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)

Feedback control between flux through the phosphorylating electron transport chain and the coordination of flux through individual steps of the citric acid cycle have been investigated under a number of different conditions of substrate availability and workloads in the isolated perfused rat heart. The transition from substrate-free perfusion to perfusion with glucose and insulin with no change of workload was associated with increases in the pool sizes of citric acid cycle intermediates except for oxaloacetate, but with an initial imbalance of flux through individual steps in the cycle and transport of anions of the malate-aspartate cycle across the mitochondrial membrane. Flux through citrate synthase initially increased while that through alpha-ketoglutarate dehydrogenase decreased. Of the components of the malate-aspartate cycle, flux through the malate-alpha-ketoglutarate exchange was increased prior to that through the glutamate-aspartate exchange and intramitochondrial aspartate aminotransferase. These changes can be accounted for on the basis of known kinetic controls of the enzyme and transport steps in response to increased pyruvate, acetyl-CoA, and NADH delivery at an approximately constant rate of ATP turnover.
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PMID:Coordination of citric acid cycle activity with electron transport flux. 126 91

Cytoplasmic aspartate aminotransferase from beef kidney loses 25% of its activity on nitration with tetranitromethane while the apoenzyme about 95%. In the holoenzyme 0.5 tyrosine residue and 1.0 tyrosine residue in the apoenzyme are nitrated per enzyme protomer. In addition 1 cysteine residue per protomer is oxidized in both. The presence of substrates, alpha-ketoglutarate and glutamate, both at ten times their Km values, does not change these results. Mercaptoethanol does not affect the residual activity of either the nitrated holo or apoenzyme. Dithionite abolishes the activity of the nitrated holoenzyme by reducing tha coenzyme moiety. It has no effect on the native holoenzyme or on either the native or nitroapoenzyme.
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PMID:Role of tyrosine residues in cytoplasmic aspartate aminotransferase from beef kidney. 127 58

The activities of aspartate and alanine aminotransferases in biological samples were assessed through a novel and sensitive procedure, based on the conversion of [U-14C]2-ketoglutarate to L-[U-14C]glutamate. In human plasma, the generation of L-[U-14C]glutamate was proportional to the volume of plasma (20-60 microL) and to the length of incubation (30-90 min). The reaction velocity was related to the temperature with a Q10 close to 1.7 for aspartate aminotransferase and 2.0 for alanine aminotransferase. At 37 degrees C, the 95% confidence interval in healthy subjects ranged from 5.1-18.8 U/mL (mean value 11.9 U/L) for aspartate aminotransferase and from zero to 20.1 U/L (mean value 9.9 U/L) for alanine aminotransferase. The intra-assay coefficient of variation did not exceed 2.5%. The present method was also applied to homogenates prepared from rat pancreatic islets, liver, heart, parotid glands, and erythrocytes, using no more than 40 micrograms wet weight of tissue per sample, and could thus be used in small biological samples, such as those obtained by needle biopsy.
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PMID:Radioisotopic assay of aspartate and alanine aminotransferase. 135 85

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.
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PMID:Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate. 161 Aug 31

The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and alpha-ketoglutarate. The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for alpha-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with beta-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F C alpha-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7). Conversely, the Y70F value of D(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5). These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionally of the deleted hydroxyl group is possibly replaced by a water molecule.
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PMID:Tyrosine 70 fine-tunes the catalytic efficiency of aspartate aminotransferase. 167 69

The Y70F mutant of aspartate aminotransferase has reduced affinity for coenzymes compared to the wild type. The equilibrium dissociation constants for pyridoxamine phosphate (PMP) holoenzymes, KPMPdiss, were determined from the association and dissociation rate constants to be 1.3 nM and 30 nM for the wild type and mutant, respectively. This increase in KPMPdiss for Y70F is due to a 27-fold increase in the dissociation rate constant. Pyridoxal phosphate (PLP) association kinetics are complex, with three kinetic processes detectable for wild type and two for Y70F. A directly determined, accurate value of KPLPdiss for wild type enzyme has been difficult to obtain because of the low value of this constant. The values of KPLPdiss for the holoenzymes were determined indirectly through the measured values for KPMPdiss, glutamate-alpha-ketoglutarate half-reaction equilibrium constants, and the equilibrium constant for the transamination of PLP by glutamate catalyzed by Y70F. The values of KPLPdiss obtained by this procedure are 0.4 pM for wild type and 40 pM for Y70F. The increases in KPMPdiss and KPLPdiss for Y70F correspond to delta delta G values of 1.9 and 2.7 kcal/mol, respectively, and are directly attributed to the loss of the hydrogen bond from the phenolic hydroxyl group of Tyr70 to the coenzyme phosphate. The delta G for association of PLP with wild type enzyme is 4.7 kcal/mol more favorable than that for PMP.
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PMID:Kinetics and equilibria for the reactions of coenzymes with wild type and the Y70F mutant of Escherichia coli aspartate aminotransferase. 167 70

