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)

The gene encoding aspartate aminotransferase of a thermophilic Bacillus species, YM-2, has been cloned and expressed efficiently in Escherichia coli. The primary structure of the enzyme was deduced from nucleotide sequences of the gene and confirmed mostly by amino acid sequences of tryptic peptides. The gene consists of 1,176 base pairs encoding a protein of 392 amino acid residues; the molecular mass of the enzyme subunit is estimated to be 42,661 daltons. The active site lysyl residue that binds the coenzyme, pyridoxal phosphate, was identified as Lys-239. Comparison of the amino acid sequence with those of aspartate aminotransferases from other organisms revealed very low overall similarities (13-14%) except for the sequence of the extremely thermostable enzyme from Sulfolobus solfataricus (34%). Several amino acid residues conserved in all the compared sequences include those that have been reported to participate in binding of the coenzyme in three-dimensional structures of the vertebrate and E. coli enzymes. However, the strictly conserved arginyl residue that is essential for binding of the distal carboxyl group of substrates is not found in the corresponding region of the sequences of the thermostable enzymes from the Bacillus species and S. solfataricus. The Bacillus aspartate aminotransferase has been purified from the E. coli clone cell extracts on a large scale and crystallized in the buffered ammonium sulfate solution by the hanging drop method. The crystals are monoclinic with unit cell dimensions a = 121.2 A, b = 110.5 A, c = 81.8 A, and beta = 97.6 degrees, belonging to space group C2, and contain two molecules in the asymmetric unit. The crystals of the enzyme-alpha-methylaspartate complex are isomorphous with those without the substrate analog.
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PMID:Thermostable aspartate aminotransferase from a thermophilic Bacillus species. Gene cloning, sequence determination, and preliminary x-ray characterization. 199 6

Apoenzyme samples of aspartate aminotransferase (AspAT) purified from the cytosolic fraction of pig heart were reconstituted with [4'-13C]pyridoxal 5'-phosphate (pyridoxal-P). The 13C NMR spectra of AspAT samples thus generated established the chemical shift of 165.3 ppm for C4' of the coenzyme bound as an internal aldimine with lysine 258 of the enzyme at pH 5. In the absence of ligands the chemical shift of C4' was shown to be pH dependent, shifting 5 ppm upfield to a constant value of 160.2 ppm above pH 8, the resulting pKa of 6.3 in agreement with spectrophotometric titrations. The addition of the competitive inhibitor succinate to the internal aldimine raises the pKa of the imine to 7.8, consistent with the theory of charge neutralization in the active site. In the presence of saturating concentrations of 2-methylaspartic acid the C4' signal of the coenzyme was shown to be invariant with pH and located at 162.7 ppm, midway between the observed chemical shifts of the protonated and unprotonated forms of the internal aldimine. The intermediate chemical shift of the external aldimine complex is thought to reflect the observation of an equilibrium mixture composed of roughly equal populations of the protonated ketoenamine and a dipolar anion species, corresponding to their respective spectral bands at 430 and 360-370 nm. Conversion to the pyridoxamine form was accomplished via reaction of the internal aldimine with L-cysteinesulfinate or by reduction with sodium borohydride, and the resulting C4' chemical shifts were identified by difference spectroscopy. Finally, the line widths of the C4' resonance under the various conditions were measured and qualitatively compared. The results are discussed in terms of the current mechanism and molecular models of the active site of AspAT.
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PMID:Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy. 200 79

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.
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PMID:Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence. 210 17

The structure of Escherichia coli aspartate aminotransferase complex with the inhibitor 2-methylaspartate, and that of the mutant enzyme in which an arginine was substituted for a lysine residue thereby forming a Schiff base with the coenzyme pyridoxal 5'-phosphate, were determined at 2.5 A resolution, by the molecular replacement method using the known structure of pig cytosolic aspartate aminotransferase. The enzyme catalyzes the reversible transamination between L-aspartate and alpha-ketoglutarate, and forms a dimeric structure of two identical subunits. Each subunit comprises two domains, a small and a large one. Although, in general, the overall and secondary structure of E. coli enzyme are similar to those of higher animals, some differences of enzymatic action between the enzyme from E. coli and those from higher animals could be explained on the basis of the X-ray structures and molecular mechanics calculation based on them.
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PMID:Three-dimensional structures of aspartate aminotransferase from Escherichia coli and its mutant enzyme at 2.5 A resolution. 212 25

