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)

Cysteine aminotransferase (L-cysteine: 2-oxoglutarate aminotransferase, EC 2.6.1.3) was purified over 400-fold from the high-speed supernatant fraction of rat liver. The purified enzyme was homogeneous as judged by gel filtration, isoelectric focusing and disc electrophoresis. The molecular weight of the enzyme was about 74,000 by gel filtration and the isoelectric point was 6.2 (4 degrees C). The enzyme catalyzed transamination between L-cysteine and 2-oxoglutarate and the reverse reaction. The optimum pH was 9.7. The Km value for L-cysteine was 22.2 mM, and that for 2-oxoglutaric acid was 0.06 mM. L-Aspartate was a potent inhibitor of the cysteine aminotransferase reaction. The enzyme was very active toward L-alanine 3-sulfinic acid at pH 8.0, and was also very active toward L-aspartic acid (Km = 1.6 mM). Ratios of activities for L-aspartic acid and L-cysteine were essentially constant during the purification of the enzyme. Evidence based on substrate specificity, enzyme inhibition, and physicochemical properties indicates that cytosolic cysteine aminotransferase is identical with cytosolic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1).
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PMID:Purification and characterization of cysteine aminotransferase from rat liver cytosol. 711 43

Alkylation of the K258C mutant of the wild-type aspartate aminotransferase (AATase) with bromoethylamine to give gamma-thialysine 258 was complicated by partial reaction with the five native cysteines [Planas, A., & Kirsch, J. F. (1991) Biochemistry 30, 8268-8276]. This problem is now overcome by carrying out the alkylation with K258CQ, in which Cys-258 is a unique cysteine residue in Quint, an engineered AATase in which the five cysteines have been converted to alanine [Gloss, L.M., et al. (1992) Biochemistry 31, 32-39]. The kinetics and spectral properties of the resulting enzyme, K258CQ-EA, have been examined and compared to those of WT and Quint. The replacement of Lys-258 by gamma-thia-Lys results in an acidic shift of 1.3 pH units in the pKa of the internal aldimine. The C alpha hydrogen kinetic isotope effects for Quint are 2.1 and 1.5 on D(kcat/KMAsp) and Dkcat, respectively. Replacement of Lys-258 by the weaker base, gamma-thia-Lys, increases these values to 3.3 and 2.6, respectively The changes of K258CQ-EA in ligand affinities and the keto acid half-reaction are minor; however, the kcat/KM values for amino acids are decreased by an order of magnitude. The KD values for PMP of K258CQ-EA and Quint are equal to each other (0.2 nM) and are 7-fold lower than that of WT. These combined effects are illustrated in the free energy diagrams of the reaction with L-Asp with K258CQ-EA, relative to WT (and Quint). The E.PLP and E.PMP complexes of Quint are 0.9 and 1.1 kcal/mol, respectively, more stable than those of WT.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Decreasing the basicity of the active site base, Lys-258, of Escherichia coli aspartate aminotransferase by replacement with gamma-thialysine. 769 64

The pH dependence of Escherichia coli aspartate aminotransferase (AATase) has been investigated by the use of site-directed mutants and alternative substrates. Inhibition of the enzyme by CHES and variations in ionic strength are proposed to explain some of the qualitative differences in the published pH dependence of pig cytosolic AATase kinetics [Velick, S. F., & Vavra, J. (1962) J. Biol. Chem. 237, 2109-2122; Kiick, D.M., & Cook, P.F. (1983) Biochemistry 22, 375-382]. The pKa values of the basic limbs in the kcat/KM profiles for the amino acids, L-Asp and L-cysteinesulfinate (L-CS), are identical, within error, to those of free substrates, (L-Asp, pKa = 9.6; L-CS, pKa = 9.0). This pKa therefore is assigned to the alpha-amino group of the substrate. Replacement of the active site base, Lys-258, with the weaker base, gamma-thia-Lys, does not alter the intrinsic pKa for the profiles of the Ki values for the maleate-E.PMP complexes or the kcat/K alpha-KGM values. The mutation Y225F results in an alkaline shift of the pKa in the kcat/K alph-KGM profile. This pKa is assigned to the C4' amino group of PMP. E. coli AATase, unlike pig cytosolic AATase, shows a pH dependence on kcat between pH 5 and 10 that arises from a change in the rate-determining step at pH extremes. C alpha proton abstraction is partially rate-determining at neutral pH values, but not at pH extremes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Use of site-directed mutagenesis and alternative substrates to assign the prototropic groups important to catalysis by Escherichia coli aspartate aminotransferase. 769 65

