Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P17174 (aspartate aminotransferase)
 14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A fluorescent derivative of lysine-258 isolated from the active site of aspartate aminotransferase modified by treatment of the apoenzyme with pyridoxal 5'-sulfate has been characterized as a substituted 2H-pyrrolo[3,4-c]pyridine. Similar pyrrolopyridines are produced in up to 20% yield by reaction of pyridoxal sulfate with simple alkylamines or with amino acids including lysine. The latter forms two products, one of which is identical with that isolated from the enzyme. The pyrrolopyridine derived from ethylamine has been characterized by proton and 13C NMR, ultraviolet-visible, and mass spectroscopy and by its chemical reactions.
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PMID:Pyrrolopyridine derivatives from pyridoxal 5'-sulfate. 717 50

A notable feature of porcine cytosolic aspartate aminotransferase is the closure of the active site cleft by a mobile amino-terminal segment (residues 15-40) upon binding substrate. The functional roles of Val17 and Phe18, residues that are part of the mobile loop, have been studied in the site-directed mutants in which the size and hydrophobic nature of these residues have been changed. Absorption, circular dichroism spectra, susceptibility to protease 401, and thermal stability did not differ appreciably between wild type and mutant enzymes. In the overall transamination between aspartate and 2-oxoglutarate, V17A represented a typical Km mutant while V17I retained the substrate binding affinity fairly well. In contrast, replacement of Phe18 by Ala resulted in a large decrease in both catalytic rate and binding affinity for substrates. F18W, F18Y, and F18H showed a moderate decrease in kcat and a considerable increase in Km values. Single-turnover reactions with four individual substrates yielded analogous results to those obtained for the overall reaction and, in addition, revealed that k/Kd values of mutants F18A and F18H were over 10 times lower for C5 substrates (glutamate and 2-oxoglutarate) than those for C4 substrates (aspartate and oxalacetate). All mutant enzymes showed variously increased Kd values for substrate analogs such as 2-methylaspartate, succinate, and glutarate. 1H NMR observations of F18H, in which His18 served as a built-in probe, were in accord with the behavior that would be expected from the conformational transition. We conclude that, although Val17 and Phe18 may not be essential for catalysis, the presence of a bulky residue of appropriate size at each position is critical for productive binding of substrate.
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PMID:Functional role of the amino-terminal mobile segment in catalysis by porcine cytosolic aspartate aminotransferase. Critical importance of Val17 and Phe18 for productive binding of substrates. 792 45

Continuing a previous investigation (Kintanar, A., Metzler, C. M., Metzler, D. E., and Scott, R. D. (1991) J. Biol. Chem. 266, 17222-17229), we have recorded 1H NMR spectra at 500 MHz in the 10-18-ppm range for the 93-kDa porcine cytosolic aspartate aminotransferase and for four specific mutant forms of the enzyme in which histidine 68 has been replaced by lysine or histidine 143, 189, or 193 has been replaced by glutamine. We have correlated resonances for apoenzyme, pyridoxamine and pyridoxal phosphate forms, and dicarboxylate complexes and have assigned imidazole NH resonances of active site histidines. The chemical shifts of several resonances undergo pH-dependent changes around the pKa of the Schiff base proton at the active site. Other resonances shift upon binding of dicarboxylates or other ligands. Phosphate or carboxylate ions, which can also occupy the site of the substrate's alpha-carboxylate, cause rapid exchange of the Schiff base proton. Although most resonances in the 10-18-ppm range disappear rapidly in D2O, a few are retained for months in the presence of the dicarboxylate inhibitor glutarate. We demonstrate that changes in chemical shifts and in exchange rates are sensitive indicators of electronic interactions of the enzyme with ligands and of conformational change. Nuclear Overhauser effects from NH protons have allowed us to identify resonances of CH protons of the imidazole rings of histidines 143, 189, and 193. Observed and predicted chemical shifts have been compared. We conclude that the net charge on this histidine cluster is zero but that some negative charge from the aspartate 222 carboxylate is donated inductively into the histidine 143 ring. Studies of the related enzyme from Escherichia coli are provided in an accompanying paper (Metzler, D. E., Metzler, C. M., Scott, R. D., Mollova, E. T., Kagamiyama, H., Yano, T., Kuramitsu, S., Hayashi, H., Hirotsu, K., and Miyahara, I. (1994) J. Biol. Chem. 269, 28027-28033). Our approach should be applicable to the study of active sites of a broad range of relatively large proteins.
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PMID:NMR studies of 1H resonances in the 10-18-ppm range for cytosolic aspartate aminotransferase. 796 36

