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)

3-Phenylpyruvate evoked a monophasic insulin release from perifused mouse islets. L-Phenylalanine was not an insulin secretagogue and was oxidized by islets at a very low rate, suggesting that 3-phenylpyruvate does not trigger insulin release by enhancing production of reducing equivalents. Moreover, allosteric activation of glutamate dehydrogenase does not play a role in 3-phenylpyruvate-induced insulin secretion.
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PMID:Mechanism of 3-phenylpyruvate-induced insulin release from isolated pancreatic islets. 703 29

This review is an exhaustive description of the biochemistry and enzymology of all 17 known NAD(P)(+)-amino acid dehydrogenases. These enzymes catalyze the oxidative deamination of an amino acid to its keto acid and ammonia, with the concomitant reduction of either NAD+ or NADP+. These enzymes have many important applications in industrial and medical settings and have been the object of prodigious enzymological research. This article describes all that is known about the poorly characterized members of the family and contains detailed information on the better characterized enzymes, including valine, phenylalanine, leucine, alanine, and glutamate dehydrogenases. The latter three enzymes have been the subject of extensive enzymological experimentation, and, consequently, their chemical mechanisms are discussed. The three-dimensional structure of the Clostridium symbiosum glutamate dehydrogenase has been determined recently and remains the only structure known of any amino acid dehydrogenase. The three-dimensional structure and its implications to the chemical mechanisms and rate-limiting steps of the amino acid dehydrogenase family are discussed.
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PMID:The biochemistry and enzymology of amino acid dehydrogenases. 770 1

The hyperthermophilic archaeon (formerly archaebacterium) Thermococcus litoralis grows at temperatures up to 98 degrees C using peptides and proteins as the sole sources of carbon and nitrogen. Cell-free extracts of the organism contained two distinct types of aromatic aminotransferases (EC 2.6.1.57) which were separated and purified to electrophoretic homogeneity. Both enzymes are homodimers with subunit masses of approximately 47 kDa and 45 kDa. Using 2-oxoglutarate as the amino acceptor, each catalyzed the pyridoxal-5'-phosphate-dependent transamination of the three aromatic amino acids but showed virtually no activity towards aspartic acid, alanine, valine or isoleucine. From the determination of Km and kcat values using 2-oxoglutarate, phenylalanine, tyrosine and tryptophan as substrates, both enzymes were shown to be highly efficient at transaminating phenylalanine (kcat/Km approximately 400 s-1 mM-1); the 47-kDa enzyme showed more activity towards tyrosine and tryptophan compared to the 45-kDa one. Kinetic analyses indicated a two-step mechanism with a pyridoxamine intermediate. Both enzymes were virtually inactive at 30 degrees C and exhibited maximal activity between 95-100 degrees C. They showed no N-terminal sequence similarity with each other (approximately 30 residues), nor with the complete amino acid sequences of aromatic aminotransferases from Escherichia coli and rat liver. The catalytic properties of the two enzymes are distinct from bacterial aminotransferases, which have broad substrate specificities, but are analogous to two aromatic aminotransferases which play a biosynthetic role in a methanogenic archaeon. In contrast, it is proposed that one or both play a catabolic role in proteolytic T. litoralis in which they generate glutamate and an arylpyruvate. These serve as substrates for glutamate dehydrogenase and indolepyruvate ferredoxin oxidoreductase in a novel pathway for the utilization of aromatic amino acids.
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PMID:Characterization of aromatic aminotransferases from the hyperthermophilic archaeon Thermococcus litoralis. 812 13

We have analysed the sequence homology between glutamate, leucine and phenylalanine dehydrogenases in the light of the solution of the structure of the glutamate dehydrogenase from Clostridium symbiosum. This analysis indicates that the elements of secondary structure comprising the core of the two domains in glutamate dehydrogenase are conserved in the other two enzymes. There is a striking conservation of the residues responsible for the recognition of the nicotinamide ring of the nucleotide cofactor and the backbone of the amino acid substrates. Furthermore, residues involved in a major conformational rearrangement on amino acid binding are preserved, as are those implicated in the catalytic chemistry. In contrast, the pattern of insertions/deletions between these enzymes is consistent with possible differences in quaternary structure. Differential substrate specificity between these enzymes is achieved by critical substitutions at the base of the binding pocket, which accommodates the side-chain of the amino acid substrate. This provides insights into the mutations necessary to produce new catalysts for the chiral synthesis of novel amino acids.
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PMID:Evolution of substrate diversity in the superfamily of amino acid dehydrogenases. Prospects for rational chiral synthesis. 826 39