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

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana. Four classes of cDNAs representing four distinct AspAT genes (ASP1-ASP4) have been cloned from Arabidopsis. Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1-AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis. These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.
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PMID:The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments. 789 12

The flux through different segments of the tricarboxylic acid cycle was measured in rat brain synaptosomes with gas chromatography-mass spectrometry using either deuterated glutamine or [13C]aspartate. The flux between 2-oxoglutarate and oxaloacetate was estimated to be 3.14 and 4.97 nmol/min/mg protein with and without glucose, respectively. These values were 3-5-fold faster than the flux between oxaloacetate and 2-oxoglutarate (0.92 nmol/min per mg protein) measured in the presence of glucose. The pattern of intermediates labeling suggests that the overall rate-controlling reaction involves either citrate synthase or pyruvate dehydrogenase but not 2-oxoglutarate or isocitrate dehydrogenase. The enrichment in [3,3,4,4-2H4]glutamate from [2,3,3,4,4-2H5]glutamine was as rapid as in [2,3,3,4,4-2H5]glutamate, which indicates that the aspartate aminotransferase reaction is severalfold faster than the flux through the tricarboxylic acid cycle. [13C]Aspartate was rapidly converted to [13C]malate, suggesting that in intact synaptosomes aspartate entry into the mitochondrion is very slow. The finding that aspartate is taken up by mitochondria as malate, along with the observed high enrichment in [3-2H]malate (from [2,3,3,4,4-2H5]glutamine), is consistent with the substantial synaptosomal activity of the malate/aspartate shuttle.
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PMID:Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle. 796 53

The aspartate and tyrosine aminotransferases from Escherichia coli have 43% sequence identity and nearly identical active sites. Both are equally good enzymes for dicarboxylate substrates, but the latter transaminates aromatic amino acids 1000 times faster. In an attempt to discover the critical residues for this differential substrate specificity, the aspartate aminotransferase mutant V39L has recently been prepared. It showed improved Kcat/Km values for aspartate, glutamate and tyrosine and the corresponding oxo acids, mainly due to two to ten times lower Km values. For example, the Km values of V39L (wild type) for Asp and Glu are 0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallized with 30 mM maleate from both polyethylene glycol and ammonium sulfate. Both structures were solved and refined to R-factors of 0.22 and 0.20 at 2.85 and 2.5 A resolution respectively. They bear strong resemblance to the closed structure of the wild type enzyme complexed with maleate. The unexpected feature is that, for the first time, the closed form was produced in crystals grown from ammonium sulfate. It is concluded that the mutation has shifted the conformational equilibrium towards the closed form, which leads to generally reduced substrate Kms.
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PMID:Three-dimensional structure of a mutant E. coli aspartate aminotransferase with increased enzymic activity. 807 30

The pathways of nitrogen transfer from 50 microM [15N]aspartate were studied in rat brain synaptosomes and cultured primary rat astrocytes by using gas chromatography-mass spectrometry technique. Aspartate was taken up rapidly by both preparations, but the rates of transport were faster in astrocytes than in synaptosomes. In synaptosomes, 15N was incorporated predominantly into glutamate, whereas in glial cells, glutamine and other 15N-amino acids were also produced. In both preparations, the initial rate of N transfer from aspartate to glutamate was within a factor of 2-3 of that in the opposite direction. The rates of transamination were greater in synaptosomes than in astrocytes. Omission of glucose increased the formation of [15N]-glutamate in synaptosomes, but not in astrocytes. Rotenone substantially decreased the rate of transamination. There was no detectable incorporation of 15N from labeled aspartate to 6-amino-15N-labeled adenine nucleotides during 60-min incubation of synaptosomes under a variety of conditions; however, such activity could be demonstrated in glial cells. The formation of 15N-labeled adenine nucleotides was marginally increased by the presence of 1 mM aminooxyacetate, but was unaffected by pretreatment with 1 mM 5-amino-4-imidazolecarboxamide ribose. It is concluded that (1) aspartate aminotransferase is near equilibrium in both synaptosomes and astrocytes under cellular conditions, but the rates of transamination are faster in the nerve endings; (2) in the absence of glucose, use of amino acids for the purpose of energy production increases in synaptosomes, but may not do so in glial cells because the latter possess larger glycogen stores; and (3) nerve endings have a very limited capacity for salvage of the adenine nucleotides via the purine nucleotide cycle.
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PMID:Cerebral aspartate utilization: near-equilibrium relationships in aspartate aminotransferase reaction. 809 34