Four aminotransferases were identified and characterized from Methanococcus aeolicus. Branched-chain aminotransferase (BcAT, EC 2.6.1.42), aspartate aminotransferase (AspAT, EC 2.6.1.1), and two aromatic aminotransferases (EC 2.6.1.57) were partially purified 175-, 84-, 600-, and 30-fold, respectively. The apparent molecular weight, substrate specificity, and kinetic properties of the BcAT were similar to those of other microbial BcATs. The AspAT had an apparent molecular weight of 162,000, which was unusually high. It had also a broad substrate specificity, which included activity towards alanine, a property which resembled the enzyme from Sulfolobus solfataricus. An additional alanine aminotransferase was not found in M. aeolicus, and this activity of AspAT could be physiologically significant. The apparent molecular weights of the aromatic aminotransferases (ArAT-I and ArAT-II) were 150,000 and 90,000, respectively. The methanococcal ArATs also had different pIs and kinetic constants. ArAT-I may be the major ArAT in methanococci. High concentrations of 2-ketoglutarate strongly inhibited valine, isoleucine, and alanine transaminations but were less inhibitory for leucine and aspartate transaminations. Aromatic amino acid transaminations were not inhibited by 2-ketoglutarate. 2-Ketoglutarate may play an important role in the regulation of amino acid biosynthesis in methanococci.
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PMID:Characterization of amino acid aminotransferases of Methanococcus aeolicus. 172 42

We have isolated an alfalfa leaf cDNA clone that encodes aspartate aminotransferase (AAT, EC 2.6.1.1) by direct complementation of an Escherichia coli aspartate auxotroph with a plasmid cDNA library. DNA sequence analysis of the recombinant plasmid, pMU1, revealed that a 1514 bp cDNA was inserted in the correct orientation and in-frame with the start of the lacZ coding sequence in the vector, pUC18. The resulting fusion protein is predicted to be 424 amino acids in length with a molecular weight of 46387 Daltons. The cDNA-encoded protein has a characteristic pyridoxal phosphate attachment site motif and has substantial amino acid sequence homology to both animal and bacterial AATs. Plasmid pMU1 encodes an AAT with a Km for aspartate of 3.3 mM, a Km for 2-oxoglutarate of 0.28 mM, and a pH optimum between 8.0 and 8.5. Several lines of evidence including Western blot analysis, the isoelectric point of the encoded protein, and the effect of pH on the activity of the fusion protein, suggest that the cDNA encodes the isozyme AAT-1 rather than AAT-2. Northern blot analysis showed that the aat-1 clone hybridized to a 1.6 kb transcript present in alfalfa leaves, roots and nodules. The relative concentrations of aat-1 mRNA in these tissues were 1:2:5, respectively. Thus, transcription of aat-1 appears to be induced during nodule development. Southern blot analysis suggested that AAT-1 in alfalfa is encoded by either a single-copy gene or a small, multigene family.
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PMID:Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase. 175 49

Site-directed mutagenesis of Tyr70 in the active site of Escherichia coli aspartate aminotransferase (AspAT) followed by kinetic studies has elucidated the roles of the hydroxyl group and benzene ring of Tyr70. X-ray crystallographic analysis showed that replacement of Tyr70 by Phe did not alter the active-site conformation of the enzyme. Comparison of the kinetic parameters of the four half-transamination reactions (the pyridoxal 5'-phosphate form of the enzyme with L-aspartate or L-glutamate and the pyridoxamine 5'-phosphate form with oxalacetate or 2-oxoglutarate) between the wild-type and [Tyr70----Phe]AspATs showed that the mutation increases the energy level of the transition state by 2 kcal.mol-1 for all the four substrates, suggesting some contribution of the hydroxyl group of Tyr70 to the transition state. When Phe70 was further replaced by Ser, the energy level of the transition state for L-glutamate or 2-oxoglutarate, but not for L-aspartate or oxalacetate, was further increased by 2-3 kcal.mol-1, suggesting that the presence of a benzene ring at position 70 is essential for recognizing the L-glutamate-2-oxoglutarate pair as substrates.
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PMID:Site-directed mutagenesis of Escherichia coli aspartate aminotransferase: role of Tyr70 in the catalytic processes. 186 57

Tyr225 in the active site of Escherichia coli aspartate aminotransferase (AspAT) was replaced by phenylalanine or arginine by site-directed mutagenesis. X-ray crystallographic analysis of Y225F AspAT showed that the benzene ring of Phe225 was situated at the same position as the phenol ring of Tyr225 in wild-type AspAT. The mutations resulted in a great decrease in the rate of the transamination reaction, suggesting that Tyr225 is important for efficient catalysis. The kinetic analysis of half-transamination reactions of Y225F AspAT with four substrates (aspartate, glutamate, oxalacetate, and 2-oxoglutarate) and some analogues (2-methylaspartate, succinate, and glutarate) revealed a considerable increase in the affinities for all these compounds. In contrast, affinity for the amino acid substrates was decreased by mutation to arginine, but affinities for the keto acid substrates and the two dicarboxylates (succinate and glutarate) were increased. The electrostatic interaction between O(3') of the coenzyme [pyridoxal 5'-phosphate (PLP)] and the residue at position 225 affected the pKa value of the Schiff base, which is formed between the epsilon-amino group of Lys258 and the aldehyde group of PLP; based on the spectrophotometric titration the pKa values were determined to be 6.8 for wild-type AspAT, 8.5 for Y225F AspAT, and 6.1 for Y225R AspAT in the absence of substrate. The absorption spectra of the three AspATs were almost identical in the acidic pH region, but the spectrum of Y225F AspAT differed from that of wild-type or Y225R AspAT in the alkaline pH region.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tyr225 in aspartate aminotransferase: contribution of the hydrogen bond between Tyr225 and coenzyme to the catalytic reaction. 186 10


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