Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction.

Enzymes activity in a cell is determined by many factors, among which viscosity of the microenvironment plays a significant role. Various cosolvents can imitate intracellular conditions in vitro, allowing to reduce a combination of different regulatory effects. The aim of the study was to analyze the media viscosity effects on the rate constants of the separate stages of the bacterial bioluminescent reaction. Non-steady-state reaction kinetics in glycerol and sucrose solutions was measured by stopped-flow technique and analyzed with a mathematical model developed in accordance with the sequence of reaction stages. Molecular dynamics methods were applied to reveal the effects of cosolvents on luciferase structure. We observed both in glycerol and in sucrose media that the stages of luciferase binding with flavin and aldehyde, in contrast to oxygen, are diffusion-limited. Moreover, unlike glycerol, sucrose solutions enhanced the rate of an electronically excited intermediate formation. The MD simulations showed that, in comparison with sucrose, glycerol molecules could penetrate the active-site gorge, but sucrose solutions caused a conformational change of functionally important αGlu175 of luciferase. Therefore, both cosolvents induce diffusion limitation of substrates binding. However, in sucrose media, increasing enzyme catalytic constant neutralizes viscosity effects. The activating effect of sucrose can be attributed to its exclusion from the catalytic gorge of luciferase and promotion of the formation of the active site structure favorable for the catalysis.

Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca2+ -regulated Photoproteins of Different Organisms.

Upon binding their metal ion cofactors, Ca2+ -regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca2+ -regulated photoproteins-aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata-demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca2+ -regulated photoproteins.