期刊
FEBS JOURNAL
卷 276, 期 10, 页码 2801-2810出版社
WILEY
DOI: 10.1111/j.1742-4658.2009.07003.x
关键词
competitive substrates; enzyme kinetics; flavin; oxidase; thymidylate synthase
资金
- NIH [R01 GM065368]
- NSF [CHE- 0715448]
- Iowa Center for Biocatalysis and Bioprocessing Predoctoral Fellowships
Flavin-dependent thymidylate synthases (FDTS) catalyze the production of dTMP from dUMP and N-5,N-10-methylene-5,6,7,8-tetrahydrofolate (CH(2)H(4)folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a FAD coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an attractive target for novel antibiotics. Like many other flavoenzymes, FDTS can function as an oxidase, which catalyzes the reduction of O-2 to H2O2, using reduced NADPH or other reducing agents. In this study, we exploit the oxidase activity of FDTS from Thermatoga maritima to probe the binding and release features of the substrates and products during its synthase activity. Results from steady-state and single-turnover experiments suggest a sequential kinetic mechanism of substrate binding during FDTS oxidase activity. CH(2)H(4)folate competitively inhibits the oxidase activity, which indicates that CH(2)H(4)folate and O-2 compete for the same reduced and dUMP-activated enzymatic complex (FDTS-FADH(2)-NADP(+)-dUMP). These studies imply that the binding of CH(2)H(4)folate precedes NADP(+) release during FDTS activity. The inhibition constant of CH(2)H(4)folate towards the oxidase activity was determined to be rather small (2 mu m), which indicates a tight binding of CH(2)H(4)folate to the FDTS-FADH(2)-NADP(+)-dUMP complex.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据