Publications

• Spectral and kinetic studies on Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

  Koyama H., Suzuki H.

  Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) contains two FAD molecules in one molecule of the enzyme (Koyama, H. (1983) J. Biochem. 93, 1313-1319). When the enzyme was mixed anaerobically with L-phenylalanine, beta-2-thienylalanine, L-tyrosine, or L-methionine, a spectral ... >> More

  Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) contains two FAD molecules in one molecule of the enzyme (Koyama, H. (1983) J. Biochem. 93, 1313-1319). When the enzyme was mixed anaerobically with L-phenylalanine, beta-2-thienylalanine, L-tyrosine, or L-methionine, a spectral species (purple intermediate) with a broad absorption band around 540 nm was observed with each substrate, and decayed slowly. From the data on the overall reaction kinetics, the rate of the L-phenylalanine oxidase reaction was expressed as follows. e/v = e/Vm + A/[S] + B/[O2] where e represents the concentration of enzyme unit, v the rate of the overall reaction, Vm the maximum velocity, and A and B are constants. Furthermore, the reactions of the enzyme with beta-2-thienylalanine (mostly an oxygenase substrate) and L-methionine (an oxidase substrate) were analyzed by the "stopped flow" method. The following scheme for the mechanism of L-phenylalanine oxidase reaction with both substrates is proposed, based on the data obtained. (formula; see text) Where Eox represents the oxidized form of the enzyme unit, EoxS the enzyme unit (oxidized form)-substrate compound, X the purple intermediate with a characteristic broad absorption band around 540 nm, S the substrate and P the product. << Less

  J. Biochem. 100:859-866(1986) [PubMed] [EuropePMC]

• Oxidation and oxygenation of L-amino acids catalyzed by a L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501.

  Koyama H.

  A number of L-amino acids and derivatives were tested as substrates for the purified Pseudomonas L-phenylalanine oxidase. The reaction products of these amino acids were analyzed by high performance liquid chromatography and the kinetic properties of the reactions were partially characterized. In ... >> More

  A number of L-amino acids and derivatives were tested as substrates for the purified Pseudomonas L-phenylalanine oxidase. The reaction products of these amino acids were analyzed by high performance liquid chromatography and the kinetic properties of the reactions were partially characterized. In addition to L-phenylalanine, L-tyrosine, DL-o-tyrosine, DL-m-tyrosine, p-fluoro-DL-phenylalanine and beta-2-thienyl-DL-alanine served as substrates for both oxidation and oxygenation catalyzed by the enzyme. On the other hand, L-methionine and L-norleucine were enzymically converted to the corresponding alpha-keto acids with the consumption of oxygen and with the formation of ammonia and hydrogen peroxide in stoichiometric amounts. Kinetic studies showed that the Km values for oxidation and oxygenation of L-phenylalanine by the enzyme were 2.04 mM and 1.96 mM for oxygen, and 13.3 microM and 11.1 microM for L-phenylalanine, respectively. omega-Phenyl fatty acids such as phenylacetic acid, 3-phenylpropionic acid and 4-phenylbutyric acid were competitive inhibitors of the enzyme towards L-phenylalanine. Both oxidation and oxygenation of L-phenylalanine by the enzyme were also inhibited by phenylacetic acid competitively. << Less

  J. Biochem. 96:421-427(1984) [PubMed] [EuropePMC]

• Further characterization of a novel L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501.

  Koyama H.

  L-Phenylalanine oxidase, purified to homogeneity from Pseudomonas sp. P-501, had a molecular weight of about 140,000 and consisted of two subunits identical in molecular weight (about 68,000). The sedimentation coefficient (S020,w) of the enzyme was determined to be 8.18S by ultracentrifugation. T ... >> More

