Department of Chemistry, 418 SGM Building, University of Southern California, 3620 McClintock Avenue, Los Angeles, CA 90089-1062, USA.
Proc Natl Acad Sci U S A. 2010 Sep 28;107(39):16869-74. doi: 10.1073/pnas.1010381107. Epub 2010 Sep 9.
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 previously 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.
在生物技术和生物化学中，一个基本的挑战是设计有效酶的能力。这样做将是对酶催化起源的充分理解的一个令人信服的体现。尽管取得了令人印象深刻的进展，但在这方面的大多数进展都是通过将反应片段放置在适当的位置来实现的，而不是通过优化环境的预组织来实现，预组织是酶催化的关键因素。合理地改善预组织需要能够可靠地评估实际催化效果的方法。这项工作以前设计的 Kemp 消除酶作为计算机辅助酶设计的基准，使用经验价键作为主要筛选工具。观察到的绝对催化效果和定向进化的效果被复制和分析（假设底物在设计的位置）。结果发现，在 Kemp 消除酶的情况下，过渡态电荷分布使得很难利用活性位点的极性，即使能够量化不同突变的影响。出乎意料的是，发现定向进化突变体导致反应物状态的溶剂化程度降低，而不是像自然进化酶那样更常见的过渡态稳定模式。最后指出，我们在改进 Kemp 消除酶方面的困难不是由于忽略了奇特的效应，而是由于设计一个预组织的环境的挑战，这个环境将利用在过渡态形成过程中电荷分布的微小变化。
