A mutagenesis study of the putative luciferin binding site residues of firefly luciferase
A mutagenesis study of the putative luciferin binding site residues of firefly luciferase
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DOI：
10.1021/bi030099x
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发表时间：
2003-09-09
期刊：
影响因子：
2.9
通讯作者：
Fleet, SE
中科院分区：
文献类型：
--
作者：
Branchini, BR;Southworth, TL;Fleet, SE
萤火虫荧光素酶催化底物萤火虫荧光素通过一系列需要镁-三磷酸腺苷和分子氧的反应高效地发出黄绿光。我们以前已经开发了[Branchini，B.R.，Magyar，R.A.，Murtiashaw，M.H.，Anderson，S.M.和Zimmer，M.(1998年)生物化学；37,15311-15319]基于分子图形的荧光素酶活性部位的工作模型，从酶的第一个X射线结构[Conti，E.，Franks，N.P.，and Brick，P.(1996年)结构4,287298]开始。在我们的模型中，荧光素结合位点包含15个残基，这些残基位于底物5A以内。利用定点突变，我们对所有这些残基进行了改变，并在此报告了相应的表达和纯化蛋白的特性。在所研究的15个残基中，12个残基与荧光素的结合亲和力显著改变(大于或等于4倍的K-m差)，7个残基在突变时具有显著(大于或等于30 nm)红移的生物发光最大值，这些残基位于初级序列区域Arg218-Ala348。我们在这里报告了突变残基在底物结合和生物发光颜色测定中的作用。这项研究的结果总体上证实了我们模型的准确性，并为未来设计改变萤火虫荧光素酶底物专一性的实验奠定了基础。
Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed [Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry; 37, 15311-15319] a molecular graphics-based working model of the luciferase active site starting with the first X-ray structure [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287298] of the enzyme without bound substrates. In our model, the luciferin binding site contains 15 residues that are within 5 A of the substrate. Using site-directed mutagenesis, we made changes at all of these residues and report here the characterization of the corresponding expressed and purified proteins. Of the 15 residues studied, 12 had a significantly (greater than or equal to4-fold K-m difference) altered binding affinity for luciferin and seven residues, spanning the primary sequence region Arg218-Ala348, had substantially (greater than or equal to30 nm) red-shifted bioluminescence emission maxima when mutated. We report here an interpretation of the roles of the mutated residues in substrate binding and bioluminescence color determination. The results of this study generally substantiate the accuracy of our model and provide the foundation for future experiments designed to alter the substrate specificity of firefly luciferase.