Publications

• The aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction.

  Mahon M.M., Graber R., Christen P., Malthouse J.P.

  1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of as ... >> More

  1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes even larger decreases of 25,000- and 100,000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site. << Less

  Biochim. Biophys. Acta 1434:191-201(1999) [PubMed] [EuropePMC]

• Glutamic aspartic transaminase. I. Assay, purification, and general properties.

  JENKINS W.T., YPHANTIS D.A., SIZER I.W.

  J Biol Chem 234:51-57(1959) [PubMed] [EuropePMC]

• KINETIC STUDIES OF GLUTAMIC OXALOACETIC TRANSAMINASE ISOZYMES.

  HENSON C.P., CLELAND W.W.

  Biochemistry 3:338-345(1964) [PubMed] [EuropePMC]

• Chicken heart soluble aspartate aminotransferase. Purification and properties.

  Bertland L.H., Kaplan N.O.

  Biochemistry 7:134-142(1968) [PubMed] [EuropePMC]

• Aspartate: 2-oxoglutarate aminotransferase from bakers' yeast: crystallization and characterization.

  Yagi T., Kagamiyama H., Nozaki M.

  Aspartate: 2-oxoglutarate aminotransferase [EC 2.6.1.1] was purified and crystallized from bakers' yeast. The crystalline preparation gave a single band on polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate. However, in the absence of sodium dodecyl sulfate, the prep ... >> More

  Aspartate: 2-oxoglutarate aminotransferase [EC 2.6.1.1] was purified and crystallized from bakers' yeast. The crystalline preparation gave a single band on polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate. However, in the absence of sodium dodecyl sulfate, the preparation gave one major band with two faint bands, all of which showed the same specific activity, molecular weight and serological properties. These faint bands appeared to be modified forms produced from the major band during the purification. The enzyme showed a molecular weight of 90,000 +/-8,000 and 92,000 +/-8,000 by gel filtration and sedimentation equilibrium analysis, respectively. The molecular weight of a subunit was estimated to be 45,000 by sodium dodecyl sulfate slab gel electrophoresis. Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate. The bound pyridoxal 5'-phosphate showed an absorption maximum at 360 nm (epsilon M: 11,500) and 430 nm (epsilon M: 8,200) in alkaline and acidic conditions, respectively. Its proteolytic pK was pH 6.3. The enzyme showed an optimum pH of 8.0-9.0, and fairly high amino donor and acceptor specificities; aromatic amino acids and their corresponding 2-oxoacids were catalyzed at rates of 0.2-0.8% of those for aspartate and oxalacetate, respectively. Michaelis constants for various substrate were: L-aspartate (0.11 mM), L-glutamate (20.0 mM), oxalacetate (0.006 mM), and 2-oxoglutarate (0.16 mM). The antiserum against yeast aspartate aminotransferase did not form precipitin bands with homogeneous aspartate aminotransferases from pig heart cytosol, pig heart mitochondria or Escherichia coli B. << Less

  J. Biochem. 92:35-43(1982) [PubMed] [EuropePMC]

• Recombinant expression, purification, and characterization of three isoenzymes of aspartate aminotransferase from Arabidopsis thaliana.

  Wilkie S.E., Warren M.J.

  Five different genes encoding isoenzymes of aspartate aminotransferase (AAT) have been identified in the plant Arabidopsis thaliana. cDNA sequences encoding three of these AAT isoenzymes, asp1 (mitochondrial), asp2 (cytosolic), and asp5 (plastid), were manipulated into bacterial expression vectors ... >> More

  Five different genes encoding isoenzymes of aspartate aminotransferase (AAT) have been identified in the plant Arabidopsis thaliana. cDNA sequences encoding three of these AAT isoenzymes, asp1 (mitochondrial), asp2 (cytosolic), and asp5 (plastid), were manipulated into bacterial expression vectors and the recombinant proteins expressed were purified from liquid culture using conventional methods. Yields of the purified isoenzymes varied from 11.5 mg/g wet wt cells (AAT5) to 0.95 mg/g wet wt cells (AAT2), an improvement of more than 1000-fold over typical yields of native isoenzymes obtained from plant tissues of other species. Analysis of the recombinant proteins on denaturing PAGE gels indicated subunit Mrs of between 44 and 45 K. Kinetic parameters (Km and kcat) obtained for all four substrates (aspartate, alpha-ketoglutarate, glutamate, and oxaloacetate) were consistent with values obtained for native AAT isoenzymes from other plant species. Further characterization of the purified recombinant enzymes alongside native enzymes from A. thaliana leaf tissue on AAT activity gels confirmed the identity of asp1 and asp2 as the mitochondrial and cytosolic AAT genes but indicated that asp5 may encode an amyloplastic rather than the chloroplastic enzyme. << Less

  Protein Expr. Purif. 12:381-389(1998) [PubMed] [EuropePMC]