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)

Native mitochondrial aspartate aminotransferase (AATase) is cleaved selectively by trypsin at the peptide bonds after Arg 26 or after Lys 31 yielding two shortened enzyme derivatives, AATase 27-410, and AATase 32-410. Recent x-ray crystallographic determination of the spatial structure of AATase has shown that the NH2-terminal segments of the two polypeptide chains of this dimeric enzyme pass in front of the active site clefts and form two separate junctions with the neighboring subunit which are not contiguous with the main subunit interface (Eichele, G., Ford, G. C., Glor, M., Jansonius, J. N., Mavrides, C., and Christen, P. (1979) J. Mol. Biol. 133, 161-180). The peptide bonds cleaved by trypsin are situated in the following stretch of the polypeptide chain which runs in exposed position on the surface of the subunit. The split-off peptide is lost during gel filtration. The molecular activity of AATase 27/32-410 (a mixture of about equal amounts of the two not readily separable derivatives) is about 3% of that of the native enzyme. In contrast, the K'm values for aspartate and 2-oxoglutarate are unchanged, indicating an unaltered geometry of the substrate binding site. A substantially diminished syncatalytic response of the reactivity of Cys 166 toward 5,5'-dithiobis-(2-nitrobenzoate) suggests that the decrease in catalytic activity is due to an interference with the syncatalytic conformational dynamics observed previously in AATase (Gehring, H., and Christen, P. (1978) J. Biol. Chem. 253, 3158-3163). Consonant with a role of the NH2-terminal segment in propagating the syncatalytic conformational rearrangements the rate of the tryptic cleavage is retarded 4-fold in the presence of the transaminating substrate pair aspartate and oxalacetate.
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PMID:Mitochondrial aspartate aminotransferase 27/32-410. Partially active enzyme derivative produced by limited proteolytic cleavage of native enzyme. 743 Jan 25

Nitric oxide synthase produces NO, citrulline, water, and NADP at the expense of arginine, NADPH, and dioxygen. While citrulline has been considered to be an inert by-product of the high output inducible isoform of NO synthase (iNOS), we show here that immunostimulants induce a metabolic pathway in vascular smooth muscle cells, which enables them to regenerate arginine from citrulline. Regeneration of arginine from citrulline is accomplished by two urea cycle enzymes: arginino-succinate synthetase (AS) and argininosuccinate lyase (AL). Whereas AL is constitutive to vascular smooth muscle cells, AS mRNA and enzyme activity is markedly induced in cells by treatment with bacterial lipopolysaccharide (LPS). The induction of AS mRNA and activity by LPS follows a time course which mirrors that for iNOS but lags 1-2 h behind. As shown for iNOS, interferon-gamma does not itself induce AS but is synergistic with LPS. AS induction is suppressed by glucocorticoids, actinomycin D, and, to a lesser extent, cycloheximide. On the other hand, AS induction is unaffected by an excess of citrulline or the inhibitor of iNOS, N omega-methyl-L-arginine. Our results show the urea cycle enzymes AS and AL confer cells with the capacity to produce NO without a need for exogenous arginine. In conjunction with NOS, citric acid cycle enzymes that covert fumarate to oxaloacetate (fumarase and malate dehydrogenase) and oxaloacetate to aspartate (aspartate transaminase), AS and AL form a novel arginine-citrulline cycle that enables high output NO production by cells.
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PMID:Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration or arginine for nitric oxide synthesis. 751 85

The electron distribution in the coenzyme-substrate adduct of aspartate aminotransferase was changed by replacing active-site Arg386 with alanine and introducing a new arginine residue nearby. [Y225R, R386A]Aspartate aminotransferase decarboxylates L-aspartate to L-alanine (kcat = 0.04 s-1), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (kcat = 0.19 s-1). Molecular-dynamics simulations based on the crystal structure of the mutant enzyme suggest that a new hydrogen bond to the imine N atom of the pyridoxal-5'-phosphate- aspartate adduct and an altered electrostatic potential around its beta-carboxylate group underlie the 650,000-fold increase in the ratio of beta-decarboxylase/transaminase activity.
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PMID:Changing the reaction specificity of a pyridoxal-5'-phosphate-dependent enzyme. 755 24

