The role of active site residue arginine 218 in firefly luciferase bioluminescence
The role of active site residue arginine 218 in firefly luciferase bioluminescence
复制标题
DOI：
10.1021/bi002246m
复制
发表时间：
2001-02-27
期刊：
影响因子：
2.9
通讯作者：
Portier, NC
中科院分区：
文献类型：
--
作者：
Branchini, BR;Magyar, RA;Portier, NC
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光。我们以前开发了一个工作模型的荧光素酶活性位点的基础上的X-射线结构的酶没有结合底物。在我们的模型中，Arg 218的侧链胍基似乎位于靠近底物的羟基的底部的Bronzin结合口袋。Arg 337也有类似的作用。我们在这里报告的构建，纯化，和突变体酶R218 A，R218 Q，R218 K，R337 Q，和R337 K的特性。Arg 218侧链的改变产生的酶具有15-20倍的K-m值增加，对于胡萝卜素。与Arg 337酶测定的相对接近正常的K-m值支持我们的建议，Arg 218（而不是Arg 337）是一个重要的精氨酸结合位点残基。生物发光研究表明，在没有带正电荷的基团在位置218，红色的生物发光产生。基于这一结果和其他荧光实验的结果，我们推测Arg 218保持了P. pyralis荧光素酶正常黄绿色发射所必需的发射体结合位点的极性和刚性。在萤火虫荧光素酶的X-射线结构和基于计算的模型的活性位点的上下文中解释这里报道的研究结果。
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 a working model of the luciferase active site based on the X-ray structure of the enzyme without bound substrates. In our model, the side chain guanidinium group of Arg218 appears to be located in close proximity to the substrate's hydroxyl group at the bottom of the luciferin binding pocket. A similar role for Arg337 also has been proposed. We report here the construction, purification, and characterization of mutant luciferases R218A, R218Q, R218K, R337Q, and R337K. Alteration of the Arg218 side chain produced enzymes with 15-20-fold increases in the K-m, values for luciferin. The contrasting near-normal K-m values for luciferin determined with the Arg337 enzymes support our proposal that Arg218 (and not Arg337) is an essential luciferin binding site residue. Bioluminescence emission studies indicated that in the absence of a positively charged group at position 218, red bioluminescence was produced. Based on this result and those of additional fluorescence experiments, we speculate that Arg218 maintains the polarity and rigidity of the emitter binding site necessary for the normal yellow-green emission of P. pyralis luciferase. The findings reported here are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.