrolysis of racemic epoxide RS-1-[2',3'-dihydro benzo[b]furan-4'-yl]-1,2-oxirane 29 the corresponding R-diol 30 and unreacted chiral S-epoxide 28 was demonstrated using R. glutinis and A. niger. Dynamic resolution of racemic diol RS-1-[2',3'-dihydrobenzo[b]furan-4'-yl]-ethane-1,2-diol 32 to corresponding S-diol S-1-[2',3'-dihydrobenzo[b]furan-4'-yl]-ethane-1,2-diol 31 was demonstrated using C. boidinii and P. methanolica. Chiral (S)-epoxide 28 and (S)-diol 31 are key intermediates for a new prospective circadian modulator drug. Enzymatic resolution of racemic 2-pentanol and 2-heptanol by lipase B from Candida antarctica was demonstrated. S-(+)-2-pentanol is a key chiral intermediate required for synthesis of anti-Alzheimer's drugs.
...
PMID:Microbial/enzymatic synthesis of chiral drug intermediates. 1287 94


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---