Kinetic isotope effect of the L-phenylalanine oxidase from Pseudomonas sp. P-501 |
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Authors: | Ohta Yui Mukouyama Etsuko B Suzuki Haruo |
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Institution: | Division of Biosciences, Graduate School of Fundamental Life Science, Kitasato University, Sagamihara, Kanagawa. |
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Abstract: | 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. |
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