UNIPROT:P17174 - FACTA Search

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Query: UNIPROT:P17174 (aspartate aminotransferase)
14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase are homologous proteins. Both are encoded by nuclear DNA and synthesized on free polysomes. The organization of their genes is very similar, five out of a total of eight introns are located at the same nucleotide position. A variant consensus sequence was observed at the 3' splice site of introns of genes of imported mitochondrial proteins which may reflect the existence of splicing factors specific for the genes of this particular group of nuclear-encoded proteins. To date the amino acid sequences of 22 aminotransferases are known. A rigorous analysis yielded clear evidence that aspartate, tyrosine, and histidinol-phosphate aminotransferases are homologous proteins despite their low degree of sequence identity. The evolutionary relationship among the vitamin B6-dependent enzymes in general appears less clear. Conceivably, their common structural and mechanistic features are dictated by the chemical properties of pyridoxal 5'-phosphate rather than being due to a common ancestor of their protein moieties. In agreement with this notion, the ubiquitous active-site lysine residue that forms a Schiff base with the coenzyme can be replaced in the case of aspartate aminotransferase by a histidine residue without complete loss of catalytic competence.
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PMID:Evolutionary and biosynthetic aspects of aspartate aminotransferase isoenzymes and other aminotransferases. 219 17

We report a case of fatal theophylline overdose in a 16-year-old asthmatic boy who presented with seizures, respiratory arrest, and a theophylline concentration of 117 mg/L in serum. His hospital course was complicated by refractory hypotension and severe ischemic necrosis of skeletal muscle, bowel, and liver. The metabolic abnormalities observed early in his hospital course included severe hyperkalemia, hyperphosphatemia, hypermagnesemia, hypocalcemia, and profound metabolic acidosis. These metabolic abnormalities differ from those previously reported in cases of massive theophylline overdose. The metabolic abnormalities observed in this patient probably reflected his extensive ischemic tissue damage with release of intracellular ions and associated acidemia. Markedly increased catalytic activities of creatine kinase, aspartate aminotransferase, and alanine aminotransferase in serum were also noted.
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PMID:Massive theophylline overdose with atypical metabolic abnormalities. 230 99

The release of isoenzymes, i.e. aspartate aminotransferase (ASAT), lactic dehydrogenase (LDH), malic dehydrogenase (MDH), and of creatine kinase (CK) after electric His bundle ablation is presented. The enzyme release is increasing with the amount of energy applied during the intervention. Mitochondrial MDH and ASAT were found in six of the seven patients. Therefore, the size of myocardial necrosis by ablation can be estimated by the sequence of the release of the enzymes, and the isoenzymes, respectively.
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PMID:[Enzyme liberation after electrical His bundle ablation]. 237 36

A protease from Streptomyces violaceochromogenes (Murao, S., Nishino, Y., & Maeda, Y. (1984) Agric. Biol. Chem. 48, 2163-2166) is known to inactivate pig heart aspartate aminotransferase [EC 2.6.1.1]. Chemical analysis of the core proteins and peptide fragments produced upon proteolysis of the aminotransferase revealed that peptide bond cleavage occurred specifically at Leu 20 with concomitant inactivation. Neither inactivation nor peptide bond cleavage was observed with the mitochondrial isoenzyme. The proteolytically produced derivative 21-412 of the cytosolic isoenzyme retained approximately 0.1% enzymic activity for transamination with natural dicarboxylic substrates. The pyridoxal form of the derivative 21-412 was fully converted by cysteinesulfinate or alanine to the pyridoxamine form and conversely the pyridoxamine form of the derivative was also fully converted by 2-oxoglutarate or pyruvate into the pyridoxal form, indicating that the derivative was still catalytically competent. However, the rates of reaction with dicarboxylic substrates were much reduced whereas the rates with monocarboxylic substrates remained at an order of magnitude similar to that observed with the native enzyme. Thus the NH2-terminal segment appears to be an import structural component which determines the substrate specificity of aspartate aminotransferase for dicarboxylic keto and amino acids. A substantial alteration in the molecular structure accompanying the loss of the NH2-terminal 20 residues was also reflected by the decrease in heat stability and in the lowering of the pKa value for His 68, which is involved in the intersubunit interaction of this dimeric enzyme.
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PMID:Selective proteolysis of cytosolic aspartate aminotransferase by a new microbial protease. 351 98