A total of 150 amino acid sequences of vitamin B6-dependent enzymes are known to date, the largest contingent being furnished by the aminotransferases with 51 sequences of 14 different enzymes. All aminotransferase sequences were aligned by using algorithms for sequence comparison, hydropathy patterns and secondary structure predictions. The aminotransferases could be divided into four subgroups on the basis of their mutual structural relatedness. Subgroup I comprises aspartate, alanine, tyrosine, histidinol-phosphate, and phenylalanine aminotransferases; subgroup II acetylornithine, ornithine, omega-amino acid, 4-aminobutyrate and diaminopelargonate aminotransferases; subgroup III D-alanine and branched-chain amino acid aminotransferases, and subgroup IV serine and phosphoserine aminotransferases. (N-1) Profile analysis, a more stringent application of profile analysis [Gribskov, M., McLachlan, A. D. and Eisenberg, D. (1987) Proc. Natl Acad. Sci. USA 84, 4355-4358], established the homology among the enzymes of each subgroup as well as among all subgroups except subgroup III. However, similarity of active-site segments and the hydropathy patterns around invariant residues suggest that subgroup III, though most distantly related, might also be homologous with the other aminotransferases. On the basis of the comprehensive alignment, a new numbering of amino acid residues applicable to aminotransferases (AT) in general is proposed. In the multiply aligned sequences, only four out of a total of about 400 amino acid residues proved invariant in all 51 sequences, i.e. Gly(314AT)197, Asp/Glu(340AT)222, Lys(385AT)258 and Arg(562AT)386, the number not in parentheses corresponding to the structure of porcine cytosolic aspartate aminotransferase. Apparently, the aminotransferases constitute a group of homologous proteins which diverged into subgroups and, with some exceptions, into substrate-specific individual enzymes already in the universal ancestor cell.
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PMID:Aminotransferases: demonstration of homology and division into evolutionary subgroups. 851 4

The mechanism of transamination catalyzed by Escherichia coli wild-type aspartate aminotransferase (AATase) and the mutant AAtase in which Tyr-225 is converted to Phe (Y225F) was investigated. The absorbance spectrum of wild-type AATase in the presence of excess L-Asp and oxalacetate is dominated by species absorbing near 330 nm. The primary C alpha 2H-Asp kinetic isotope effects (KIEs) on reactions catalyzed by wild-type AAtase at pH 8.9 and 7.5 on kcat/KMAsp are approximately 2, and the KIEs on kcat are 1.9 (pH 8.9) and 1.4 (pH 7.5). The C alpha 2H-Asp KIEs on reactions catalyzed by Y225F are near unity at both pH values. The solvent deuterium KIEs (SKIEs) on kcat for reactions with L-Asp catalyzed by wild-type AATase and Y225F at their pH/pD maxima approximately 2, and the SKIE on kcat/kMAsp is increased from 1.3 to 2.3 by the mutation. The C4' (S)-2H-pyridoxamine 5'-phosphate KIE values on reactions of alpha-ketoacids with both enzymes are near unity. The viscosity effects on kcat/KMAsp and kcat for wild-type AAtase at pH 9 are 0.10 and 0.31, respectively, indicating that the reaction is partially diffusion limited. The viscosity effects on kcat/KMAsp and kcat for Y225F are reduced to -0.02 and 0.06, respectively, indicating that the mutant catalyzed reaction is almost fully chemistry-limited. A free-energy profile for the L-Asp-to-oxalacetate half-reaction was constructed for wild-type AAtase. C alpha H abstraction, ketimine hydrolysis, and oxalacetate dissociation are partially rate-determining. Ketimine hydrolysis is the sole rate-determining step for the corresponding Y225F- catalyzed reaction.
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PMID:The reaction catalyzed by Escherichia coli aspartate aminotransferase has multiple partially rate-determining steps, while that catalyzed by the Y225F mutant is dominated by ketimine hydrolysis. 861 15


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