Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents.
Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents.
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DOI：
10.1021/ja806996q
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发表时间：
2009-04-01
影响因子：
15
通讯作者：
Clark, Douglas S.
中科院分区：
文献类型：
--
作者：
Hudson, Elton P.;Eppler, Ross K.;Beaudoin, Julianne M.;Dordick, Jonathan S.;Reimer, Jeffrey A.;Clark, Douglas S.
枯草杆菌蛋白酶嘉士伯的表面活性剂溶解到两种有机溶剂，异辛烷和四氢呋喃，并通过逐步变化的热力学水活度，aw水合。在这些体系中，表观转换数kcatapp的范围为0.2 - 80 s-1，随着aw的增加，在异辛烷中增加11倍，在四氢呋喃中增加50倍。采用活性位点抑制剂的19 F-NMR弛豫实验用于评估活性位点运动对aw的依赖性。NMR推导的快（k > 107 s-1）和慢（k < 104 s-1）活性位点运动的速率在两种溶剂中水合后增加，但只有慢运动与kcat相关。19 F化学位移是局部电子环境的灵敏探针，并提供了活性位点介电常数εas的经验测量，其在每种溶剂中随水合作用增加至εas ε 13。在两种溶剂中，过渡态自由能数据和ε遵循柯克伍德的偶极连续溶剂化模型，表明水也通过改变活性中心的电子环境和增加其极性来更好地稳定过渡态来增强催化作用。这些结果表明，有利的动力学和静电效应都有助于加速催化溶解枯草杆菌蛋白酶嘉士伯在有机溶剂中水合。
The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, aw. The apparent turnover number kcatapp in these systems ranged from 0.2 to 80 s−1 and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing aw. 19F-NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on aw. The rates of NMR-derived fast (k > 107 s−1) and slow (k < 104 s−1) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with kcat. The 19F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant εas, which increased with hydration to εas ≈ 13 in each solvent. In both solvents the transition state free energy data and εas followed Kirkwood’s model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site’s electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents.
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影响因子：
2.9
作者：
CARTER, P;WELLS, JA
通讯作者：
WELLS, JA
影响因子：
2.2
作者：
CARVER, JP;RICHARDS, RE
通讯作者：
RICHARDS, RE
影响因子：
56.9
作者：
FRAUENFELDER, H;SLIGAR, SG;WOLYNES, PG
通讯作者：
WOLYNES, PG
影响因子：
4.4
作者：
Kirkwood, John G.
通讯作者：
Kirkwood, John G.
DOI：
10.1021/je050333h
发表时间：
2006-03-01
期刊：
JOURNAL OF CHEMICAL AND ENGINEERING DATA
影响因子：
--
作者：
Gómez-Díaz, D;Mejuto, JC;Navaza, JM
通讯作者：
Navaza, JM