Aspartate: 2-oxoglutarate aminotransferase from bakers' yeast: crystallization and characterization.
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.
D000596:Amino Acids / Q000032:analysis; D001219:Aspartate Aminotransferases / Q000302:isolation & purification* / Q000378:metabolism; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D001219:Aspartate Aminotransferases