200 MHz proton nuclear magnetic resonance spectra were compared between the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from pig heart. The pattern of signal distribution in the whole spectral region differed considerably between the two isoenzymes, reflecting the difference in their amino acid sequences. A group of distinct signals were resolved at elevated temperatures (50 to 70 degrees C) in the low field region (9.0 to 7.5 ppm) of the spectra of both isoenzymes in the pyridoxal form. Most of these signals were also observable at 28 degrees C although some showed considerable line broadening. Among resonance lines in this spectral region, cAAT in the pyridoxal form showed four pH-titratable resonances with pKa of 9.54, 6.72, 5.69, and 4.87 at 28 degrees C. Variation in pK and line width of these signals indicated differences in the microenvironment of histidyl residues. On the other hand, mAAT showed six pH-titratable resonances with pKa of 6.73 (peak 2), 6.77 (peak 3), 6.07 (peak 4), 4.71 (peak 5), 4.54 (peak 6), and 4.33 (peak 7). Peaks 2, 3, and 4 were narrow and others were considerably broad. Thus, only part of the histidyl residues present in each isoenzyme (8 and 10 His/monomeric unit of cAAT and mAAT, respectively) appeared on the spectra as pH-titratable resonances. With both isoenzymes, chemical shift and pKa values of these signals obtained for the pyridoxal form were indistinguishable from those for the pyridoxamine form and the borohydride-reduced form. None of the observable signals were affected upon the interaction of cAAT with glutarate. By contrast, peaks 2 and 4 in mAAT showed subtle but distinct chemical shift changes upon complex formation with succinate, suggesting that these two resonances are due to histidyl residues located at the part of the enzyme molecule which undergoes a conformational change upon the interaction with the dicarboxylate.
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PMID:1H NMR studies of aspartate aminotransferase. Histidyl residues of cytosolic and mitochondrial isoenzymes. 670 84

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

In an attempt to change the reaction and substrate specificity of aspartate aminotransferase, several apolar active-site residues were substituted in turn with a histidine residue. Aspartate aminotransferase W140H (of Escherichia coli) racemizes alanine seven times faster (Kcat' = 2.2 x 10(-4) s-1) than the wild-type enzyme, while the aminotransferase activity toward L-alanine was sixfold decreased. X-ray crystallographic analysis showed that the structural changes brought about by the mutation are limited to the immediate environment of H140. In contrast to the tryptophan side chain in the wild-type structure, the imidazole ring of H140 does not form a stacking interaction with the coenzyme pyridine ring. The angle between the two ring planes is about 50 degrees. Pyridoxamine 5'-phosphate dissociates 50 times more rapidly from the W140H mutant than from the wild-type enzyme. A model of the structure of the quinonoid enzyme substrate intermediate indicates that H140 might assist in the reprotonation of C alpha of the amino acid substrate from the re side of the deprotonated coenzyme-substrate adduct in competition with si-side reprotonation by K258. In aspartate aminotransferase I17H (of chicken mitochondria), the substituted residue also lies on the re side of the coenzyme. This mutant enzyme slowly decarboxylates L-aspartate to L-alanine (Kcat' = 8 x 10(-5) s-1). No beta-decarboxylase activity is detectable in the wild-type enzyme. In aspartate aminotransferase V37H (of chicken mitochondria), the mutated residue lies besides the coenzyme in the plane of the pyridine ring; no change in reaction specificity was observed. All three mutations, i.e. W140-->H, I17-->H and V37--H, decreased the aminotransferase activity toward aromatic amino acids by 10-100-fold, while decreasing the activity toward dicarboxylic substrates only moderately to 20%, 20% and 60% of the activity of the wild-type enzymes, respectively. In all three mutant enzymes, the decrease in aspartate aminotransferase activity at pH values lower than 6.5 was more pronounced than in the wild-type enzyme, apparently due to the protonation of the newly introduced histidine residues. The study shows that substitutions of single active-site residues may result in altered reaction and substrate specificities of pyridoxal-5'-phosphate-dependent enzymes.
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PMID:Substitution of apolar residues in the active site of aspartate aminotransferase by histidine. Effects on reaction and substrate specificity. 785 26