  L-Phenylalanine oxidase, purified to homogeneity from Pseudomonas sp. P-501, had a molecular weight of about 140,000 and consisted of two subunits identical in molecular weight (about 68,000). The sedimentation coefficient (S020,w) of the enzyme was determined to be 8.18S by ultracentrifugation. The enzyme showed absorption maxima at 276, 390, and 466 nm and a shoulder around 490 nm and contained 2 mol of FAD per mol of enzyme. Oxygen-18 supplied as molecular oxygen was incorporated into the carbonyl group of alpha-phenylacetamide formed by the enzymic oxidation of L-phenylalanine. Michaelis constants of the enzyme were 1.07 X 10(-2) mM for L-phenylalanine and 1.82 mM for oxygen. Maximum activities in oxidation and oxygenation (catalyzed simultaneously by the enzyme) were observed at different pHs and different temperatures. Several metal ions inhibited the oxidase activity preferentially. << Less

  J. Biochem. 93:1313-1319(1983) [PubMed] [EuropePMC]

• A simple and rapid enzymatic determination of L-phenylalanine with a novel L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501.

  Koyama H.

  A simple and rapid method for the determination of L-phenylalanine using Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) has been developed. This method involves the oxidative deamination of L-phenylalanine using L-phenylalanine oxidase from Pseudomonas sp. P-501, and the col ... >> More

  A simple and rapid method for the determination of L-phenylalanine using Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) has been developed. This method involves the oxidative deamination of L-phenylalanine using L-phenylalanine oxidase from Pseudomonas sp. P-501, and the colorimetric determination of hydrogen peroxide formed from L-phenylalanine with 4-aminoantipyrine and N,N'-dimethylaniline in the presence of peroxidase. This method requires a smaller quantity of sample and is less time-consuming than those previously reported. The method can be used for the direct assay of serum L-phenylalanine levels without pretreatment. << Less

  Clin Chim Acta 136:131-136(1984) [PubMed] [EuropePMC]

• Kinetic isotope effect of the L-phenylalanine oxidase from Pseudomonas sp. P-501.

  Ohta Y., Mukouyama E.B., Suzuki H.

  Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) mainly catalyzes oxygenation when L-phenylalanine is used as the substrate, but oxidation when L-methionine is used as the substrate. Using [C(alpha)-H]-DL-methionine and [C(alpha)-D]-DL-methionine as substrate, the reductive ha ... >> More

  Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) mainly catalyzes oxygenation when L-phenylalanine is used as the substrate, but oxidation when L-methionine is used as the substrate. Using [C(alpha)-H]-DL-methionine and [C(alpha)-D]-DL-methionine as substrate, the reductive half reaction of FAD cofactor of enzyme has been studied by stopped-flow spectrophotometry. The rate of reduction of FAD cofactor has a kinetic isotope effect (KIE) of 5.4 and 4.1 in the absence and presence of 30% glycerol, respectively. The KIE is independent of temperature, but the rates of the reductive half reaction are dependent on temperature, indicating that thermally induced motion at the active site drives the H-transfer reaction by H-tunneling. << Less

  J. Biochem. 139:551-555(2006) [PubMed] [EuropePMC]

• Purification and characterization of a novel L-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501.

  Koyama H.

  L-Phenylalanine oxidase from Pseudomonas sp. P-501 has been purified to homogeneity as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme produced both beta-phenylpyruvate and alpha-phenylacetamide from L-phenylalanine. Balance studies demonstrated that consumption of 1 m ... >> More

  L-Phenylalanine oxidase from Pseudomonas sp. P-501 has been purified to homogeneity as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme produced both beta-phenylpyruvate and alpha-phenylacetamide from L-phenylalanine. Balance studies demonstrated that consumption of 1 mol each of L-phenylalanine and oxygen resulted in the formation of 0.2 mol each of beta-phenylpyruvate, ammonia, and hydrogen peroxide and 0.8 mol each of alpha-phenylacetamide and carbon dioxide under aerobic conditions. Thus, the same enzyme preparation catalyzed simultaneous oxidative deamination and oxygenative decarboxylation of L-phenylalanine. Besides L-phenylalanine, the enzyme oxidized L-tyrosine, L-methionine, and L-tryptophan at lower reaction rates. << Less

  J. Biochem. 92:1235-1240(1982) [PubMed] [EuropePMC]