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:2.6.1.1 (aspartate aminotransferase)
21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Four nonapeptides that inhibit juvenile hormone synthesis have been isolated by four high performance liquid chromatographic steps from extracts of the brain of the field cricket, Gryllus bimaculatus. The primary structures of these peptides were assigned by Edman degradation and mass spectrometry as Gly-Trp-Gln-Asp-Leu-Asn-Gly-Gly-Trp-NH2 (Grb-AST B1), Gly-Trp-Arg-Asp-Leu-Asn-Gly-Gly-Trp-NH2 (Grb-AST B2), Ala-Trp-Arg-Asp-Leu-Ser-Gly-Gly-Trp-NH2 (Grb-AST B3), and Ala-Trp-Glu-Arg-Phe-His-Gly-Ser-Trp-NH2 (Grb-AST B4). Each of the peptides shows high sequence similarity to the locustamyoinhibiting peptide (Lom-MIP), but is structurally different from all the allatostatins so far identified. The synthetic allatostatins Grb-AST B1-4 are potent inhibitors (50% inhibition at 10(-8) to 7 x 10(-8) M) of juvenile hormone III biosynthesis by corpora allata from 3-day-old virgin females of G. bimaculatus using an in vitro bioassay. At 10(-7) M, Grb-AST B1 also strongly inhibits juvenile hormone III biosynthesis by corpora allata from 2-day-old adult males and 1-day-old (males and females) and 4-day-old (females) last instar larvae of G. bimaculatus. The inhibitory effect of Grb-AST B1 was also evident on corpora allata from a related species, Acheta domesticus. Inhibition of juvenile hormone synthesis by Grb-AST B1-4 is reversible.
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PMID:A family of neuropeptides that inhibit juvenile hormone biosynthesis in the cricket, Gryllus bimaculatus. 767 41

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

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylated O-linked carbohydrate chains (Konami Y, Yamamoto K. Osawa T, Irimura T (1994) FEBS Lett 342:334-38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinated Maackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.
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PMID:Cloning and sequence analysis of the Maackia amurensis haemagglutinin cDNA. 769 60

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 production of juvenile hormone III (JH III) by the corpora allata of the cockroach Diploptera punctata is regulated in part by peptides originating from the brain. One group of these peptides, termed allatostatins, reversibly inhibits the biosynthesis of JH in vitro. Allatostatin 4 (AST4: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide) is the smallest member of the AST family yet defined and was used as the benchmark peptide for these initial structure-activity studies. Two initial analog series of AST4 were examined for the ability of each analog to inhibit JH biosynthesis by corpora allata in vitro. Each analog series consisted of analogs that contained a single amino acid change from the native AST4 sequence. The first series contained Ala replacement analogs and the second contained analogs with D-amino acid replacements. The first analog series used Ala replacements to help indicate which amino acid side chains were most important for inhibition of JH biosynthesis. The most important side chain appeared to be Leu8 followed by Phe6 and Tyr4. Additionally, the D-amino acid series suggested that a secondary structural element(s) at the C-terminus of AST4 could be important to the biological activity.
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PMID:Structure-activity studies of allatostatin 4 on the inhibition of juvenile hormone biosynthesis by corpora allata: the importance of individual side chains and stereochemistry. 785 67

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

Sixteen Escherichia coli clinical isolates which were resistant to ampicillin and amoxicillin-clavulanate but susceptible to cephalothin were studied. Eight strains showed the presence of a beta-lactamase which comigrates with reference OXA-1 enzyme. The eight other strains produced different TEM-1 derivatives which had in common a higher Km for penicillins and a higher 50% inhibitory concentration for the beta-lactamase inhibitors. By oligotyping and sequencing of PCR products, it was shown that Ser (AGC) (TEM-30; also called TRI-1) in three strains and Cys (TGC) (TEM-31; also called TRI-2) in one strain were substituted for Arg-241 (CGC), that Leu (CTG) (TEM-33) and Val (GTG) (TEM-34) in one strain each were substituted for Met-67 (ATG), and that in other mutants the two latter substitutions occurred together with the substitution of Asp (GAT) (TEM-35 and TEM-36) for Asn-272 (AAT). Therefore, different sets of amino acid substitutions of TEM-1 can be found in clinical isolates and lead to resistance to beta-lactamase inhibitors.
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PMID:Emergence of clinical isolates of Escherichia coli producing TEM-1 derivatives or an OXA-1 beta-lactamase conferring resistance to beta-lactamase inhibitors. 806 42

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


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