hisH encodes imidazole acetol phosphate (IAP) aminotransferase in Zymomonas mobilis and is located immediately upstream of tyrC, a gene which codes for cyclohexadienyl dehydrogenase. A plasmid containing hisH was able to complement an Escherichia coli histidine auxotroph which lacked the homologous aminotransferase. DNA sequencing of hisH revealed an open reading frame of 1,110 bp, encoding a protein of 40,631 Da. The cloned hisH product was purified from E. coli and estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of 40,000 Da. Since the native enzyme had a molecular mass of 85,000 Da as determined by gel filtration, the active enzyme species must be a homodimer. The purified enzyme was able to transaminate aromatic amino acids and histidine in addition to histidinol phosphate. The existence of a single protein having broad substrate specificity was consistent with the constant ratio of activities obtained with different substrates following a variety of physical treatments (such as freeze-thaw, temperature inactivation, and manipulation of pyridoxal 5'-phosphate content). The purified enzyme did not require addition of pyridoxal 5'-phosphate, but dependence upon this cofactor was demonstrated following resolution of the enzyme and cofactor by hydroxylamine treatment. Kinetic data showed the classic ping-pong mechanism expected for aminotransferases. Km values of 0.17, 3.39, and 43.48 mM for histidinol phosphate, tyrosine, and phenylalanine were obtained. The gene structure around hisH-tyrC suggested an operon organization. The hisH-tyrC cluster in Z. mobilis is reminiscent of the hisH-tyrA component of a complex operon in Bacillus subtilis, which includes the tryptophan operon and aroE. Multiple alignment of all aminotransferase sequences available in the database showed that within the class I superfamily of aminotransferases, IAP aminotransferases (family I beta) are closer to the I gamma family (e.g., rat tyrosine aminotransferase) than to the I alpha family (e.g., rat aspartate aminotransferase or E. coli AspC). Signature motifs which distinguish the IAP aminotransferase family were identified in the region of the active-site lysine and in the region of the interdomain interface.
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PMID:Imidazole acetol phosphate aminotransferase in Zymomonas mobilis: molecular genetic, biochemical, and evolutionary analyses. 788 15

Molecular modeling suggested that the large and small domain of mitochondrial aspartate aminotransferase might be linked by an engineered disulfide bond that could be expected to interfere with ligand-induced and syncatalytic changes in conformation and thus to assist in the elucidation of their significance for the catalytic mechanism. His-352, which is situated in the small domain close to Cys-166 of the large domain, was replaced with a cysteine residue by oligonucleotide-directed mutagenesis. Aspartate aminotransferase H352C, that had not been exposed to reducing conditions, in part contained a disulfide bond between Cys-166 and Cys-352. Exposure to a reducing agent cleaved the crosslink completely and produced an enzyme derivative with 8% of the activity of the wild type enzyme. Cu2+-mediated autoxidation resulted in complete formation of the disulfide bond and a decrease in enzymic activity to 2%. Independently of the redox state of the disulfide bond, the H352C substitution seems to shift the equilibrium from the open toward the closed conformation of the enzyme. This change in conformation was accompanied by an increase in the binding affinity for both the amino and oxo acid substrate by one order of magnitude. Apparently, 1-2 kcal/mol of the binding energy of the substrates are no longer diverted to shift the conformational equilibrium toward the closed conformation. The kcat/Km values were unchanged or even increased in the reduced form of the mutant enzyme and only slightly decreased in its oxidized form. Both the disulfide-independent decrease in enzymic activity, as observed in reduced aspartate aminotransferase H352C and also in two other mutant enzymes (C166H/H352C and H352Q), and the redox-dependent modulation of activity indicate that unhindered domain movements are essential for full catalytic competence of aspartate aminotransferase.
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PMID:Modulation of the activity of mitochondrial aspartate aminotransferase H352C by the redox state of the engineered interdomain disulfide bond. 792 41

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

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