Rapid kinetics of dehalogenation promoted by iodotyrosine deiodinase from human thyroid.
Rapid kinetics of dehalogenation promoted by iodotyrosine deiodinase from human thyroid.
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DOI：
10.1021/acs.biochem.5b00410
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发表时间：
2015-07-28
期刊：
影响因子：
2.9
通讯作者：
Rokita SE
中科院分区：
文献类型：
--
作者：
Bobyk KD;Ballou DP;Rokita SE
由碘酪氨酸脱碘酶（IYD）催化的还原性脱卤在需氧生物中是非常罕见的，但对于甲状腺素生物合成过程中产生的碘酪氨酸的碘化物回收是必要的。同样不寻常的是这一过程对黄素的依赖。快速动力学已经被用来定义IYD催化的基本过程。利用黄素荧光随时间猝灭来监测卤代酪氨酸与IYD的结合。底物氯、溴和碘酪氨酸的结合速率常数（kon）相似，在1.3 × 106 ~ 1.9 × 106 M-1 s-1之间。只有惰性底物类似物氟酪氨酸表现出明显（5倍）慢于（0.3 × 106 M-1 s-1）。所有数据都符合标准的双态模型，表明在底物诱导的活性位点盖闭合过程中没有中间复合物积累。随后的卤化物消除似乎并没有限制溴酪氨酸和碘酪氨酸的反应，因为尽管它们的碳-卤素键的强度不同，但它们都以几乎相等的二级速率常数（分别为7.3 × 103和8.6 × 103 M-1 s-1）完全氧化了还原酶。与这些底物相比，氯酪氨酸与还原酶的反应速度大约慢20倍，并显示出与溴酪氨酸和碘酪氨酸反应速度大致相同的光谱中间体。
Reductive dehalogenation such as that catalyzed by iodotyrosine deiodinase (IYD) is highly unusual in aerobic organisms but necessary for iodide salvage from iodotyrosine generated during thyroxine biosynthesis. Equally unusual is the dependence of this process on flavin. Rapid kinetics have now been used to define the basic processes involved in IYD catalysis. Time-dependent quenching of flavin fluorescence was used to monitor halotyrosine association to IYD. The substrates chloro-, bromo-, and iodotyrosine bound with similar rate constants (kon) ranging from 1.3 × 106 to 1.9 × 106 M–1 s–1. Only the inert substrate analogue fluorotyrosine exhibited a significantly (5-fold) slower kon (0.3 × 106 M–1 s–1). All data fit a standard two-state model and indicated that no intermediate complex accumulated during closure of the active site lid induced by substrate. Subsequent halide elimination does not appear to limit reactions of bromo- and iodotyrosine since both fully oxidized the reduced enzyme with nearly equivalent second-order rate constants (7.3 × 103 and 8.6 × 103 M–1 s–1, respectively) despite the differing strength of their carbon–halogen bonds. In contrast to these substrates, chlorotyrosine reacted with the reduced enzyme approximately 20-fold more slowly and revealed a spectral intermediate that formed at approximately the same rate as the bromo- and iodotyrosine reactions.