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)

The binding site of Pyridoxal-5-P in 4-aminobutyrate aminotransferase was studied by using analogs of the cofactor. A phosphorothioate analog (PLP(S] recognizes the catalytic site; it forms a stable complex with the apoenzyme (KD = 1nM) and serves as cofactor during catalysis. Replacement of a non-bridged oxygen by sulfur in the phosphate side chain renders a compound which preserves the negative charges needed for correct alignment of the cofactor at the catalytic site. This phosphorothioate analog of PLP can be used to investigate the catalytic site of vitamin B6 dependent enzymes by means of 31P NMR spectroscopy. A bulky P-pyridoxamine derivative, ie, N-4-azido-2-nitrophenyl pyridoxyl-5-P (NANP) competes with natural cofactor for its binding site. Upon illumination, the arylazide of P-pyridoxamine acts as an efficient photolabeling reagent of the protein. A characteristic feature of this photolabeling reagent, ie, its ability to recognize the cofactor binding site, can be exploited to ascertain the chemical nature of amino acid residues at the catalytic site.
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PMID:Binding of new PLP analogs to the catalytic domain of GABA transaminase. 383 74

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

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

A selective 1H NMR spin-echo editing method was used to detect the 4-CH2 of GABA in rat brain in vivo before and after intravenous administration of the highly selective GABA transaminase inhibitor, gabaculine (3-amino-2,3-dihydrobenzoic acid-HCl; 100 mg/kg, intravenously). The effects of the inhibitor on high energy phosphates and pHi were determined by 31P NMR. GABA levels increased approximately linearly (r = 0.81 to 0.94; P < 0.0005) from 1.9 +/- 0.4 mumol/g (pre-gabaculine; mean +/- SD) to between 6 and 8 mumol/g after 4 hr at rates of accumulation of 1.1 to 2.9 mumol/hr/g. 1H NMR spectroscopic measurements of cerebral GABA and its rate of turnover offers a new approach in the study of GABA-mediated processes in vivo.
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PMID:Measurement of GABA following GABA-transaminase inhibition by gabaculine: a 1H and 31P NMR spectroscopic study of rat brain in vivo. 791 62

Localized 1H NMR spectroscopy in conjunction with J editing was used to measure the concentration of gamma-aminobutyric acid (GABA) in the occipital lobe of four control human volunteers and four epileptic volunteers who were receiving the drug vigabatrin. The GABA concentration measured in four nonepileptic subjects was 1.1 +/- 0.1 mumol/cm3 of brain, which is in good agreement with previous values measured in surgically removed human cortex. A dose-dependent elevation of GABA concentration was measured in patients receiving the GABA transaminase inhibitor vigabatrin, with the maximum measured level of 3.7 mumol/cm3 of brain measured at the highest dose (6 g per day) studied. 1H NMR measurements of GABA in those patients receiving GABA-elevating agents such as vigabatrin will be of importance in establishing the relationship between seizure suppression and the concentration of brain GABA.
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PMID:Localized 1H NMR measurements of gamma-aminobutyric acid in human brain in vivo. 851 15

The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by 13C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2(-13)C2]acetate (15 mumol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2(-13)C2]glucose (15 mumol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by 13C NMR. In vigabatrin-treated animals [13C]glutamine, a common intermediate for [13C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2(-13)C2]glucose or [1,2(-13)C2]acetate. However, [13C]GABA accumulation was sevenfold higher during [1,2(-13)C2]glucose infusions or twofold higher during [1,2(-13)C2]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [13C]GABA accumulation occurs initially in the neuronal compartment.
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PMID:Metabolic precursors and compartmentation of cerebral GABA in vigabatrin-treated rats. 885 58

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.
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PMID:The tricarboxylic acid cycle of Helicobacter pylori. 1009 6

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid, 1), a naturally occurring neurotoxin isolated from Streptomyces toyocaenis, has been shown to be a mechanism-based inactivator of gamma-aminobutyric acid aminotransferase (GABA-AT) (Rando, R. R. Biochemistry 1977, 16, 4604). Inactivation results from reaction of gabaculine with the pyridoxal 5'-phosphate (PLP) cofactor. Two HPLC systems for isolating this inactivator-PLP adduct are described as well as a detailed characterization of the adduct, including the ultraviolet-visible spectrum, electrospray mass spectra, and NMR spectrum. The same spectral characterization of the chemically synthesized gabaculine-PLP adduct is also reported.
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PMID:Isolation and characterization of the product of inactivation of gamma-aminobutyric acid aminotransferase by gabaculine. 1048 50

Editing of GABA by (1)H MRS in a specific brain area is a unique tool for in vivo non-invasive investigation of neurotransmission disorders. Selective GABA detection is achieved using sequences based on double quantum coherence (DQC). Our pulse sequence makes accurate measurements without artefacts due to spatial localization. The sequence was tested on a phantom solution. The effect of vigabatrin, a specific inhibitor of GABA transaminase, was measured in rat brain and GABA detection was performed in vivo in monkey brain using this procedure. Rats were split into two groups. In the control group, the rats had access to water and, in the other group (vigabatrin, VGB, rats), animals were allowed free access to drinking water containing vigabatrin. After 3 weeks of treatment, rats were anesthetized for in vivo NMR spectroscopy investigation. At the end of the experiment, brains were quickly removed, freeze-clamped and extracted with 4% perchloric acid. One part of the acid extract was used for GABA concentrations assessment by ion exchange chromatography with ninhydrin detection. The second was used for high-resolution NMR analysis. By chromatography measurements, the GABA concentration was 1.23+/-0.06 micromol/g for controls, while for vigabatrin-treated rats the GABA concentration was 4.89+/-1.60 micromol/g. The NMR in vivo results were closely correlated with the NMR ex vivo (r=0.99, p<0.01) and chromatography results (r=0.98, p<0.01). The correlation between ex vivo results and chromatography results was also high (r=0.99, p<0.001). This pulse sequence performed GABA editing from a 376 microl voxel located on the right basal ganglia area in a non-human primate brain. This in vivo GABA editing scheme can thus be proposed for accurate measurement of brain GABA concentrations.
NMR Biomed 2004 Apr
PMID:Brain GABA editing by localized in vivo (1)H magnetic resonance spectroscopy. 1505 53

Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the alpha-carboxylate of alpha-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the delta-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.
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PMID:Determinants of substrate specificity in omega-aminotransferases. 1609 75


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