Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and 15N Kinetic Isotope Effects on Alanine Oxidation by an L-Amino Acid Oxidase

详细信息    查看全文

• 作者：Erik C. Ralph ; Mark A. Anderson ; W. Wallace Cleland ; Paul F. Fitzpatrick
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：December 26, 2006
• 年：2006
• 卷：45
• 期：51
• 页码：15844 - 15852
• 全文大小：110K
• 年卷期：v.45,no.51(December 26, 2006)
• ISSN：1520-4995

文摘

Tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi catalyzes the oxidativedecarboxylation of L-tryptophan during the biosynthesis of indoleacetic acid. Structurally and mechanistically, the enzyme is a member of the family of L-amino acid oxidases. Deuterium and ¹⁵N kinetic isotopeeffects were used to probe the chemical mechanism of L-alanine oxidation by TMO. The primary deuteriumkinetic isotope effect was pH independent over the pH range 6.5-10, with an average value of 6.0 ± 0.5,consistent with this being the intrinsic value. The deuterium isotope effect on the rate constant for flavinreduction by alanine was 6.3 ± 0.9; no intermediate flavin species were observed during flavin reduction.The k_cat/K_ala value was 1.0145 ± 0.0007 at pH 8. NMR analyses gave an equilibrium ¹⁵N isotope effectfor deprotonation of the alanine amino group of 1.0233 ± 0.0004, allowing calculation of the ¹⁵N isotopeeffect on the CH bond cleavage step of 0.9917 ± 0.0006. The results are consistent with TMO oxidationof alanine occurring through a hydride transfer mechanism.