Branchini Bruce R, Southworth Tara L, Murtiashaw Martha H, Boije Henrik, Fleet Sarah E
Department of Chemistry, Connecticut College, New London, Connecticut 06320, USA.
Biochemistry. 2003 Sep 9;42(35):10429-36. doi: 10.1021/bi030099x.
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, 287-298] 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 (>or=4-fold K(m) difference) altered binding affinity for luciferin and seven residues, spanning the primary sequence region Arg218-Ala348, had substantially (>or=30 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.
萤火虫荧光素酶通过一系列需要Mg-ATP和分子氧的反应，催化底物萤火虫荧光素高效发射黄绿色光。我们之前已经开发出[Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311 - 15319]一种基于分子图形的荧光素酶活性位点工作模型，该模型起始于该酶无结合底物时的首个X射线结构[Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287 - 298]。在我们的模型中，荧光素结合位点包含15个与底物距离在5埃以内的残基。利用定点诱变，我们对所有这些残基进行了改变，并在此报告相应表达和纯化蛋白质的特性。在所研究的15个残基中，12个对荧光素的结合亲和力有显著改变（K(m)差异≥4倍），并且跨越一级序列区域Arg218 - Ala348的7个残基在突变时生物发光发射最大值有大幅红移（≥30纳米）。我们在此报告对突变残基在底物结合和生物发光颜色确定中作用的解释。这项研究的结果总体上证实了我们模型的准确性，并为未来旨在改变萤火虫荧光素酶底物特异性的实验奠定了基础。
