Title
Probing the emitter site of Renilla luciferase using small organic molecules; an attempt to understand the molecular architecture of the emitter site
Author
Farajollah Salehi, Rahman Emamzadeh, Mahboobeh Nazari, Seyed Mohammad Mahdi Rasa
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
Renilla luciferase is a sensitive enzyme and has wide applications in biotechnology such as drug screening. Previous studies 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 Renillaluciferase was studied using molecular dynamics simulations. Kinetics studies showed that at low concentration of DMSO (16.6 to 66 mM) and isopropanol (19.3 to 76 mM) theKm changed and a competitive inhibition pattern was observed. Moreover, spectroscopy studies reveled that the alteration of activity of Renilla luciferase in the presence of low concentrations of organic small 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.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Chemical stability, Biochemistry