Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Structural organization of the entire mouse mitochondrial aspartate aminotransferase (EC 2.6.1.1) gene was determined by analyzing the overlapping genomic clones obtained from a Charon 4A DNA library. The gene is 25 X 10(3) base-pairs long and contains ten exons interrupted by nine introns of various sizes. The 5' and 3'-flanking regions, the exact sizes and boundaries of the exon blocks including the transcription-initiation sites were determined. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements, such as TATA and CAAT boxes, but contains G + C-rich sequences, two putative binding sites for a cellular transcription factor, Sp1, and multiple transcription-initiation sites. Moreover, the sequences around the transcription-initiation sites are compatible with the formation of a number of potentially stable stem-loop structures. The leader sequence, which is essential for the transport of the protein into the mitochondria, is coded by the first exon and is separated from the mature protein by the first intron. The pyridoxal 5'-phosphate-binding domain, consisting of seven alternating beta-sheets and alpha-helical polypeptide strands, is separated by four introns present at the ends of alpha-helices. These genomic DNA structures suggest that the introns were not inserted into a previously uninterrupted coding sequence, but rather are products of evolution of the ancestral gene. However, a further correlation between the positions of introns relative to the well-defined structural domains of the mature protein was not obvious.
J Mol Biol 1987 Nov 05
PMID:Structural organization of the mouse mitochondrial aspartate aminotransferase gene. 282 32

The amino acid pool sizes of Trichomonas vaginalis are reported. Alanine, glutamic acid, proline and leucine account for 72% of the measured amino acids. Growth of T. vaginalis was unaffected by gostatin, an irreversible inhibitor of aspartate aminotransferase, when the enzyme activity within the cell had been completely inhibited and a specific elevation of the aspartate pool had occurred. In media lacking aspartate and glutamate, the amino acid substrates of the aspartate aminotransferase reaction, gostatin caused a larger increase in the aspartate pool. During incubation of cells with or without gostatin, aspartate and glutamate were produced in the medium, presumably by proteolysis of medium proteins. Hence any requirement for the aspartate aminotransferase reaction might have been bypassed. Glutamate-gamma-hydroxamate and aminooxyacetate inhibited growth of T. vaginalis but caused large changes in the pool-sizes of aspartate, glutamate, pyruvate plus oxaloacetate and 2-oxoglutarate, suggesting a more general interference with amino acid metabolism.
Mol Biochem Parasitol 1986 Oct
PMID:Modulation of amino acid and 2-oxo acid pools in Trichomonas vaginalis by aspartate aminotransferase inhibitors. 287 95

Measurement of the stereospecific release of the pro-S proton from C-4' of enzyme-bound pyridoxamine 5'-phosphate provides an experimental means to probe parts of the active site of aspartate aminotransferase independently of substrate turnover (Tobler, H. P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). The release of pro-S 3H from enzyme-bound [3H]pyridoxamine 5'-phosphate is 30,000 times faster than from free coenzyme. Enzyme-bound [3H]pyridoxine 5'-phosphate is not detritiated suggesting an essential role of the 4'-amino group. Formation of the unproductive complex of the [3H]pyridoxamine 5'-phosphate-enzyme with aspartate or glutamate results in a 400-fold acceleration of 3H release. In contrast, addition of borohydride or cyanoborohydride immediately stops 3H release. Experiments with a fluorescent reporter group and with differential chemical modifications indicate that the activating effect of aspartate on the release of 3H is accompanied by a shift of the so-called open/closed conformational equilibrium of the enzyme (Kirsch, J.F., Eichele, G., Ford, G. C., Vincent, M.G., Jansonius, J.N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) toward the closed conformation; the inhibiting effect of borohydride and cyanoborohydride appears to be accompanied by a shift toward the open conformation. Apparently, at least part of the catalytic apparatus of aspartate aminotransferase becomes fully operative only in the closed conformation of the enzyme.
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PMID:Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase. Activators and inhibitors. 288 26

