Uncovering the role of sorbitol in Renilla luciferase kinetics: Insights from spectroscopic and molecular dynamics studies.

Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide, resulting in the emission of a photon of light. This study investigated the impact of sorbitol on the structural and kinetic properties of Renilla luciferase using circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations. Our investigation, carried out using circular dichroism and fluorescence analyses, as well as a thermal stability assay, has revealed that sorbitol induces conformational changes in the enzyme but does not improve its thermal stability. Moreover, through kinetic studies, it has been demonstrated that at a concentration of 0.4 M, sorbitol enhances the catalytic efficiency of Renilla luciferase. However, at higher concentrations, sorbitol results in a decrease in catalytic efficiency. Additionally, molecular dynamics simulations have shown that sorbitol increases the presence of hydrophobic pockets on the enzyme's surface. These simulations have also provided evidence that at a concentration of 0.4 M, sorbitol facilitates substrate access to the active site of the enzyme. Nevertheless, at higher concentrations, sorbitol obstructs substrate trafficking, most likely due to its impact on the gateway to the active site. This study may provide insights into the kinetic changes observed in enzymes with buried active sites, such as those with α/ß hydrolase fold.

Kinetics, structure, and dynamics of Renilla luciferase solvated in binary mixtures of glycerol and water and the mechanism by which glycerol obstructs the enzyme emitter site.

Renilla Luciferase is a bioluminescent enzyme which is broadly implemented as protein reporter in biology-related researches. In this study, new evidences on the kinetics, structure, and dynamics of Renilla luciferase solvated in binary mixtures of glycerol and water using MD simulation along with experimental procedures including fluorescence and CD spectroscopy were obtained. The results indicated that the Renilla luciferase activity decreased at 0.8 and 1.2 M of glycerol through the obstruction of enzyme emitter site. The present study may describe a new molecular mechanism of decreasing enzyme activity in the presents of glycerol.