Department of Biology, University of Isfahan, Isfahan, Iran.
Department of Biology, University of Isfahan, Isfahan, Iran.
Int J Biol Macromol. 2016 Dec;93(Pt A):1253-1260. doi: 10.1016/j.ijbiomac.2016.09.060. Epub 2016 Sep 17.
Renilla luciferase is a sensitive enzyme and has wide applications in biotechnology such as drug screening. Previous studies have tried to show the catalytic residues, nevertheless, the accurate architecture and molecular behavior of its emitter site remains uncharacterized. In this study, the activity of Renilla luciferase, in the presence of two small organic molecules including dimethyl sulfoxide (DMSO) and isopropanol was considered and the structure was studied by circular dichroism (CD) and fluorescence spectroscopy. Moreover, the interaction of small organic molecules with the Renilla luciferase was studied using molecular dynamics simulations. Kinetics studies showed that at low concentration of DMSO (16.6-66mM) and isopropanol (19.3-76mM) the K changed and a competitive inhibition pattern was observed. Moreover, spectroscopy studies reveled that the changes of activity of Renilla luciferase in the presence of low concentrations of small organic molecules was not associated with structural collapse or severe changes in the enzyme conformation. Molecular dynamics simulations indicated that DMSO and isopropanol, as probing molecules, were both able to bind to the emitter site and remained with the residues of the emitter site. Based on the probing data, the architecture of the emitter site in the "non-binding" model was proposed.
海肾荧光素酶是一种灵敏的酶，在药物筛选等生物技术中有广泛的应用。先前的研究试图揭示其催化残基，但对于其发射部位的精确结构和分子行为仍未得到阐明。在本研究中，考虑了海肾荧光素酶在两种小分子有机化合物（二甲基亚砜（DMSO）和异丙醇）存在下的活性，并通过圆二色性（CD）和荧光光谱研究了其结构。此外，还通过分子动力学模拟研究了小分子与海肾荧光素酶的相互作用。动力学研究表明，在低浓度的 DMSO（16.6-66mM）和异丙醇（19.3-76mM）下，K 值发生了变化，观察到竞争抑制模式。此外，光谱研究表明，在低浓度小分子存在下，海肾荧光素酶活性的变化与结构崩溃或酶构象的严重变化无关。分子动力学模拟表明，DMSO 和异丙醇作为探测分子，均能够结合到发射部位，并与发射部位的残基保持结合。基于探测数据，提出了“非结合”模型中发射部位的结构。
