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Observed crowding effects on Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase enzyme activity are not due to excluded volume only.
Scientific Reports ( IF 3.8 ) Pub Date : 2017-Jul-28 , DOI: 10.1038/s41598-017-07266-w Mariane Rotta 1, 2 , Luis F S M Timmers 3, 4 , Carlos Sequeiros-Borja 3, 4 , Cristiano V Bizarro 1 , Osmar N de Souza 3, 4 , Diogenes S Santos 1 , Luiz A Basso 1, 2
Scientific Reports ( IF 3.8 ) Pub Date : 2017-Jul-28 , DOI: 10.1038/s41598-017-07266-w Mariane Rotta 1, 2 , Luis F S M Timmers 3, 4 , Carlos Sequeiros-Borja 3, 4 , Cristiano V Bizarro 1 , Osmar N de Souza 3, 4 , Diogenes S Santos 1 , Luiz A Basso 1, 2
Affiliation
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The cellular milieu is a complex and crowded aqueous solution. Macromolecular crowding effects are commonly studied in vitro using crowding agents. The aim of the present study was to evaluate the effects, if any, of macromolecular synthetic crowding agents on the apparent steady-state kinetic parameters (K m , k cat , and k cat /K m ) of Mycobacterium tuberculosis 2-trans-enoyl-ACP (CoA) reductase (InhA). Negligible effects on InhA activity were observed for ficoll 70, ficoll 400 and dextran 70. A complex effect was observed for PEG 6000. Glucose and sucrose showed, respectively, no effect on InhA activity and decreased k cat /K m for NADH and k cat for 2-trans-dodecenoyl-CoA. Molecular dynamics results suggest that InhA adopts a more compact conformer in sucrose solution. The effects of the crowding agents on the energy (E a and E η ), enthalpy (∆H # ), entropy (∆S # ), and Gibbs free energy (∆G # ) of activation were determined. The ∆G # values for all crowding agents were similar to buffer, suggesting that excluded volume effects did not facilitate stable activated ES # complex formation. Nonlinear Arrhenius plot for PEG 6000 suggests that "soft" interactions play a role in crowding effects. The results on InhA do not unequivocally meet the criteria for crowding effect due to exclude volume only.
中文翻译：
观察到的对结核分枝杆菌 2-反式烯酰基-ACP (CoA) 还原酶活性的拥挤效应不仅仅归因于排除体积。
细胞环境是一个复杂且拥挤的水溶液。通常使用拥挤剂在体外研究大分子拥挤效应。本研究的目的是评估大分子合成拥挤剂对结核分枝杆菌 2-反式烯酰表观稳态动力学参数（K m 、 k cat和 k cat /K m ）的影响（如果有） -ACP (CoA) 还原酶 (InhA)。观察到聚蔗糖 70、聚蔗糖 400 和葡聚糖 70 对 InhA 活性的影响可忽略不计。观察到 PEG 6000 的复杂影响。葡萄糖和蔗糖分别对 InhA 活性没有影响，并且降低了 NADH 和 k cat 的k cat /K m对于2-反式十二碳烯酰辅酶A。分子动力学结果表明，InhA 在蔗糖溶液中采用更紧凑的构象异构体。确定了拥挤剂对活化能量（E a和 E η ）、焓（ΔH # ）、熵（ΔS # ）和吉布斯自由能（ΔG # ）的影响。所有拥挤剂的 ΔG #值与缓冲液相似，表明排除的体积效应不会促进稳定的活化 ES #复合物形成。 PEG 6000 的非线性阿伦尼乌斯图表明“软”相互作用在拥挤效应中发挥作用。由于仅排除体积，InhA 的结果并未明确满足拥挤效应的标准。
更新日期：2017-07-30
中文翻译：
观察到的对结核分枝杆菌 2-反式烯酰基-ACP (CoA) 还原酶活性的拥挤效应不仅仅归因于排除体积。
细胞环境是一个复杂且拥挤的水溶液。通常使用拥挤剂在体外研究大分子拥挤效应。本研究的目的是评估大分子合成拥挤剂对结核分枝杆菌 2-反式烯酰表观稳态动力学参数（K m 、 k cat和 k cat /K m ）的影响（如果有） -ACP (CoA) 还原酶 (InhA)。观察到聚蔗糖 70、聚蔗糖 400 和葡聚糖 70 对 InhA 活性的影响可忽略不计。观察到 PEG 6000 的复杂影响。葡萄糖和蔗糖分别对 InhA 活性没有影响，并且降低了 NADH 和 k cat 的k cat /K m对于2-反式十二碳烯酰辅酶A。分子动力学结果表明，InhA 在蔗糖溶液中采用更紧凑的构象异构体。确定了拥挤剂对活化能量（E a和 E η ）、焓（ΔH # ）、熵（ΔS # ）和吉布斯自由能（ΔG # ）的影响。所有拥挤剂的 ΔG #值与缓冲液相似，表明排除的体积效应不会促进稳定的活化 ES #复合物形成。 PEG 6000 的非线性阿伦尼乌斯图表明“软”相互作用在拥挤效应中发挥作用。由于仅排除体积，InhA 的结果并未明确满足拥挤效应的标准。
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