Exploring challenges in rational enzyme design by simulating the catalysis in artificial kemp eliminase

被引:101
作者
Frushicheva, Maria P. [1 ]
Cao, Jie [1 ]
Chu, Zhen T. [1 ]
Warshel, Arieh [1 ]
机构
[1] Univ So Calif, Dept Chem, Los Angeles, CA 90089 USA
基金
美国国家卫生研究院;
关键词
computer aided enzyme design; empirical valence bond; directed evolution; ANTIBODY; ENERGIES; ENZYMOLOGY; PROTEINS; DYNAMICS; CHARGE;
D O I
10.1073/pnas.1010381107
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
One of the fundamental challenges in biotechnology and in biochemistry is the ability to design effective enzymes. Doing so would be a convincing manifestation of a full understanding of the origin of enzyme catalysis. Despite an impressive progress, most of the advances on this front have been made by placing the reacting fragments in the proper places, rather than by optimizing the environment preorganization, which is the key factor in enzyme catalysis. Rational improvement of the preorganization would require approaches capable of evaluating reliably the actual catalytic effect. This work takes apreviously designed kemp eliminases as a benchmark for a computer aided enzyme design, using the empirical valence bond as the main screening tool. The observed absolute catalytic effect and the effect of directed evolution are reproduced and analyzed (assuming that the substrate is in the designed site). It is found that, in the case of kemp eliminases, the transition state charge distribution makes it hard to exploit the active site polarity, even with the ability to quantify the effect of different mutations. Unexpectedly, it is found that the directed evolution mutants lead to the reduction of solvation of the reactant state by water molecules rather that to the more common mode of transition state stabilization used by naturally evolved enzymes. Finally it is pointed out that our difficulties in improving Kemp eliminase are not due to overlooking exotic effect, but to the challenge in designing a preorganized environment that would exploit the small change it charge distribution during the formation of the transition state.
引用
收藏
页码:16869 / 16874
页数:6
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