Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase
Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase
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DOI：
10.1021/bi050935y
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发表时间：
2005-10-04
期刊：
影响因子：
2.9
通讯作者：
Tu, SC
中科院分区：
文献类型：
--
作者：
Li, CH;Tu, SC
哈维氏弧菌荧光素酶是一种含有单一活性位点的α β异源二聚体，较早提出位于α亚基中的裂缝处。在这项工作中，6个保守的苯丙氨酸残基在这个拟议的活性位点进行定点突变，以调查他们可能的功能作用，并描绘的萤光素酶活性位点的组成。在初始筛选Phe -> Ala突变体后，选择aLF 46、aF 49、aF 114和aF 117用于另外突变为Asp、Ser和Tyr。野生型和突变型荧光素酶的一般动力学性质的比较表明，α F46、α F49、α F114和α F117的疏水性质对于荧光素酶V-max和V-max/K-m是重要的，对于Phe -> Asp突变体，其降低了3-5个数量级。α F46和α F117似乎也参与了还原型黄素底物的结合。对4 α-氢过氧黄素中间体II的稳定性和产率的额外研究以及通过α F46 D、α F49 D、α F114 D和α F117 D对癸醛底物氧化的测量揭示，它们的总量子产率（phi（o））的显著降低是荧光素酶中间体产率降低的结果，并且除了α F114 D之外，激发的发射体的发射量子产率，由于由阴离子Asp的疏水性Phe的替代。这四个关键的苯丙氨酸残基的位置与其他必要的和/或疏水性的残基描绘在一个细化的地图的活性位点。这些残基的功能影响进行了讨论。
Vibrio harveyi luciferase is an alpha beta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe -> Ala mutants, alpha LF46, alpha F49, alpha F114, and alpha F117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alpha F46, alpha F49, alpha F114, and alpha F117 was important to luciferase V-max and V-max/K-m, which were reduced by 3-5 orders of magnitude for the Phe -> Asp mutants. Both alpha F46 and alpha F117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alpha F46D, alpha F49D, alpha F114D, and alpha F117D revealed that their marked reductions in the overall quantum yield (phi(o)) were a consequence of diminished yields of luciferase intermediates and, with the exception of alpha F114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed.