Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.6.1.19 (GABA transaminase)
 808 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Rat retinae pre-incubated and incubated at 37 degrees C in media containing amino-oxyacetic acid (AOAA) (0.1 muM to 1 mM) accumulated more (3)H-gamma-aminobutyric acid ((3)H-GABA) than control retinae incubated in the absence of AOAA. This increased accumulation of (3)H-GABA by tissue exposed to AOAA was not apparent at short incubation times (0-20 min), but became significant after incubations of 30 min, and maximal after incubation for 60 minutes.2. At a concentration of 10 muM, AOAA did not alter the apparent K(m) for (3)H-GABA uptake or V(max) for either the low or the high affinity GABA uptake systems present in retina.3. The potentiation of (3)H-GABA accumulation produced by AOAA appeared to parallel the inhibitory effect of this compound on 2-oxoglutarate-4-aminobutyrate aminotransferase (GABA-T). Similarly, hydrazinopropionic acid inhibited retinal GABA-T and potentiated the accumulation of (3)H-GABA, but hydroxylamine and thiosemicarbazide which did not affect GABA-T, were also without effect on the retinal accumulation of (3)H-GABA.4. In vitro incubation with AOAA did not increase the endogenous levels of GABA or other amino acids in the retina.5. AOAA did not significantly increase the retinal accumulation of radioactive L-glutamate, L-glutamine, taurine, glycine, alpha-aminoisobutyrate or dopamine: the accumulation of L-aspartate was increased by approximately 30%.6. The inhibition of retinal GABA-T by AOAA was time-dependent and was not reversed by pyridoxal-5'-phosphate or by repeated washing of the tissue with fresh medium.7. AOAA also inhibited glutamate decarboxylase (GAD) in retinae incubated in vitro. This inhibitory effect was partially reversed by pyridoxal-5'-phosphate.8. Efflux of radioactivity from the retina was strikingly reduced in the presence of AOAA at concentrations sufficient to inhibit GABA-T by 100%.9. These findings suggest that AOAA potentiates the accumulation of (3)H-GABA by isolated retina, not by increasing the exchange of (3)H-GABA with the endogenous GABA pools, but by reducing the metabolism of the amino acid and hence reducing the loss of radioactivity from the tissue in the form of tritiated metabolites.
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PMID:Effect of inhibitors of -aminobutyrate aminotransferase on the accumulation of 3H- -aminobutyric acid by the retina. 473 Aug 31

Among uracil derivatives investigated, 6-azauracil, 6-azathymine, and 5-iodouracil were found to be potent inhibitors of purified rabbit liver 4-aminobutyrate aminotransferase while 6-azauridine and 6-azauridine 5'-phosphate were not. The enzyme inhibited by 6-azauracil was reactivated by dialysis but not by addition of pyridoxal 5'-phosphate. 6-Azauracil acted as a non-competitive inhibitor with respect to beta-alanine as well as 2-oxoglutaric acid, and had a K1 of approximately 0.7 mM at pH 7.3. The kinetic data suggested that 2-oxoglutaric acid acted as an inhibitor as well as an amino acceptor for the enzyme; a catalytic site was associated with an apparent Km of 0.15 mM for 2-oxoglutaric acid and a low affinity site was associated with an I50 of approximately 5 mM for the 2-oxo acid. With inhibitory concentrations of 2-oxoglutaric acid as substrate the inhibitory effect of 6-azauracil was considerably diminished. From these findings, the inhibitory effect of 6-azauracil was revealed to be different from that of structural analogs of 4-aminobutyric acid showing that 6-azauracil is a new type of 4-aminobutyrate aminotransferase inhibitor.
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PMID:Inhibitory effect of 6-azauracil on purified rabbit liver 4-aminobutyrate aminotransferase. 668 75

beta-Alanine aminotransferase from rabbit liver has been purified 1,700-fold over the initial liver extract. The purified enzyme was shown to be homogeneous by disc electrophoresis and SDS polyacrylamide electrophoresis. The molecular weight of the purified enzyme determined by gel filtration was 95,000 +/- 5,700 and the subunit molecular weight was 48,000 +/- 2,100. The enzyme showed absorption maxima at 282, 330, and 414 nm and contained only 1 mol of pyridoxal 5'-phosphate/mol of dimer. The pH optimum for enzyme activity was 8.8 and the Km values for beta-alanine and 2-oxoglutaric acid were calculated to be 3.9 and 1.4 mM, respectively. The enzyme catalyzed transamination of various omega-amino acids with 2-oxoglutaric acid, which was a favourable amino acceptor. beta-Alanine, gamma-aminobutyric acid, and beta-aminoisobutyric acid, which are naturally occurring substrates, were preferred amino donors, but taurine, alanine, ornithine, spermine, and spermidine were not. 6-Azauracil inhibited the enzyme activity with a Ki of approximately 1.5 mM. From the above properties, beta-alanine aminotransferase from rabbit liver was seen to closely resemble with 4-aminobutyrate aminotransferase from liver and brain.
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PMID:Purification and properties of beta-alanine aminotransferase from rabbit liver. 681 89