Mutation of six residues of Escherichia coli aspartate aminotransferase results in substantial acquisition of the transamination properties of tyrosine amino-transferase without loss of aspartate transaminase activity. X-ray crystallographic analysis of key inhibitor complexes of the hexamutant reveals the structural basis for this substrate selectivity. It appears that tyrosine aminotransferase achieves nearly equal affinities for a wide range of amino acids by an unusual conformational switch. An active-site arginine residue either shifts its position to electrostatically interact with charged substrates or moves aside to allow access of aromatic ligands.
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PMID:Alternating arginine-modulated substrate specificity in an engineered tyrosine aminotransferase. 766 15

Nitric oxide (NO) has been implicated as a mediator of hemodynamic and metabolic changes associated with endotoxemia and inflammation. In vitro studies suggest that NO inhibits hepatocyte protein synthesis but the role of NO in the regulation of hepatic protein synthesis in vivo is not known. In this study, rats were given endotoxin or saline after pretreatment with the NO synthase inhibitor NG-nitro-L-arginine or solvent, and plasma levels of nitrite (NO2), nitrate (NO3), and aspartate aminotransferase and hepatic protein synthesis rate in vivo were measured after 4 and 10 hours. The NG-nitro-L-arginine effectively blocked the increase in serum NO2/NO3 seen in endotoxemia and also inhibited the increase in hepatic protein synthesis in endotoxemic rats. The aspartate aminotransferase levels were elevated in endotoxemic rats pretreated with NG-nitro-L-arginine. Results support previous reports of a protective effect of NO on the liver in endotoxemia and suggest that NO may upregulate hepatic protein synthesis in vivo. Further study is needed to clarify the reason for the apparent difference between the effect of NO on hepatic protein synthesis in vivo and in vitro.
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PMID:Nitric oxide may upregulate in vivo hepatic protein synthesis during endotoxemia. 767 68

Ornithine decarboxylases from Trypanosoma brucei, mouse, and Leishmania donovani share strict specificity for three basic amino acids, ornithine, lysine, and arginine. To identify residues involved in this substrate specificity and/or in the reaction chemistry, six conserved acidic resides (Asp-88, Glu-94, Asp-233, Glu-274, Asp-361, and Asp-364) were mutated to alanine in the T. brucei enzyme. Each mutation causes a substantial loss in enzyme efficiency. Most notably, mutation of Asp-361 increases the Km for ornithine by 2000-fold, with little effect on kcat, suggesting that this residue is an important substrate binding determinant. Mutation of the only strictly conserved acidic residue, Glu-274, decreases kcat 50-fold; however, substitution of N-methylpyridoxal-5'-phosphate for pyridoxal-5'-phosphate as the cofactor in the reaction restores the kcat of E274A to wild-type levels. These data demonstrate that Glu-274 interacts with the protonated pyridine nitrogen of the cofactor to enhance the electron withdrawing capability of the ring, analogous to Asp-222 in aspartate aminotransferase (Onuffer, J. J., and Kirsch, J. F. (1994) Protein Eng. 7, 413-424). Eukaryotic ornithine decarboxylase is a homodimer with two shared active sites. Residues 88, 94, 233, and 274 are contributed to each active site from the same subunit as Lys-69, while residues 361 and 364 are part of the Cys-360 subunit.
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PMID:Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis. 774 28

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC and 5'-methylthioadenosine, the committed step in ethylene biosynthesis in plants. Apple ACC synthase was overexpressed in Escherichia coli (3 mg/liter) and purified to near homogeneity. A continuous assay was developed by coupling the ACC synthase reaction to the deamination of 5'-methylthioadenosine by adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Aspergillus oryzae. The enzyme is dimeric, with kcat = 9s-1 per monomer and Km = 12 microM for AdoMet. The pyridoxal phosphate-binding site of ACC synthase appears to be highly homologous to that of aspartate aminotransferase, suggesting similar roles for corresponding residues. Site-directed mutagenesis of Lys-273, Arg-407, and Tyr-233 (corresponding to residues 258, 386, and 225 in aspartate aminotransferase) and kinetic analyses of the mutants confirms their importance in the ACC synthase mechanism. The Lys-273 to Ala mutant has no detectable activity, supporting the identification of this residue as the base catalyzing C alpha proton abstraction. Mutation of Arg-407 to Lys results in a precipitous drop in kcat/Km and an increase in Km for AdoMet of at least 20-fold, in accordance with its proposed role as principal ligand for the substrate alpha-carboxylate group. Replacement of Tyr-233 with Phe causes a 24-fold increase in the Km for AdoMet and no change in kcat, suggesting that this residue plays a role in orienting the pyridoxal phosphate cofactor in the active site.
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PMID:Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms. 780 54

