Abstracts

At present, there is a state fact that the interior of the real cellular pattern is quite different from the classical one, which is based on buffer solutions. The impact of crowding agents on enzymatic activities is one of the hot topics for the plenty of research devoted to shedding light on mechanisms of the cellular organization of the metabolic pathways. Numerous articles have been published and new knowledge about enzyme behaviour in the presence of different crowding agents and cosolvents has been received. Nevertheless, there is an issue that requires attention - whether the crowded and/or viscous medium affects, in the same way, the kinetics of a particular enzyme functioning alone and as part of a conjugation chain (metabolic pathway). To shed the light on this issue, the coupled enzyme system based on NAD(P)H:FMN-oxidoreductase and luciferase from luminous bacteria (Red + Luc) seems the unique research tool. Firstly, the activities as single enzymes as the coupled one are easy to monitor without sophisticated equipment. Secondly, the Luc activity in the presence of cosolvents has been studied by different authors. But there is little known about how the Red activity changes in the presence of cosolvents. This work is aimed to investigate the kinetic features of Red in the presence of glycerol and sucrose. The obtained data demonstrate that the Red activity decreases in the viscous medium. In the presence of glycerol, the Red activity was reduced in a diffusion-controlled manner, whereas the effect of sucrose solutions was weaker. In addition, the effect of the cosolvents on K_m (FMN) of Red was estimated. It has been shown that the presence of 20% sucrose leads to a 0.7-fold lower K_m(FMN) value compared to one in the buffer. In contrast, in the medium with 20% glycerol 1.9-fold higher K_m (FMN) has been obtained compared to one in the buffer. This could be caused by different relative solvent accessibility values of glycerol and sucrose to Red’s active centre. If we compare the Red activities obtained in the presence of the cosolvents with previously published data for Luc and Red + Luc activities, there could be stated that the influence of the cosolvents on the activity of the coupled enzyme system is close to additive manner. Thus, the residual bioluminescence intensity of the Red + Luc enzyme system in the presence of the glycerol and sucrose is close to a sum of individual effects of the cosolvents on the kinetic features of both Luc and Red. The investigation of the cosolvents effects on the kinetics of a single enzyme is relevant for figuring out the cellular enzyme's behaviour of multienzyme systems within luminous bacteria.