Computer Simulation of Enzymatic Reactions

描述（由申请人提供）：酶催化生物化学反应，并在执行和控制大多数生命过程中发挥重要作用。因此，对生物系统的详细了解需要了解相应酶的作用。许多疾病可以通过开发药物来控制，这些药物可以阻断导致这些疾病的病原体的关键生物途径中的酶的作用，这一事实突出了这种理解的重要性。至少在原则上，我们也有可能开发出药物来恢复与毁灭性疾病有关的有缺陷的酶的活性。另一个重要的发展是酶设计领域的出现，在定向进化和计算机辅助设计方面取得了可喜的进展。然而，这一进展尚未导致设计酶可以与天然酶竞争。因此，这一重要领域的潜力可以通过计算方法来提高，这些方法实际上确定了被催化反应的活化势垒。在以前的资助期间，我们开发了改进和应用强大的方法来模拟酶的反应，并检查了它们的性能。使用这些方法帮助我们量化了关键的催化因子，并使我们进入了一个阶段，在这个阶段，我们可以为酶设计的新前沿和催化景观的探索做出重大贡献。在此，我们提出以下计划：（i）我们将主要致力于计算机辅助酶设计：（a）推进EVB作为酶设计最后阶段的定量工具的使用;（B）为不同的筛选阶段开发粗粒度的方法，以及（c）在实际的酶设计项目中使用我们的方法，包括改变混杂酶的作用，改进可用的设计酶并帮助设计新的酶。(ii)我们将继续发展从头算自由能微扰方法，使其能够有效地用于酶促反应的研究。这将包括：（a）改进QM（ai）/MM自由能模拟中EV B参考势的使用;（B）发展和改进我们的具有平均势和基于Langevin动力学的平均力势的加速QM/MM方法;（c）改进CDFT方法在金属酶研究和自由能映射中的使用。(iii)我们将通过推进以下项目来量化折叠和稳定性之间的关系：（a）探索活性位点的预组织与蛋白质的局部稳定性之间的关系;（B）探索热稳定性与催化之间的关系;以及（c）使用简化模型来评估总稳定性和相应的化学活化自由能。(iv)我们将对几类重要的酶促反应进行研究。(v)我们将继续对不同的非静电催化方案进行系统的研究。1 酶催化生化反应，在执行和控制大多数生命过程中发挥重要作用。因此，对生物系统的详细了解需要对决定相应酶的作用的因素进行定量描述。这是至关重要的，因为许多疾病可以通过开发药物来控制，这些药物可以阻断引起这些疾病的细菌或病毒的关键生物途径中的酶的作用。至少在原则上，我们也有可能开发出药物来恢复与毁灭性疾病有关的有缺陷的酶的活性。此外，量化不同残基对酶的原始活性和药物结合的贡献应有助于对抗耐药性。定向进化和酶设计的出现开辟了该领域的另一个令人兴奋的前沿，具有很大的实用性（例如生物化学相关分子的新合成）和概念上的重要性。然而，尽管最近在计算机辅助酶设计方面取得了成功，但这一进展尚未导致与天然酶竞争的设计酶。因此，很明显，这一重要领域的潜力可以通过计算方法以主要方式增强，这些方法实际上可以准确有效地评估被催化反应的活化势垒。在以前的资助期间，我们开发了强大的计算方法来模拟酶促反应和阐明相关的催化因素。现在，我们准备推动酶的结构/功能相关性研究的前沿，重点是（i）大量的酶设计研究，这些研究的进展应该为理解酶催化提供最终证据，（ii）更深入地了解蛋白质折叠和催化之间的关系，以及（iii）开发和验证用于酶系统计算的强大策略。即使是适度的成功也将帮助我们改进我们的方法，并最终帮助开发有效的药物，这些药物将有助于对抗与缺陷酶相关的疾病，并帮助设计专门的酶，最后，作为对抗耐药性的指南。1

DESCRIPTION (provided by applicant): Enzymes catalyze biochemical reactions and play a major role in performing and controlling most life processes. Therefore, a detailed understanding of biological systems requires an understanding of the action of the corresponding enzymes. The importance of such an understanding is highlighted by the fact that many diseases can be controlled by developing drugs that block the action of enzymes in the crucial biological pathways of the pathogens that cause these diseases. It is also possible, at least in principle, to develop drugs that restore the activity of defective enzymes that are involved in devastating diseases. Another important development has been the emergence of the field of enzyme design, with promising advances in directed evolution and in computer aided design. However, this progress has not yet led to designer enzymes that can rival native enzymes. Thus, the potential of this important field can be enhanced in a major way by computational approaches that actually determine the activation barriers of the reactions that are being catalyzed. During previous grant periods, we developed refined and applied powerful methods for simulating reactions in enzymes and examined their performance. Using these methods helped us to quantify key catalytic factors and brought us to a stage where we can make significant contributions to the new frontiers of enzyme design and the exploration of catalytic landscapes. Here, we propose the following projects: (i) We will invest major effort into computer-aided enzyme design by: (a) advancing the use of the EVB as a quantitative tool in the final stage of enzyme design; (b) developing coarse grained approaches for the different screening stages, and (c) using our approaches in actual enzyme design projects, including changing the action of promiscuous enzymes, improving available designer enzymes and helping in the design of new enzymes. (ii) We will continue to develop ab