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:2.6.1.19 (GABA transaminase)
808 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Physical interactions between the enzymes involved in the catabolism of the neurotransmitter 4-aminobutyrate were detected by means of affinity chromatography and fluorescence techniques. By immobilizing one enzyme (4-aminobutyrate aminotransferase) indirectly through antibodies bound to protein A-Sepharose, it was possible to demonstrate that succinic semialdehyde dehydrogenase interacts with the aminotransferase at neutral pH and ionic strength values higher than 0.2. Increasing the ionic strength of the medium results in dissociation of the "enzyme cluster." Binding of succinic semialdehyde dehydrogenase to the aminotransferase tagged with a fluorescent probe was detected by polarization of fluorescence measurements at neutral pH. Upon saturation of the aminotransferase with succinic semialdehyde dehydrogenase, the polarization of fluorescence increases from 0.13 to 0.21. The results are consistent with a model in which one molecule of succinic semialdehyde dehydrogenase is bound to one molecule of 4-aminobutyrate aminotransferase with an equilibrium dissociation constant of 0.1 microM. Since the concentrations of both enzymes in the mitochondrial matrix have been estimated to be around 2 microM, the results obtained with the purified mitochondrial enzymes strongly suggest that the aminotransferase is saturated with succinic semialdehyde dehydrogenase to form a stable enzymatic complex under in vivo conditions.
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PMID:Interactions between 4-aminobutyrate aminotransferase and succinic semialdehyde dehydrogenase, two mitochondrial enzymes. 647 7

4-Aminobutyrate aminotransferase (EC 2.6.1.19, 4 aminobutyrate:2-oxoglutarate aminotransferase) is cleaved by trypsin, yielding an enzymatically active species which can be separated from the split peptides by gel filtration. The shortened enzyme derivative gives one band (Mr = 95,000) on polyacrylamide gradient gel electrophoresis. Changes in protein conformation induced by tryptic digestion were studied by fluorescence spectroscopy. The native enzyme tagged with the chromophore fluorescein yields a rotational relaxation time of 106 ns, whereas the trypsin-digested enzyme gives a rotational relaxation time of 33 ns. The decrease in rotational relaxation time is attributed to flexibility of the polypeptide chain with enhanced rotational freedom of the probe covalently linked to one thiol group. The reactivity of sulfhydryl groups toward 5,5'-dithiobis(2-nitrobenzoic acid) is also affected by trypsin cleavage. More--SH groups (2.6/dimer) become reactive toward 5,5'-dithiobis(2-nitrobenzoic acid) as a result of trypsin digestion. Local conformational fluctuations are induced as a result of tryptic cleavage, but the catalytic sites remain intact. The peptides released from 4-aminobutyrate aminotransferase were characterized by fingerprint analysis and their amino acid composition determined.
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PMID:Catalytic and structural properties of trypsin-treated 4-aminobutyrate aminotransferase. 661 42

It was previously shown that 5-hexyne-1,4-diamine is a potent enzyme-activated irreversible inhibitor of mammalian ornithine decarboxylase. However this compound has secondary pharmacological effects owing to its in vivo oxidation to 4-aminohex-5-ynoic acid, an irreversible inhibitor of 4-aminobutyrate aminotransferase. The first step of this oxidation is catalysed by mitochondrial monamine oxidase. The monomethyl and dimethyl analogues of 5-hexyne-1,4-diamine, i.e. 6-heptyne-2,5-diamine and 2-methyl-6-heptyne-2,5-diamine, which cannot be substrate of monoamine oxidase, were tested as selective irreversible inhibitors of ornithine decarboxylase. Our results demonstrate that (2R,5R)-6-heptyne-2,5-diamine is greater than 10 times more potent, both in vitro and in vivo, than alpha-difluoromethylornithine, the most widely used irreversible inhibitor of this enzyme.
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PMID:(2R,5R)-6-heptyne-2,5-diamine, an extremely potent inhibitor of mammalian ornithine decarboxylase. 663 60

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

The analogues 5-phospho-pyridoxal-aminooxyacetate and 4-vinyl-pyridoxal 5-phosphate inhibit reduced 4-aminobutyrate aminotransferase reconstituted with pyridoxal 5-phosphate, but they do not affect the catalytic activity of the holoenzyme. The binding of the 5-phosphopyridoxal adducts to the holoenzyme was monitored by measuring the fluorescence properties of the protein and the ligands. The presence of nonequivalent binding sites in reduced samples of enzyme containing 1 mol and 1.8 mol P-pyridoxyl residues/mol dimer was detected by steady and nanosecond fluorescence spectroscopy. The spectroscopic properties (fluorescence lifetime tau = 1 ns, polarization = 0.36 and quantum yield = 0.01) of the first molecule of 5-phospho-pyridoxyl are different from the properties of the second molecule of 5-phospho-pyridoxyl covalently bound to the enzyme (tau = 2.1 ns, P = 0.16, QT = 0.1). The spectroscopic properties of pyridoxal 5-phosphate and 5-phospho-pyridoxal-aminooxyacetate bound to different sites on the holoenzyme can be used to deduce proximity relationships between the catalytic sites of 4-aminobutyrate aminotransferase. On the basis of resonance energy transfer measurements, it is proposed that a distance of 27 A (2.7 nm) separates the two catalytic binding sites.
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PMID:4-Aminobutyrate aminotransferase. Different susceptibility to inhibitors, microenvironment of the cofactor binding site and distance of the catalytic sites. 714 Jul 43

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

The enzyme-induced or 'suicide' step by which the substrate analogue ethanolamine O-sulphate inactivates 4-aminobutyrate aminotransferase occurs at a rate that is one-tenth that observed for the release of the products of beta-elimination, namely ammonia, acetaldehyde and sulphate. An additional reversible reaction not leading to inactivation can be detected spectrally and this decreases the rates of both beta-elimination and inactivation. This reaction is ascribed to a step on the normal transamination path, although complete transamination does not occur significantly. The 14C moiety of radiolabelled ethanolamine O-sulphate is stably bound to the inactive enzyme in the proportion of 1 mol/mol of active site. The 35S-labelled sulphate moiety is not bound after inactivation, showing that beta-elimination must precede inactivation.
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PMID:The reaction of ethanolamine O-sulphate with 4-aminobutyrate aminotransferase. 731 26

The enzyme 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) from pig brain is inactivated by incubation with 5-5'-dithiobis-2-nitrobenzoic acid at pH 7.4. The reaction of one SH group per dimer brings about 95% loss of aminotransferase activity. The reaction with 5-5'-dithiobis-2-nitrobenzoic acid under pseudo-first-order conditions proceeds at a slow rate (Kobs = 0.05 min-1) and the substrate 4-aminobutyrate has no effect on the rate of inactivation. The inactivation of the enzyme cannot be related to dissociation of the cofactor (pyridoxal-P and pyridoxamine-P) from the catalytic site. One thiol group of the enzyme reacts with N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid at pH 7.4. The reaction of approximately one SH group per dimer causes 95% loss of aminotransferase activity. The absorption and fluorescence properties of the enzyme tagged with the chromophore were used to gain information on the microenvironment of the reactive SH group critically connected with catalytic activity. The fluorescent properties, emission maximum (470 nm), fluorescence lifetime (19 ns) and degree of exposure to the collisional quencher acrylamide (Kq = 1.8 . 10(8) M-1 . s-1) indicate that the chromophore is shielded by the protein matrix.
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PMID:Reactivity of one thiol group in the dimeric protein, 4-aminobutyrate aminotransferase. 744 94


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