We report here the x-ray studies of the complex cytosolic aspartate aminotransferase from chicken heart with D-aspartate at 2,7 A resolution. Crystals of the complex was prepared by diffusing D-aspartate into free enzyme crystals; their space group is P 2(1)2(1)2(1) with cell dimensions (A): a = 62.59; b = 117.83; c = 124.38. They contain one dimeric molecule in the asymmetric unit. The x-ray crystallographic analysis proves that the connection of the D-aspartate induces small conformational changes in the active site of two subunits of the enzyme: considerable conformational changes are determined for His 189, Phe 360, Tyr 70, Arg 292, Phe 18 and Glu 141.
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PMID:[The complex of aspartate aminotransferase with D-aspartate]. 781 5

Two refined crystal structures of aspartate aminotransferase from E. coli are reported. The wild type enzyme is in the pyridoxal phosphate (PLP) form and its structure has been determined to 2.4 A resolution, refined to an R-factor of 23.2%. The structure of the Arg292Asp mutant has been determined at 2.8 A resolution, refined to an R-factor of 20.3%. The wild type and mutant crystals are isomorphous and the two structures are very similar, with only minor changes in positions of important active site residues. As residue Arg292 is primarily responsible for the substrate charge specificity in the wild type enzyme, the mutant containing a charge reversal at this position might be expected to catalyze transamination of arginine as efficiently as the wild type enzyme effects transamination of aspartate [Cronin, C.N. and Kirsch, J.F. (1988) Biochemistry, 27, 4572-4579]. This mutant does in fact prefer arginine over aspartate as a substrate, however, the rate of catalysis is much slower than that of the wild type enzyme with its physiological substrate, aspartate. A comparison of these two structures indicates that the poorer catalytic efficiency of R292D, when presented with arginine, is not due to a gross conformational difference, but is rather a consequence of both small side chain and main chain reorientations and the pre-existing active site polar environment, which greatly favors the wild type ion pair interaction.
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PMID:The structural basis for the altered substrate specificity of the R292D active site mutant of aspartate aminotransferase from E. coli. 790 46

Nitric oxide (NO) is a readily diffusible, short-lived free radical with a multitude of organ-specific regulatory functions. Within the hepatocyte, NO production is associated with inhibition of mitochondrial electron transport enzyme activity, activation of soluble guanylyl cyclase, and inhibition of glyceraldehyde-3-phosphate dehydrogenase. However, while NO can regulate a number of hepatocyte functions, it is unknown whether NO production is hepatoprotective or hepatotoxic. Using isolated rat hepatocytes in primary short-term culture, we investigated the role of cytokine-mediated NO production in toxin-induced hepatocyte injury. In a model of acetaminophen (AM) hepatotoxicity, inhibition of cytokine-mediated NO production potentiated AM injury. In the presence of an inhibitor of NO synthesis, NG-monomethyl-L-arginine (L-NMMA), hepatocyte release of aspartate aminotransferase was increased twofold in the presence of 4.0 and 8.0 mM AM (P < 0.01). In addition, in the presence of AM, cytokine-mediated NO production was increased by 75% over baseline (P < 0.01). Maximum NO synthesis occurred at an AM concentration of 2 mM. A potential mechanism for the hepatoprotective effect of NO centers on its role in glutathione (GSH) homeostasis. In the presence of increasing concentrations of AM, hepatocyte GSH stores decreased in parallel in both control and cytokine-stimulated hepatocytes (ANOVA, P < 0.01). When cytokine-stimulated hepatocytes were exposed to 50 microM L-NMMA, NO release was ablated, while glutathione levels decreased by threefold in comparison to controls (P < 0.01). In the presence of increasing concentrations of AM, cytokine-treated cells exposed to 50 microM L-NMMA exhibited significant decremental decreases in GSH levels (P < 0.05). These data suggest that inhibition of cytokine-mediated NO production potentiates AM hepatoxicity by modulation of hepatocyte glutathione stores.
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PMID:Nitric oxide decreases oxidant-mediated hepatocyte injury. 801 16


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