Purification and characterization of a dimeric phenylalanine dehydrogenase from Rhodococcus maris K-18.
NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.
Misono
H
H
Department of Agricultural Chemistry, Kochi University, Japan.
Yonezawa
J
J
Nagata
S
S
Nagasaki
S
S
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012241:Rhodopseudomonas / Q000145:classification / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D017886:Durapatite; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase