title: Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii.
abstract: An enzyme which catalyzes the transamination of L-aspartate with 2-oxoglutarate has been purified 400-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138,000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65,000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5-phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-aspartate. L-Aspartate or pyridoxal 5-phosphate, but not 2-oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L-phenylalanine and L-tyrosine with 2-oxoglutarate, and L-serine, L-alanine and L-glutamine with oxaloacetate. L-Aspartate aminotransferase exhibited hyperbolic kinetics for 2-oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L-aspartate and L-glutamate. Apparent Km values were 0.55 mM for 2-oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L-aspartate and 3.88 mM for L-glutamate. Transamination of L-aspartate in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Ciencias, Universidad de Córdoba, Spain.
authors.forename: B
authors.initials: B
authors.lastname: Lain-Guelbenzu
authors.forename: J
authors.initials: J
authors.lastname: Muñoz-Blanco
authors.forename: J
authors.initials: J
authors.lastname: Cárdenas
mesh_terms: D001219:Aspartate Aminotransferases / Q000302:isolation & purification*; D002384:Catalysis; D002696:Chlamydomonas / Q000201:enzymology*; D002850:Chromatography, Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007526:Isoelectric Point
chemical_list: D001219:Aspartate Aminotransferases