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

Abstract

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.

Structure-Function Relationships in Temperature Effects on Bacterial Luciferases: Nothing Is Perfect

“…A concentration of 30% was used, which corresponded to 0.99 M of sucrose and gave more than a threefold higher viscosity as compared with the buffer. From the kinetic curves in Figure 1 , one can see that the reaction noticeably slows down in a more viscous medium for both luciferases, which is due to the diffusion control of some reaction steps [ 24 ]. The change of kinetic parameters in sucrose solution over the entire temperature range is shown in Figure 2 and Figure S1 .…”

“…Thus, distinct temperature dependences of k decay for two luciferases could be caused by two reasons: (i) by a stronger destabilizing effect on peroxyflavin intermediate in V. harveyi reaction and/or (ii) by temperature influence on the others than k dd rate constants which could be more pronounced in the V. harveyi reaction due to their high contribution into k decay . Earlier, we demonstrated that for the reaction of P. leiognathi luciferase in the presence of sucrose the rate of the dark decay of peroxyflavin intermediate becomes lower, while the rate of the formation of an electronically excited intermediate increases as compared with the buffer [ 24 ]. This could underlie the contrasting changes of k decay observed for two luciferases at 40–45 °C in a sucrose solution ( Figure 2 c,d).…”

“…The reaction kinetics for the luciferases was measured at 5–45 °C in a single-turnover assay using a SX20 stopped-flow spectrometer (Applied Photophysics, Leatherhead, UK) as described elsewhere [ 24 ]. Decanal and photoreduced flavin mononucleotide in the buffer or in the sucrose solution were loaded into one drive syringe of the spectrometer and pre-incubated there for 5 min.…”

“…We studied Vibrio harveyi luciferase characterized by the slow decay kinetics and Photobacterium leiognathi defined as the “fast” enzyme. Sucrose was chosen as a model extremolyte, since its buffer solutions proved to have no inhibitory effect on bacterial luciferase activity in contrast to glycerol solutions [ 24 ].…”

Molecular modeling of osmolytes’ effect on bacterial luciferase

“…catalytic activity. For Photobacterium leiognathi luciferase it was revealed that integral intensity of the reaction changes in the presence of different cosolvents as follows: sucrose > sorbitol > glucose > glycerol > ethylene glycol that correlates with the relation of hydrodynamic radii of the cosolvent molecules: 5.2 > 3.9 > 3.6 > 3.1 > 2.6 (Å) [2]. Moreover, sucrose increases catalytic constant of excited state intermediate formation up to 5-fold unlike other cosolvents.…”