We have recorded 500-MHz 1H NMR spectra in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli and for three specific mutant forms. Histidine 143 has been replaced by either alanine or asparagine. In the third mutant, tryptophan 140 has been replaced by phenylalanine. The NMR spectrum of the native enzyme is very similar to that of porcine cytosolic aspartate aminotransferase in the most downfield region. However, the resonances of the proton on the ring nitrogen of the pyridoxal 5'-phosphate (peak A) and on the His-143 imidazole ring (peak B) of the E. coli enzyme are broader and more readily lost at low pH or higher temperatures than those of the porcine enzyme. The possible role of tautomerism in promoting such broadening is discussed. In the histidine mutant proteins, peak A of the pyridoxal 5'-phosphate form is too broad to see under most conditions but is clearly present in the pyridoxamine phosphate form. Peak B is missing in the 2 histidine mutants. Observation of nuclear Overhauser effects further confirms the identity of B as the resonance of HN epsilon 2 of His-143 and that of peak D at approximately 11.8 ppm as HN epsilon 2 of His-189. The mutant spectra also provide insight into electronic interactions between groups in and near the active site which confirm and supplement conclusions drawn from spectra of porcine cAspAT. While no clear loss of a peak was observed for the Trp-140 mutant in its free form, the spectrum of the succinate complex lacked a strong band at 11.26 ppm. This may represent the Trp-140 indole NH proton which has been shifted downfield by binding to a succinate carboxylate group. While our results confirm the basic similarity of cytosolic aspartate aminotransferase and E. coli aspartate aminotransferase 1H NMR spectra, they also point out differences that may be useful in identifying resonances. A large number of mutant proteins have been prepared for the E. coli enzyme. The present results provide essential information for future study of these mutants and for study of NMR spectra of isotopically labeled enzyme.
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PMID:NMR studies of 1H resonances in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli. 796 37

Recent studies suggest a major role played by sodium in the pathogenesis of ischemic liver injury: in these studies, sodium-free media have been shown to offer protection against hypoxic injury to isolated hepatocytes. As sodium-free perfusions of the isolated rat liver proved impossible because of extensive vasoconstriction, we assessed the effects of two inhibitors of the Na+-K+-2Cl- cotransporter, the loop diuretics furosemide and bumetanide, on ischemic liver injury. In untreated control livers lactate dehydrogenase (LDH) efflux immediately after reperfusion after 60 minutes of ischemia at 37 degrees C was 1666 +/- 473 U/L. When livers were pretreated with furosemide or bumetanide before the ischemic period, LDH efflux was only 773 +/- 292 U/L and 702 +/- 183 U/L respectively (P < .01). LDH activity in the effluent of the pretreated livers remained significantly below the values of ischemic control livers for the whole reperfusion period of 90 minutes. Bile flow in the postischemic phase was improved by pretreatment with furosemide or bumetanide. The increase in intracellular sodium, as measured by 23Na-NMR, was attenuated from 193% +/- 71% during 60 minutes of ischemia in controls to 148% +/- 80% after bumetanide application (P < .05). Also, after 120 minutes of warm ischemia, LDH and aspartate aminotransferase release were significantly decreased and bile flow increased by pretreatment with bumetanide. Thus, both furosemide and bumetanide showed a clear benificial effect on rat livers subjected to warm ischemia. These data suggest that one means by which sodium ions are accumulated during liver ischemia might be the Na+-K+-2Cl- cotransporter, which is blocked by furosemide and bumetanide.
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PMID:Decrease of ischemic injury to the isolated perfused rat liver by loop diuretics. 918 63