Sodium valproate (VPA), the salt of a branched short-chain fatty acid, is a major antiepileptic whose mode of action, as yet unclear, may involve effects on the organization of membranes. VPA was either injected into rats whose liver and kidney mitochondria were then isolated, or was preincubated with isolated mitochondria. First, liver and kidney mitochondria were studied with paramagnetic probes. The electron paramagnetic resonance spectra of proteins of VPA-treated mitochondria spin-labeled with 4-maleimido-2,2,6,6-tetramethyl-1-pyrrolidinoxyl showed that the ratio of weakly immobilized to strongly immobilized SH groups was reduced with respect to control mitochondria, more so in liver than in kidney mitochondria of VPA-injected rats, and more so in kidney than in liver mitochondria for VPA-incubated mitochondria. Spectra of mitochondrial lipids spin-labeled with 5-doxyl stearic methyl ester showed that VPA had no significant effect on order parameters S. Second, the transmembrane movement of aspartate aminotransferase was studied by incubating liver mitochondria in a sucrose-succinate medium and then fractionating them. The translocation of aspartate aminotransferase from mitoplasts, vesicles formed of inner membrane and matrix, to the intermembrane fluid, was significantly higher in VPA-treated than in control mitochondria. Thus, VPA, at concentrations in the range of those used therapeutically, interacted with membranes by modifying the structural organization of the internal mitochondrial membrane, essentially the membrane protein conformation.
Mol Pharmacol 1986 Sep
PMID:Effects of sodium valproate on mitochondrial membranes: electron paramagnetic resonance and transmembrane protein movement studies. 301 82

Crystallographic refinement by simulated annealing with molecular dynamics has been applied to a 2.8 A (1 A = 0.1 nm) resolution X-ray structure of aspartate aminotransferase. Comparison of the refined structure and a structure obtained by combined restrained least-squares refinement and manual re-fitting shows a similar R factor, stereochemistry, and mean difference from the isomorphous replacement phase centroids. Crystallographic refinement by simulated annealing accomplished structural changes and improvements of the electron density maps that were not possible by using restrained least-squares refinement without manual re-fitting. Crystallographic refinement by simulated annealing can generate an ensemble of structures, each of which agrees with the diffraction information. Regions of large variations of the ensemble indicate either erroneously fitted or disordered segments of the macromolecule.
J Mol Biol 1988 Oct 05
PMID:Crystallographic refinement by simulated annealing. Application to a 2.8 A resolution structure of aspartate aminotransferase. 306 81

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of aspartate aminotransferase. It forms a salt bridge with the beta or gamma carboxylate group of the substrate [Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525]. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity (kcat/KM) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and alpha-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9 fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration, no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (kcat/KM)cationic/(kcat/KM)anionic is in the range 2-40-fold for R292D, whereas this ratio has a range of [(0.3-6) x 10(-6)]-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis. 316

The crucial step in enzymatic transamination is the tautomerization of aldimine/ketimine intermediates, formed between the pyridoxyl coenzyme and the amino/keto acid substrate, which is catalyzed primarily by the active site residue Lys-258 (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921; W. L. Finlayson and J. F. Kirsch, in preparation). Tyr-70 is localized in close proximity to Lys-258 and, in addition, forms a hydrogen bond with the coenzyme phosphate. Tyr-70 has been postulated to have an important role in the tautomerization (Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525). This hypothesis has now been tested by the construction and analysis of a mutant Escherichia coli aspartate aminotransferase in which Tyr-70 has been changed to Phe (Y70F). Y70F retains at least 15% of the maximal activity of the wild type enzyme (WT) (kcat = 170 +/- 15 s-1 for WT versus greater than or equal to 26 +/- 3 s-1 for Y70F and shows increased Michaelis constants for both substrates (KmAsp = 2.5 +/- 0.4 mM; Km alpha Kg = 0.59 +/- 0.08 mM for WT versus KmAsp = 3.9 +/- 0.3 mM; Km alpha Kg = 2.70 +/- 0.02 mM for Y70F (where alpha Kg is alpha-ketoglutarate) ). The spectrophotometrically determined pK a values of the internal aldimines formed between pyridoxal 5'-phosphate (PLP) and Lys-258 are identical for WT and Y70F. In assays where excess L-aspartate and excess PLP are incubated with either WT or Y70F, the mutant enzyme converts the free PLP to free pyridoxamine 5'-phosphate 80-fold faster than WT (k = (3.75 +/- 0.23) X 10(-2)s-1 for Y70F versus (4.90 +/- 0.02) X 10(-4)s-1 for WT). Y70F also converts free pyridoxamine 5'-phosphate to free PLP faster than WT. Thus, Y70F dissociates coenzyme more readily than does WT. It therefore appears that the role of Tyr-70 is mainly in preventing the dissociation of the coenzyme from the enzyme. Tyr-70 does not function in an essential chemical step.
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PMID:Tyrosine 70 increases the coenzyme affinity of aspartate aminotransferase. A site-directed mutagenesis study. 330 7

