Branchini B R, Magyar R A, Murtiashaw M H, Anderson S M, Zimmer M
Department of Chemistry, Connecticut College, New London 06320, USA.
Biochemistry. 1998 Nov 3;37(44):15311-9. doi: 10.1021/bi981150d.
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., Hinz, 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 ( approximately 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.
萤火虫荧光素酶通过一系列需要Mg-ATP和分子氧的反应，催化底物荧光素高效发射黄绿色光。我们之前报道过[Branchini, B. R., Magyar, R. A., Marcantonio, K. M., Newberry, K. J., Stroh, J. G., Hinz, L. K., and Murtiashaw, M. H. (1997) J. Biol. Chem. 272, 19359 - 19364]，萤火虫荧光素类似物2-(4-苯甲酰基苯基)噻唑-4-羧酸（BPTC）是一种有效的荧光素酶光灭活试剂。我们鉴定出一种荧光素酶肽244HHGF247，其降解与光氧化过程直接相关。我们在此报告野生型和突变型荧光素酶H244F、H245F、H245A和H245D的构建与纯化。用这些蛋白质进行的光灭活以及动力学和生物发光研究结果表明，His245是BPTC催化的酶失活的主要功能靶点。H245D突变体极低的比活性（比野生型低约300倍）支持了His245在光氧化反应过程中被氧化为天冬氨酸的可能性。以无底物的荧光素酶晶体结构[Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287 - 298]以及功能相关的短杆菌肽合成酶1的苯丙氨酸激活亚基[Conti, E., Stachelhaus, T., Marahiel, M. A., and Brick, P. (1997) EMBO J. 16, 4174 - 4183]为起点，我们进行了分子建模研究，并在此提出一个结合了底物荧光素和Mg-ATP的荧光素酶活性位点模型。我们利用这个模型对244HHGF247在萤火虫生物发光中的作用进行基于结构的解释。