A new mathematical model, based on the observation of 13C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-13C]Glc or [1,2-(13)C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the 13C-enrichment of acetyl-CoA can be deduced from 13C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-13C]glucose in the absence of insulin, was investigated by 13C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate <--> 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G0) or aspartate (A0) were approximately the same for all hearts and remained constant during perfusion: G0 = (0.74 +/- 0.03) micromol/g; A0 = (1.49 +/- 0.05) micromol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min(-1) and 0.72 min(-1), respectively; the flux of this cycle is about (1.07 +/- 0.02) micromol min(-1) g(-1). Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.
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PMID:13C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts. 992 79

1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes even larger decreases of 25,000- and 100,000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site.
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PMID:The aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction. 1055 73

This study used in vitro 13C NMR spectroscopy to directly examine bidirectional reactions of the Wood-Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-13C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 micromol min(-1) g(-1) (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood-Werkman cycle was evident from the formation of intermediates such as [3-13C]pyruvate and [3-13C]malate and of products like [2-13C]acetate from [2-13C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-13C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.
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PMID:In vivo 13C NMR study of the bidirectional reactions of the Wood-Werkman cycle and around the pyruvate node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici. 1093 24

(13)C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate with deuterons from intracellular heavy water providing information on alpha-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-(13)C]alanine in buffer containing or not 50% (2)H(2)O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution (13)C NMR (150.13 MHz) with (1)H decoupling only and with simultaneous (1)H and (2)H decoupling. (13)C-(2)H couplings and (2)H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min(-1)) for (i) the reversible exchange of [2-(13)C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-(13)C]glutamate H3(proS) as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3(proR) and H3(proS) as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of (1)H-(2)H exchange for the H2 (0.4, 0.5) or H3(proR) (0.3, 0.2) or the H2 and H3(proS) hydrogens (0.20, 0.23) of [3-(13)C]aspartate isotopomers. The ubiquitous subcellular localization of (1)H-(2)H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-(13)C]glutamate and [3-(13)C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.
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PMID:Hydrogen turnover and subcellular compartmentation of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate as detected by (13)C NMR. 1174 18

Oxidative lipid metabolism as a result of acute cyanobacterial toxin-induced hepatotoxicity was monitored in male Sprague-Dawley rats using electron spin resonance (ESR) spectroscopy and image-guided proton nuclear magnetic resonance (1H-NMR) spectroscopy. ESR spectroscopy, coupled with spin trapping, was used to trap and detect lipid-derived radicals, formed in rat livers after acute in vivo exposure (LD50) to the cyanobacterial toxin, microcystin-LR (MCLR). A statistically significant increase in the levels (spectral peak integrals) of lipid radicals was detected in MCLR-treated livers (p < 0.05) (n = 8), in comparison to control livers (n = 6). In order to monitor lipid metabolism, before and for a period of 3 h, following toxin exposure, in vivo proton image-guided NMR spectroscopy was used. A statistically significant decrease in the levels of lipid methylene hydrogen resonances (spectral peak integrals) was observed from MCLR-treated livers (n = 6) 2 and 3 h post-exposure (p < 0.05), in comparison to controls (n = 6). Image-guided NMR spectroscopy was also used to detect significant decreasing levels of in vivo glutamine/glutamate, following exposure to MCLR. Biochemical assessment of perchloric extracts of liver glutamine and glutamate levels correlated with NMR spectroscopy results. Lactate levels measured as perchloric acid extracts, were also found to significantly decrease. In addition, assessment of serum enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were used to confirm hepatotoxicity (n = 20). This study strongly suggests that oxidative stress related processes are involved in in vivo microcystin-induced hepatotoxicity in mammals, and may play an integral role in MCLR-induced toxicity.
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PMID:Assessment of in vivo oxidative lipid metabolism following acute microcystin-LR-induced hepatotoxicity in rats. 1199 4


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