To date, no attempt has been made to study alterations occurring in the amino acid profile in chronic models of thioacetamide-induced liver cirrhosis. In this work, changes in serum amino acids and proteins in rats with thioacetamide-induced liver cirrhosis are reported, together with changes in enzyme activities in the liver and serum. Seventeen female Wistar rats were used. Eight rats were given 300 mg thioacetamide/l in drinking water for 4 months and nine rats were given water ad libitum during the same time-period. Significant increases in glycine, alanine, serine, methionine, glutamate, ornithine, phenylalanine, tyrosine, histidine and proline were observed in rats with the resulting experimental liver cirrhosis. Threonine, taurine, glutamine, lysine and citrulline tended to increase while isoleucine, leucine, aspartate, arginine and tryptophan tended to decrease. Total and nonessential amino acids increased significantly in cirrhotic animals. Total essential and aromatic amino acids tended to increase in the thioacetamide-treated group, whereas branched chain amino acids tended to decrease in the same group. Regarding serum proteins, a decrease in albumin concentration in the thioacetamide-treated animals was the only change detected. The liver enzyme activities under observation (aspartate and alanine aminotransferases, glutamate dehydrogenase and threonine deaminase) were lower in the thioacetamide group. Decreases were significant for both transaminases and threonine deaminase. Results for serum activities showed that transaminases did not change in thioacetamide-treated rats in comparison with controls. In contrast, alkaline phosphatase rose dramatically in cirrhotic rats. We conclude that the serum amino acid pattern in this chronic model of liver cirrhosis resembles in part that of the corresponding human disease.
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PMID:Serum amino acid changes in rats with thioacetamide-induced liver cirrhosis. 857 92

Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-phosphate active site per subunit. In addition to catalyzing the decarboxylation of -aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammonia. These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis. The Kcat and Km values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively. The NaBH4-reduced enzyme does not catalyze this reaction. Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with Ki and Kinact values of 0.40 mM and 0.023 min-1, respectively. Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine. Inactivation with [2-14C]5-HT results in the incorporation of 1 mol of label/enzyme subunit. Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21. The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized. A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented.
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PMID:Mechanism-based inactivation of dopa decarboxylase by serotonin. 879 28

By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol. consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 l/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 l/g. Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species. Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants.
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PMID:Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis. 891 16

We tested the hypothesis that nutritional state affects seawater acclimation by transferring either fed or food-deprived (2 weeks) male tilapia (Oreochromis mossambicus) from fresh water to full-strength sea water. Food-deprivation resulted in a significant increase in plasma concentrations of Na+, Cl-, cortisol, glucose, total amino acid, glutamate, serine and alanine, and in hepatic pyruvate kinase (PK) and lactate dehydrogenase (LDH) activities, whereas the prolactin-188 to prolactin-177 ratio (tPRL188:tPRL177) and plasma prolactin-188 (tPRL188), lactate, arginine and hepatic glycogen content and hepatic alanine aminotransferase (AlaAT) and 3-hydroxyacyl-Coenzyme A dehydrogenase (HOAD) activities were lower than in the fed group. Seawater transfer significantly increased the tPRL188:tPRL177 ratio and plasma concentrations of Na+, Cl-, K+, growth hormone (GH), glucose, aspartate, tyrosine, alanine, methionine, phenylalanine, leucine, isoleucine and valine levels as well as gill Na+/K+-ATPase activity and hepatic PK and LDH activities, whereas plasma tPRL177, tPRL188, glycine and lysine concentrations were significantly lower than in fish retained in fresh water. There was a significant interaction between nutritional state and salinity that affected the tPRL188:tPRL177 ratio and plasma concentrations of Cl-, GH, glucose, aspartate, tyrosine, serine, alanine, glycine, arginine and hepatic PK, LDH, AlaAT, aspartate aminotransferase, glutamate dehydrogenase and HOAD activities. These results, taken together, indicate that food-deprived fish did not regulate their plasma Cl- levels, despite an enhancement of plasma hormonal and metabolic responses in sea water. Our study also suggests the possibility that plasma prolactin and essential amino acids may be playing an important role in the seawater acclimation process in tilapia.
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PMID:Food-deprivation affects seawater acclimation in tilapia: hormonal and metabolic changes 932 Mar 94

The refolding of Clostridium symbiosum glutamate dehydrogenase (GDH) involves the formation of an inactive structured monomeric intermediate prior to its concentration-dependent association. The structured monomer obtained after removal of guanidinium chloride was stable and competent for reconstitution into active hexamers. Site-directed mutagenesis of C. symbiosum gdh gene was performed to replace the residues Arg-61 and Phe-187 which are involved in subunit-subunit interactions, as determined by three-dimensional structure analysis. Heterologous over-expression in Escherichia coli of the double mutant (R61E/F187D) led to the production of a soluble protein with a molecular mass consistent with the monomeric form of clostridial GDH. This protein is catalytically inactive but cross-reacts with an anti-wild-type GDH antibody preparation. The double mutant R61E/F187D does not assemble into hexamers. The physical properties and the stability toward guanidinium chloride and urea of R61E/F187D were studied and compared to those of the structured monomeric intermediate.
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PMID:A monomeric mutant of Clostridium symbiosum glutamate dehydrogenase: comparison with a structured monomeric intermediate obtained during refolding. 956 3

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.
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PMID:Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug. 1133 76


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