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

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 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 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/β hydrolase fold.