复杂的异质细胞内环境似乎通过改变生物分子的流动性来影响酶催化,其稳定性,和它们的构象状态,以及通过促进或阻碍不断发生的互动。细胞质基质组分对酶活性的影响的评估和描述是仍未解决的问题。在这项工作中,我们旨在确定双组分介质与各种分子大小的共溶剂在细菌荧光素酶催化的复杂多阶段生物发光反应中的作用机理。乙二醇的动力学和结构效应,甘油,山梨醇,葡萄糖,蔗糖,葡聚糖,用停流和荧光光谱技术和分子动力学模拟研究了聚乙二醇对细菌荧光素酶的影响。我们已经发现,在共溶剂存在下的扩散限制促进了黄素底物和过氧黄素中间体的稳定反应,但在生物发光量子产率方面没有任何优势,因为底物结合也减慢了。已发现细菌荧光素酶的催化常数与粘度无关,并且与水-共溶剂相互作用的参数相关(Norrish常数,范德华相互作用能)。拥挤的特工,与低分子量共溶剂相比,对过氧黄素中间衰变和酶催化常数影响不大。我们将特定的动力学效应归因于共溶剂与酶表面的优先相互作用以及它们渗透到活性位点中。
A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in
bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water-cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site.