Structural organization of the mouse mitochondrial malate dehydrogenase (EC 1.1.1.37) gene was determined by analyzing a genomic DNA fragment isolated from a cosmid library. The gene is 12,000 base-pairs long and contains nine exons interrupted by eight introns of various sizes. The 5' and 3'-flanking regions, and the exact sizes and boundaries of the exon blocks including the transcription-initiation sites were determined. In the 5'-flanking region, there is neither a TATA box nor a CAAT box. Instead of these sequences, there are six copies of the GGGCGG or CCGCCC sequence, which is a potential binding site for the transcription factor, Sp1. The 5'-flanking region up to about 600 nucleotides is G + C-rich (65%) and contains sequences compatible with the formation of a number of potentially stable stem-loop structures. S1 nuclease mapping and primer extension analysis demonstrated that transcription of the mitochondrial malate dehydrogenase gene initiates at multiple sites. Comparison of the nucleotide sequence of the promoter region of the mitochondrial malate dehydrogenase gene with that of the mitochondrial aspartate aminotransferase gene, revealed that there are several highly conserved regions between these two mitochondrial enzyme genes participating in the malate-aspartate shuttle.
J Mol Biol 1988 Mar 05
PMID:Structural organization of the mouse mitochondrial malate dehydrogenase gene. 337 35

We have cloned and characterized a mouse cytosolic aspartate aminotransferase (AspAT) (EC 2.6.1.1) gene, which is about 32,000 base-pairs long and is interrupted by eight introns. The 5' and 3'-flanking regions, and the exact sizes and boundaries of the exon blocks, including the transcription-initiation sites, were determined. The 5' end of the gene lacks the TATA and CAAT boxes characteristic of eukaryotic promoters, but contains G + C-rich sequences, three putative binding sites for a cellular transcription factor, Sp1, and multiple transcription-initiation sites. The sequences around the transcription-initiation sites are compatible with the formation of a number of potentially stable stem-loop structures. We compared the structural organization of the mouse cytosolic AspAT gene with that of the mouse mitochondrial AspAT gene, which has nine introns. We found that the promoter regions share a high level of homology and five of the introns are at identical places. This close matching leads to the tentative conclusion that the introns were in place before the divergence of cytosolic and mitochondrial isoenzyme genes.
J Mol Biol 1988 Mar 05
PMID:Structural organization of the mouse aspartate aminotransferase isoenzyme genes. Introns antedate the divergence of cytosolic and mitochondrial isoenzyme genes. 337 36

We have previously identified a unique site, pac, from which packaging of precursor concatameric viral DNA into proheads starts during the maturation process of bacteriophage CP-T1. The direction of this packaging was determined from restriction enzyme cleavage patterns of CP-T1 DNA. A restriction enzyme generated fragment containing pac was cloned and the surrounding DNA region sequenced. Analysis of the nucleotide sequence revealed numerous repeat regions related to the consensus sequence PuagttGAT.AAT.aa.t. Within the sequenced region an open reading frame encoding a 12260 Mr protein was also identified. This protein appears to share homology with the binding domains of known DNA binding proteins and may represent a putative Pac terminase possessing the specific endonuclease activity required for cleavage at the pac site. Minicell analysis of deletion derivatives of the pac-containing clone revealed a protein of approximately 12,900 Mr encoded within this same region, confirming that this Pac protein is phage encoded.
Mol Gen Genet 1988 Jun
PMID:Molecular analysis of the packaging signal in bacteriophage CP-T1 of Vibrio cholerae. 341 20


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