Defining the mechanistic determinants of catalytic bias in cofactor-based enzymatic oxidation-reduction reactions

项目摘要 酶促反应包括细胞生物化学过程所必需的基本构件， 新陈代谢.虽然大多数酶促反应是可逆的，但某些酶在一种酶促反应中加速反应。 方向明显不同于反向。这种现象的机械决定因素- 通常被称为“催化偏差”-通常是未知的。我们这个项目的长期目标是确定 氧化还原酶（参与氧化还原反应的酶）采用的基本原理， 提供催化偏置的分子水平控制。在这样做的时候，我们将提供一个更大的基础， 了解所有生命中控制代谢过程的因素。具体而言，本提案的目标 描述影响含铁氢化酶模型定向反应性的决定因素， 结构上相关，但对特定可逆氢（H2）的催化偏置表现出很大差异 电子和质子的氧化反应。中心假设是，在氧化还原反应中， 酶的结果从各自的氧化态的相对差异稳定和不稳定 氧化还原辅因子对决定催化循环的限速步骤至关重要。的基本原理， 建议是，实施将导致定义拟议模型的机械决定因素 系统拟议的工作将最终阐明普遍的，机械因素的催化偏见， 控制大量的氧化还原酶，如果不是全部的话。中心假设将通过追求三个测试 具体的目的是研究1）活性位点的还原电位如何接近辅助氧化还原簇; 2） 活性位点簇蛋白环境的特征; 3）次级配位球 结构动力学可以控制基于辅因子的氧化还原催化中的反应性和催化偏差。到 为了实现这些目标，我们将采用一种创新的战略，将生物化学、结构、光谱和 在一个独特的，独一无二的模型氢化酶平台上的计算方法。拟议的研究是 意义重大，因为它将描绘影响氧化还原辅因子微调的分子决定因素 通过氧化态的相对稳定化或不稳定化来提供一定的反应性， 引导细胞中的能量和物质。这项工作将提供额外的基础资源， 整合多种生物物理和计算方法的蓝图。

PROJECT SUMMARY Enzymatic reactions comprise the basic building blocks essential to the biochemical processes of cellular metabolism. While most enzymatic reactions are reversible, certain enzymes accelerate a reaction in one direction significantly differently than the reverse direction. The mechanistic determinants of this phenomenon – often referred to as “catalytic bias” – are generally unknown. Our long-term goal of this project is to identify the fundamental principles adopted by redox enzymes (enzymes involved in oxidation-reduction reactions) that provide molecular level control of the catalytic bias. In doing so, we will provide a greater fundamental understanding of the factors that control metabolic processes in all life. Specifically, the objective of this proposal is to delineate determinants that influence the directional reactivity of model Fe containing hydrogenases that are structurally related but exhibit large differences in catalytic bias for the specific reversible hydrogen (H2) oxidation reaction from electron and protons. The central hypothesis is that catalytic bias commonly in redox enzymes results from the relative differential stabilization and destabilization of oxidation states of the respective redox cofactors critical to determining the rate limiting step of the catalytic cycle. The rationale underlying the proposal is that the implementation will result in defining mechanistic determinants of the proposed model system. The proposed work will culminate in elucidating universal, mechanistic factors of catalytic bias that govern a large number of, if not all, redox enzymes. The central hypothesis will be tested by pursuing three specific aims that examine how 1) the reduction potential of the active site proximal accessory redox clusters; 2) the characteristics of the active site cluster protein environment; and 3) the secondary coordination sphere structural dynamics can control reactivity and catalytic bias in cofactor-based oxidation reduction catalysis. To pursue these aims, we will employ an innovative strategy integrating biochemical, structural, spectroscopic, and computational approaches on a unique, one-of-a-kind model hydrogenase platform. The proposed research is significant, because it will delineate molecular determinants that influence the fine-tuning of redox cofactors through the relative stabilization or destabilization of oxidation states to afford certain reactivity fundamental to directing energy and matter in cells. The work will provide additional foundational resources in the form of a blueprint for integrating multiple biophysical and computational approaches.