initio-free energy perturbation approaches to a level where they can be used effectively in studies of enzymatic reactions. This will include: (a) improving the use of EVB reference potentials for QM(ai)/MM free energy simulations; (b) developing and refining our accelerated QM/MM approach with average potentials and a Langevin dynamics based potential of mean force, and (c) refining the use of the CDFT method in studies of metalloenzymes and in free energy mapping. (iii) We will quantify the relationship between folding and stability by advancing the following projects: (a) exploring the relationship between the pre-organization of the active sites and the local stability of the protein; (b) exploring the relationship between thermostability and catalysis, and (c) using a simplified model to evaluate the total stability and the corresponding chemical activation free energy. (iv) We will conduct studies of several important classes of enzymatic reactions. (v) We will continue with the systematic examination of different non-electrostatic catalytic proposals. 1 PUBLIC HEALTH RELEVANCE: Enzymes catalyze biochemical reactions and play a major role in performing and controlling most life processes. Thus, the detailed understanding of biological systems requires a quantitative description of the factors that determine the action of the corresponding enzymes. This is crucial, since many diseases can be controlled by developing drugs that block the action of enzymes in the key biological pathways of the bacteria or viruses that cause these diseases. It is also possible, at least in principle, to develop drugs that restore the activity of defective enzymes that are involved in devastating diseases. Furthermore, quantifying the contributions of different residues to both the original activity of the enzyme and to the binding of drugs should help in fighting drug resistance. The emergence of directed evolution and enzyme design has opened another exciting front in the field, with great practical (e.g. the novel synthesis of biochemically relevant molecules) and conceptual importance. Nevertheless, despite recent success in computer aided enzyme design, this progress has not led yet to designer enzymes that rival native enzymes. Thus, it is clear that the potential of this important field can be enhanced in a major way by computational approaches that can actually accurately and efficiently evaluate the activation barriers of the reactions that are being catalyzed. During the previous grant periods, we developed powerful computational approaches for the simulation of enzymatic reactions and for the elucidation of the relevant catalytic factors. Now, we are ready to push the frontiers of studies on the structure/function correlation of enzymes, focusing on (i) massive studies of enzyme design where progress in such studies should provide the ultimate proof of the understanding of enzyme catalysis, (ii) gaining a deeper understanding of the relationship between protein folding and catalysis, and (iii) developing and validating powerful strategies for the computation of enzymatic systems. Even moderate successes will help us to refine our methods and, ultimately, to help in the development of effective drugs that will aid in the fight against diseases that are associated with defective enzymes as well as helping in the design of specialized enzymes, and finally, to act as a guide in fighting drug resistance. 1