Wilcox Xander E, Chung Charmaine B, Slade Kristin M
Department of Chemistry, University of California at Davis, CA, 95616, USA.
Department of Chemistry, Hobart and William Smith Colleges, 300 Pulteney St, Geneva, NY, 14456, USA.
Biochem Biophys Rep. 2021 Feb 20;26:100956. doi: 10.1016/j.bbrep.2021.100956. eCollection 2021 Jul.
In order to better understand how the complex, densely packed, heterogeneous milieu of a cell influences enzyme kinetics, we exposed opposing reactions catalyzed by yeast alcohol dehydrogenase (YADH) to both synthetic and protein crowders ranging from 10 to 550 kDa. The results reveal that the effects from macromolecular crowding depend on the direction of the reaction. The presence of the synthetic polymers, Ficoll and dextran, decrease V and K for ethanol oxidation. In contrast, these crowders have little effect or even increase these kinetic parameters for acetaldehyde reduction. This increase in V is likely due to excluded volume effects, which are partially counteracted by viscosity hindering release of the NAD product. Macromolecular crowding is further complicated by the presence of a depletion layer in solutions of dextran larger than YADH, which diminishes the hindrance from viscosity. The disparate effects from 25 g/L dextran or glucose compared to 25 g/L Ficoll or sucrose reveals that soft interactions must also be considered. Data from binary mixtures of glucose, dextran, and Ficoll support this "tuning" of opposing factors. While macromolecular crowding was originally proposed to influence proteins mainly through excluded volume effects, this work compliments the growing body of evidence revealing that other factors, such as preferential hydration, chemical interactions, and the presence of a depletion layer also contribute to the overall effect of crowding.
为了更好地理解细胞中复杂、密集堆积且异质的环境如何影响酶动力学，我们将酵母乙醇脱氢酶（YADH）催化的相反反应暴露于分子量范围为10至550 kDa的合成拥挤剂和蛋白质拥挤剂中。结果表明，大分子拥挤效应取决于反应方向。合成聚合物聚蔗糖和葡聚糖的存在会降低乙醇氧化的V和K。相反，这些拥挤剂对乙醛还原的这些动力学参数几乎没有影响，甚至会使其增加。V的这种增加可能是由于排除体积效应，而粘度阻碍NAD产物的释放会部分抵消这种效应。在大于YADH的葡聚糖溶液中存在耗尽层，这使大分子拥挤效应更加复杂，耗尽层会减弱粘度带来的阻碍。25 g/L葡聚糖或葡萄糖与25 g/L聚蔗糖或蔗糖产生的不同效应表明，还必须考虑软相互作用。葡萄糖、葡聚糖和聚蔗糖二元混合物的数据支持了这种对相反因素的“调节”。虽然最初提出大分子拥挤主要通过排除体积效应影响蛋白质，但这项工作补充了越来越多的证据，表明其他因素，如优先水合作用、化学相互作用和耗尽层的存在也对拥挤的总体效应有贡献。
