Effects of Arg90 Neutralization on the Enzyme-Catalyzed Rearrangement of Chorismate to Prephenate.
Effects of Arg90 Neutralization on the Enzyme-Catalyzed Rearrangement of Chorismate to Prephenate.
复制标题
Arg90 中和对酶催化分支酸重排至预苯酸的影响。
DOI：
10.1021/ct0500803
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发表时间：
2005
影响因子：
5.5
通讯作者：
W. L. Jorgensen
中科院分区：
文献类型：
--
作者：
C. Guimarães;Marina Udier;Ivan Tubert;W. L. Jorgensen
分支酸酯酶（CM）是催化分支酸酯克莱森重排为预苯酸酯的酶。在最近的努力，以了解催化的基础上，由CM，Kienhöfer和同事（J. Am. Soc.2003，125，3206-3207）报道了枯草芽孢杆菌CM（BsCM）中Arg 90突变为瓜氨酸（Cit）的结果，瓜氨酸是一种等排但中性的精氨酸类似物。一个CA。突变后，观察到总催化的kcat降低10（4）倍或自由能垒（ΔG（ΔG））增加5.9 kcal/mol。在这项工作中，注意力转向确定的关键因素，有助于降低Arg 90 Cit BsCM的催化效率。采用QM/MM Monte Carlo/自由能微扰法，得到ΔΔG（ε）= 3.3 kcal/mol。突变体的更高的自由能势垒完全与TS的稳定性差有关，特别是其羧酸基团之一，由中性Cit。此外，反应的放能增加了2.0千卡/摩尔。由于BsCM受到产品释放的限制，该步骤导致从Arg 90到Cit的速率常数降低10（4）倍。
Chorismate mutase (CM) is an enzyme that catalyzes the Claisen rearrangement of chorismate to prephenate. In a recent effort to understand the basis for catalysis by CM, Kienhöfer and co-workers (J. Am. Chem. Soc. 2003, 125, 3206-3207) reported results on the mutation of Arg90 in Bacillus subtilis CM (BsCM) to citrulline (Cit), an isosteric but neutral arginine analogue. An ca. 10(4)-fold decrease in kcat or 5.9 kcal/mol increase in the free-energy barrier (ΔG(‡)) for the overall catalysis was observed upon mutation. In this work, attention is turned to determining the key factors that contribute to the reduced catalytic efficiency of Arg90Cit BsCM. Using a combined QM/MM Monte Carlo/Free-Energy Perturbation method, a ΔΔG(‡) value of 3.3 kcal/mol is obtained. The higher free-energy barrier for the mutant is exclusively related to inferior stabilization of the TS, particularly one of its carboxylate groups, by neutral Cit. In addition, the reaction becomes 2.0 kcal/mol more exergonic. As BsCM is limited by product release, this step contributes to the remainder of the 10(4)-fold decrease in the rate constant in going from Arg90 to Cit.