Increased dynamic effects in a catalytically compromised variant of Escherichia coli dihydrofolate reductase.
Increased dynamic effects in a catalytically compromised variant of Escherichia coli dihydrofolate reductase.
复制标题
DOI：
10.1021/ja410519h
复制
发表时间：
2013-12-11
影响因子：
15
通讯作者：
Allemann RK
中科院分区：
文献类型：
--
作者：
Ruiz-Pernia JJ;Luk LY;García-Meseguer R;Martí S;Loveridge EJ;Tuñón I;Moliner V;Allemann RK
利用同位素取代（15N, 13C, 2H）对大肠杆菌二氢叶酸还原酶EcDHFR-N23PP/S148A的催化受损变体进行了研究，研究了这些突变对催化的影响。EcDHFR-N23PP/S148A催化反应中化学步骤速率常数的降低主要是准经典自由能垒的增加和过渡态划分面上重交轨迹数量的增加的结果。由于变体酶催化反应的条件不太好，因此需要更高程度的活性位点重组才能达到TS。虽然变体中丢失了毫秒级的活性位点运动，但在飞秒时间尺度上具有更大的灵活性。因此，“动态敲除”EcDHFR-N23PP/S148A在化学步骤层面上是“动态敲入”，与化学配位的动态偶联增加实际上对催化是有害的。这一发现很可能不仅适用于EcDHFR中的氢转移，也适用于其他酶系统。
Isotopic substitution (15N, 13C, 2H) of a catalytically compromised variant of Escherichia coli dihydrofolate reductase, EcDHFR-N23PP/S148A, has been used to investigate the effect of these mutations on catalysis. The reduction of the rate constant of the chemical step in the EcDHFR-N23PP/S148A catalyzed reaction is essentially a consequence of an increase of the quasi-classical free energy barrier and to a minor extent of an increased number of recrossing trajectories on the transition state dividing surface. Since the variant enzyme is less well set up to catalyze the reaction, a higher degree of active site reorganization is needed to reach the TS. Although millisecond active site motions are lost in the variant, there is greater flexibility on the femtosecond time scale. The “dynamic knockout” EcDHFR-N23PP/S148A is therefore a “dynamic knock-in” at the level of the chemical step, and the increased dynamic coupling to the chemical coordinate is in fact detrimental to catalysis. This finding is most likely applicable not just to hydrogen transfer in EcDHFR but also to other enzymatic systems.
登录
查看更多内容
DOI：
10.1126/science.1198542
发表时间：
2011-04-08
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Bhabha G;Lee J;Ekiert DC;Gam J;Wilson IA;Dyson HJ;Benkovic SJ;Wright PE
通讯作者：
Wright PE
影响因子：
3.9
作者：
Allemann, Rudolf K.;Evans, Rhiannon M.;Loveridge, E. Joel
通讯作者：
Loveridge, E. Joel
影响因子：
8
作者：
Khavrutskii, Ilja V.;Price, Daniel J.;Brooks, Charles L., III
通讯作者：
Brooks, Charles L., III
影响因子：
56.9
作者：
Boehr, David D.;McElheny, Dan;Wright, Peter E.
通讯作者：
Wright, Peter E.
影响因子：
56.9
作者：
FRAUENFELDER, H;SLIGAR, SG;WOLYNES, PG
通讯作者：
WOLYNES, PG