title: Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes.
abstract: Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.
authors.affiliation: Department of Chemistry, Kyoto University of Education, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ohshima
authors.forename: H
authors.initials: H
authors.lastname: Takada
authors.forename: T
authors.initials: T
authors.lastname: Yoshimura
authors.forename: N
authors.initials: N
authors.lastname: Esaki
authors.forename: K
authors.initials: K
authors.lastname: Soda
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004735:Energy Transfer; D004795:Enzyme Stability; D006358:Hot Temperature; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010649:Phenylalanine / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010649:Phenylalanine; D006859:Hydrogen; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase