Site-directed mutagenesis of histidine 245 in firefly luciferase: A proposed model of the active site
Site-directed mutagenesis of histidine 245 in firefly luciferase: A proposed model of the active site
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DOI：
10.1021/bi981150d
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发表时间：
1998-11-03
期刊：
影响因子：
2.9
通讯作者：
Zimmer, M
中科院分区：
文献类型：
--
作者：
Branchini, BR;Magyar, RA;Zimmer, M
萤火虫荧光素酶通过一系列需要镁-三磷酸腺苷和分子氧的反应，催化底物荧光素发出高效的黄绿光。我们先前报道了[Branchini，B.R.，Magyar，R.A.，Marcantonio，K.M.，Newberry，K.J.，Stroh，J.G.，Hint，L.K.，and Murtiashaw，M.H.(1997)J.Biol.化学。19359-19364]萤火虫荧光素类似物2-(4-苯甲酰苯基)噻唑-4-羧酸是一种有效的荧光素酶光灭活剂。我们鉴定了一种荧光素酶肽244HHGF247，它的降解与光氧化过程直接相关。我们报道了野生型和突变型荧光素酶H244F、H245F、H245A和H245D的构建和纯化。这些蛋白质的光灭活、动力学和生物发光研究结果与His245是BPTC催化的酶失活的主要功能靶点是一致的。His245在光氧化反应中被氧化为天冬氨酸的可能性得到了H245D突变体极低的比活(类似于WT的300倍)的支持。利用无底物荧光素酶的晶体结构[Conti，E.，Franks，N.P.，and Brick，P.(1996)Structure 4,287-298]和功能相关的革兰素合成酶1的苯丙氨酸激活亚单位[Conti，E.，Stchehaus，T.，Marahiel，M.A.，and Brick，P.(1997)EMBO J.16,4174-4183]作为起点，我们进行了分子模拟研究，并在此提出了具有底物荧光素和镁-ATP结合的荧光素酶活性部位的模型。我们使用这个模型来提供一个基于结构的解释244HHGF247在萤火虫生物发光中的作用。
Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We previously reported [Branchini, B. R., Magyar, R. A., Marcantonio, K. M., Newberry, K. J., Stroh, J. G., Hint, L. K., and Murtiashaw, M. H. (1997) J. Biol. Chem. 272, 19359-19364] that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analogue, was a potent photoinactivation reagent for luciferase. We identified a luciferase peptide 244HHGF247, the degradation of which was directly correlated to the photooxidation process. We report here the construction and purification of wild-type and mutant luciferases H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins are consistent with His245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His245 is oxidized to aspartate during the photooxidation reaction was supported by the extremely low specific activity (similar to 300-fold lower than WT) of the H245D mutant. Using the crystal structures of luciferase without substrates [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298] and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 [Conti, E., Stachelhaus, T., Marahiel, M. A., and Brick, P. (1997) EMBO J. 16, 4174-4183] as a starting point, we have performed molecular-modeling studies and propose here a model for the luciferase active site with substrates luciferin and Mg-ATP bound. We have used this model to provide a structure-based interpretation of the role of 244HHGF247 in firefly bioluminescence.