The fluorescence dye 1-anilinonaphthalene-8-sulfonate (ANS) was used as a probe of non-polar binding sites in 4-aminobutyrate aminotransferase. ANS binds to a single binding site of the dimeric protein with a Kd of 6 microM. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (phi = 65 ns) corresponds to the rotation of a rather rigid dimeric structure. The microenvironment surrounding the natural probe pyridoxal-5-P covalently bound to the dimeric structure was explored using 31P-NMR at 72.86 MHz. In the native enzyme, the pyridoxal-5-P 31P-chemical shift is pH-independent, indicating that the phosphate group is well protected from the solvent. The correlation time determined from the 31P-spectrum of the aminotransferase exceeds the value calculated for the hydrated spherical model (phi = 40 ns). It is concluded that the phosphate of the pyridoxal-5-P molecule is rigidly bound to the active site of 4-aminobutyrate aminotransferase.
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PMID:Rotational dynamics of 4-aminobutyrate aminotransferase. 688 10

A homogeneous 4-aminobutyrate aminotransferase isolated from pig brain exhibits a k(cat) of 9.6 s-1 and contains one mole of pyridoxal 5-phosphate/mole of dimer. THe reaction of the enzyme with gabaculine (5-amino-1,3-cyclohexadiene carboxylic acid) was studied by observing changes in the absorption spectrum of the bound cofactor and by monitoring loss of catalytic activity. The enzyme is completely inactivated by gabaculine, but the dialyzed inactive sample containing 0.5 mol of gabaculine/mol dimer is fully reconstituted by addition of pyridoxal 5-phosphate. Stopped-flow kinetic studied reveal that gabaculine reacts with the cofactor bound to the aminotransferase with a second-order rate constant of 2.5 X 10(3) M-1 s-1. Fluorometric titrations of the apoprotein with m-carboxyphenyl-pyridoxamine 5-phosphate show the binding of two moles of inhibitor/mole of enzyme. The binding process is reversible and the affinity of the apoprotein for the inhibitor is at least 10-fold higher than the affinity for the cofactor. It is postulated that the dimeric enzyme contains two potential active sites per dinner, but the binding site characterized by a weaker affinity constant for pyridoxal 5-phosphate becomes functional only after specific chemical modification of the molecule of cofactor tightly bound to the protein.
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PMID:Gabaculine and m-carboxyphenyl-pyridoxamine 5-phosphate as probes of the catalytic binding sites of 4-aminobutyrate aminotransferase. 728 25

Alanine-glyoxylate aminotransferase (AGT) 2 is a pyridoxal 5'-phosphate dependent, mitochondrial enzyme which, in the rat, is expressed at a high level in the kidney. The amino acid sequences of nine tryptic and seven CNBr peptides of the rat kidney AGT2 were determined. Three overlapping cDNAs encoding the AGT2 were cloned on the basis of its partial amino acid sequences by means of a polymerase chain reaction-based approach involving rat kidney poly(A)+ RNA. The complete cDNA sequence comprised 1,919 bases, and contained a 1,536-base open reading frame which encodes a polypeptide of 512 amino acid residues with a putative presequence consisting of 39 amino acid residues at the amino terminus, giving a precursor protein with a molecular mass of 57,150 Da. The sequence of AGT2 exhibits significant homology with neither peroxisomal AGT1 from human liver nor mitochondrial AGT1 from rat liver. However, the sequence of AGT2 exhibited 30.8, 29.2, and 27.1% identity with those of Escherichia coli 4-aminobutyrate aminotransferase, rat ornithine aminotransferase, and Pseudomonas cepacia 2,2-dialkylglycine decarboxylase, respectively. The active site sequences were also well conserved among these aminotransferases. AGT2, thus, is more similar to the other aminotransferases than to AGT1. The results suggest that the rat kidney AGT2 may play a biological role in amino acid metabolism distinct from that of AGT1.
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PMID:Molecular cloning and sequencing of a cDNA encoding alanine-glyoxylate aminotransferase 2 from rat kidney. 759 50

gamma-Aminobutyric acid (GABA) aminotransferase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of GABA into succinic semialdehyde. Hydrazine analogues have long been known to act as inactivators of PLP-dependent enzymes, including GABA aminotransferase, however, no studies of the molecular mechanism of inactivation of PLP-dependent enzymes by hydrazines have been reported. 3-Hydroxybenzylhydrazine is shown to be a potent in vitro time-dependent inhibitor of pig brain GABA aminotransferase. UV-visible and 1H NMR studies, both with GABA aminotransferase and with PLP as a chemical model for the enzyme-catalyzed reaction, indicate that 3-hydroxybenzylhydrazine reacts both enzymatically and nonenzymatically to form the 3-hydroxybenzylhydrazone of PLP without tautomerization.
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PMID:Time-dependent inhibition of gamma-aminobutyric acid aminotransferase, by 3-hydroxybenzylhydrazine. 764 6

