College of Chemistry and Chemical Engineering, Shaoxing University, Shaoxing, Zhejiang Province 312000, China.
College of Chemistry and Chemical Engineering, Shaoxing University, Shaoxing, Zhejiang Province 312000, China.
Int J Biol Macromol. 2023 Jun 30;241:124525. doi: 10.1016/j.ijbiomac.2023.124525. Epub 2023 Apr 20.
Macromolecular crowding can change kinetics of enzyme catalysis. How interaction between enzymes and neighboring macromolecules contributes to the crowding effect on enzyme catalysis has not been quantitatively revealed. In this study, crowding effects of dextran and poly(ethylene glycol) (PEG) on glucose oxidase (GOx) are studied. Fluorescence resonance energy transfer experiments show the high transfer efficiency and stable interaction between the dextran and GOx. Further fluorescence quenching analysis also proves that the association of the dextran-GOx pair can become stronger than that of the PEG-GOx pair. Dextrans with concentrations above or below their chain overlap concentrations (c*) reduce Michaelis constants (K) of GOx catalysis by 90 % or 45 %, respectively, through volume exclusion mechanism, and in the meantime elevate the enzymatic efficiency (k/K) by 8-fold or by 3-fold, respectively, which is more dramatic than that found in other enzymes before. Strong association between the enzyme and the dextran results in slow turnover rates (k). Intermediate crowding with weak to moderate affinity to the enzyme below the c* can tune the k higher than in the free state. Catalysis under crowded conditions is a joint effect of the enzyme-crowder nonspecific interaction, volume exclusion and overlap condition of the crowders.
大分子拥挤会改变酶催化的动力学。酶与相邻大分子之间的相互作用如何促成对酶催化的拥挤效应尚未得到定量揭示。在这项研究中，研究了葡聚糖和聚（乙二醇）（PEG）对葡萄糖氧化酶（GOx）的拥挤效应。荧光共振能量转移实验表明，葡聚糖与 GOx 之间具有高的能量转移效率和稳定的相互作用。进一步的荧光猝灭分析也证明，葡聚糖-GOx 对的结合可以比 PEG-GOx 对的结合更强。浓度高于或低于其链重叠浓度（c*）的葡聚糖通过体积排除机制将 GOx 催化的米氏常数（K）分别降低 90%或 45%，同时使酶效率（k/K）分别提高 8 倍或 3 倍，这比以前在其他酶中发现的更为显著。酶与葡聚糖之间的强烈结合导致较慢的周转率（k）。在 c*以下，与酶的弱至中等亲和力的中间拥挤可以将 k 调至高于游离状态。拥挤条件下的催化是酶-拥挤者非特异性相互作用、体积排除和拥挤者重叠条件的共同作用。