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

The method of Smith and Hartman [J. Biol. Chem., 263, 4921-4925 (1988)] for introducing the non-natural lysine analog, S-(2-aminoethyl)cysteine, into specific sites in proteins by alkylation of a genetically introduced cysteine with 2-bromoethylamine has been generalized to be applicable to proteins containing one or more endogenous cysteines. The target cysteine residue introduced at the active site of aspartate aminotransferase is protected by bound cofactor. The enzyme is partially unfolded in low concentrations of urea, and the non-active site cysteine residues derivatized by a reversible thiol protecting reagent. The active site cysteine is then exposed and alkylated in 6 M urea. Enzyme activity is regenerated by removal of the thiol protecting groups and refolding of the protein.
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PMID:Sequential protection-modification method for selective sulfhydryl group derivatization in proteins having more than one cysteine. 221 35

The three-dimensional structure of a mutant of the aspartate aminotransferase from Escherichia coli, in which the active-site lysine has been substituted by alanine (K258A), has been determined at 2.8-A resolution by X-ray diffraction. The mutant enzyme contains pyridoxamine phosphate as cofactor. The structure is compared to that of the mitochondrial aspartate aminotransferase. The most striking differences, aside from the absence of the lysine side chain, occur in the positions of the pyridoxamine group and of tryptophan 140.
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PMID:2.8-A-resolution crystal structure of an active-site mutant of aspartate aminotransferase from Escherichia coli. 251 75

A co-culture system of cerebellar granule cells (glutamatergic neurons) and hepatocytes has been developed. Petri dishes divided in halves by a temporary septum were coated with poly-L-lysine and cerebellar granule cells plated in one of the compartments. Five days later hepatocytes were plated in the other compartment and after 2 days the septum was removed and the two cell types shared the same culture medium for a period of 5 days. During this period of time cultures of neurons and hepatocytes kept separately or in co-culture exhibited identical characteristics with regard to activities of pyruvate kinase and glucokinase (hepatocytes), aspartate aminotransferase (neurons) as well as evoked transmitter release (neurons) and content of cytochrome P-450 (hepatocytes). The results show that it is possible to maintain neurons and hepatocytes in co-culture sharing the same culture medium for a prolonged period of time. Such a system may serve as a pharmacological model to study interactions between liver and brain cells with regard to neuroactive drugs.
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PMID:Characterization of a co-culture system of neurons and hepatocytes. 256 Aug 19

The primary structure of tyrosine aminotransferase, as deduced from the nucleotide sequence of complementary DNA, was confirmed by fast atom bombardment mass spectrometry of tryptic peptides derived from the purified protein. Limited digestion of the native enzyme with trypsin released an acetylated, amino-terminal peptide; the new amino terminus in the modified enzyme was Val65. Endogenous proteases generated a chromatographically separable form of tyrosine aminotransferase that began at Lys35. Neither trypsin nor the other proteases altered the catalytic activity of tyrosine aminotransferase. Reduction of the holoenzyme with sodium borohydride yielded a major tryptic peptide containing phosphopyridoxamine bound to lysine 280, which probably functions in transamination. The carboxyl terminus of tyrosine aminotransferase contains features that typify proteins with short half-lives; it includes two negatively charged, hydrophilic segments that are enriched for glutamyl residues and are similar to a PEST region in ornithine decarboxylase (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). Tyrosine aminotransferase belongs to a superfamily of enzymes which includes aspartate aminotransferase and can be aligned so that many invariant, functional residues coincide. Like the isoenzymes of aspartate aminotransferase, tyrosine aminotransferase may contain two domains, with a central, catalytic core, and a small domain made up of both amino- and carboxyl-terminal components. We speculate that the exposed small domain may confer the unusually rapid degradative rate that characterizes this enzyme.
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PMID:The structure of tyrosine aminotransferase. Evidence for domains involved in catalysis and enzyme turnover. 256 40

The effects of recombinant human interleukin-1 beta (rhIL-1 beta) on various serum constituents were studied following subcutaneous injection (12.5 or 125 micrograms/kg) in female Wistar rats. Protein electrophoresis and the determination of the serum concentrations of carboxypeptidase N (CPN), aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, aldolase, total proteins, iron, urea, creatinine, and several amino acids were performed 12, 24, and 72 hr after injection. With both doses of rhIL-1 beta, iron, albumin, CPN, and lysine were significantly decreased whereas alpha 2-globulin, urea, and creatinine were significantly increased 12 hr after administration. Iron and CPN were still low after 24 hr but returned to normal levels after 72 hr. With the higher dose of rhIL-1 beta, only alanine and phenylalanine levels were increased after 12 and 72 hr, taurine after 12 hr, and methionine after 24 hr. There were no biochemical or histological signs of hepatotoxicity. The findings indicate that rhIL-1 beta produces a reversible alteration of various biochemical plasma constituents without any apparent signs of cytotoxicity. Moreover, the decrease in CPN observed may influence the degradation of inflammatory peptides.
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PMID:Recombinant human interleukin-1 beta decreases serum carboxypeptidase N and modifies serum amino acid concentrations in rats. 278 29


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