The enzyme 4-aminobutyrate aminotransferase (EC 2.6.1.19) isolated from Pseudomonas fluorescens was inhibited by the nucleotide ATP in an apparent competitive manner (Ki = 10.4 mM). This reversible effect was antagonized by the substrate GABA, whose apparent Km was increased from 0.6 mM to 2 mM in the presence of 20 mM ATP, suggesting that ATP interferes with GABA binding to the active site of the enzyme. The apparent Km with respect to the second substrate alpha-ketoglutarate was also increased, although to a lesser extent, whereas the cofactor pyridoxal 5'-phosphate was unable to influence the inhibition by ATP. The ATP structural analogues ADP, CTP and XTP were also able to inhibit the enzyme to a similar extent. These data indicate that GABA concentrations within the bacterial cell can be regulated by the action of ATP on 4-aminobutyrate aminotransferase. In addition, because the inhibition by ATP is similar to the inhibition of the enzyme from mammalian brain, the bacterial enzyme could provide a convenient source of the enzyme for studies of drug effects on brain GABA metabolism in vitro.
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PMID:Inhibition of 4-aminobutyrate aminotransferase from Pseudomonas fluorescens by ATP. 826 Sep 45

4-Aminobutyrate aminotransferase (EC 2.6.1.19), obtained from Pseudomonas fluorescens, was irreversibly inhibited by phenylglyoxal, a reagent that specifically modifies arginyl residues. The inactivation appeared to be the result of a simple, bimolecular reaction since no evidence of a reversible complex between inhibitor and enzyme emerged. The second-order rate constant was 0.221 +/- 0.077 M-1 sec-1. The concentration of either substrate had no effect on the inhibition, but an increase in the concentration of pyridoxal 5'-phosphate reduced the rate of inactivation by phenylglyoxal. The data are consistent with the modification of amino acid residues at the cofactor binding site on the enzyme.
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PMID:Chemical modification of bacterial 4-aminobutyrate aminotransferase by phenylglyoxal. 859 41

The mechanism of inactivation of the pyridoxal 5'-phosphate (PLP)-dependent enzyme gamma-aminobutyric acid (GABA) aminotransferase by 3-amino-4-fluorobutanoic acid (2) has been investigated. As in the case of the homologue, 4-amino-5-fluoropentanoic acid (1), 2 equiv of radiolabeled inactivator become covalently attached to the enzyme, and no transamination, as determined by the lack of conversion of [1-14C] alpha-ketoglutarate into [1-14C] glutamate during inactivation, was observed. In the case of 1, the conclusion was that inactivation was completely the result of modification of the coenzyme and that there was no metabolic turnover; every enzyme molecule catalysed the conversion of one molecule of inactivator to the activated species, which inactivated the enzyme by an enamine mechanism. With 2, however, 6.7 +/- 0.7 equiv of fluoride ions were released during inactivation, and it took 7.6 +/- 0.7 inactivator molecules to inactivate each enzyme dimer. Since no transamination was occurring, another metabolic event besides inactivation must result from the PLP form of the enzyme. Inactivation of GABA amino-transferase with [1,2-14C]-2 produced [14C] acetoacetic acid (about 5.5 equiv) as the metabolite. The 1.93 +/- 0.25 equiv of radioactivity covalently bound to the enzyme after inactivation with [1,2-14C]-2 and gel filtration were completely released by base treatment. HPLC analysis showed that three radioactive compounds, identified as 2, the product of reaction of PLP with acetone (3), and the product of reaction of PLP with acetoacetate (4), were detected. The release of 3 and 4 and the prevention of release of radioactivity by treatment with sodium borohydride are consistent with the formation of covalent intermediates that have beta-carbonyl-like character, such as 6 and/or 7 (Scheme 2). Inactivation of [3H] PLP-reconstituted GABA aminotransferase with 2 followed by gel filtration then base denaturation released all of the radioactivity as a mixture of PLP, 3, and 4. Inactivation with [1,2-14C]-2 resulted in the release of 1.37 equiv of 14CO2, which was shown to be the result of decarboxylation of the acetoacetate/4 after release from the enzyme. These results are not consistent with a Michael addition mechanism (Scheme 3), but are consistent with inactivation by an enamine mechanism; release of the enamine five out of seven turnovers accounts for the formation of acetoacetate as the metabolite. To account for the detection of PLP and 2 after denaturation, it is suggested that a nonproductive formation of the Schiff base of PLP with 2 occurs in the second subunit of the enzyme; this complex is released and hydrolysed to PLP and 2 upon base denaturation.
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PMID:Mechanism-based inactivation of gamma-aminobutyric acid aminotransferase by 3-amino-4-fluorobutanoic acid. 889 9


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