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

项目总结 酶反应构成了细胞生化过程中必不可少的基本构件 新陈代谢。虽然大多数酶反应是可逆的，但某些酶在一种情况下会加速反应 方向与相反方向明显不同。这一现象的机械决定因素-- 通常被称为“催化偏向”--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)所采用的基本原理 提供催化偏置的分子水平控制。在这样做的过程中，我们将提供一个更基本的 理解所有生命中控制新陈代谢过程的因素。具体地说，这项提案的目标是 描述了影响含有氢酶的模型铁的定向反应性的决定因素 在结构上相关，但在特定可逆氢(H2)的催化偏置方面表现出很大的差异 电子和质子的氧化反应。中心假设是催化偏向通常存在于氧化还原过程中。 酶的产生是由于各自氧化状态的相对差异稳定和不稳定。 氧化还原辅助因素对确定催化循环的限速步骤至关重要。其背后的理由是 建议是，实施将导致确定拟议模式的机械性决定因素 系统。拟议的工作将最终阐明催化偏向的普遍的、机械的因素 管理大量的，如果不是全部的话，氧化还原酶。核心假设将通过追求三个方面来检验 检查1)活性部位近端附件氧化还原簇的还原潜力的特定目标；2) 活性中心簇蛋白质环境的特点；3)次级配位球体 在辅因子氧化还原催化中，结构动力学可以控制反应活性和催化偏向。至 为了实现这些目标，我们将采用一种创新的战略，将生化、结构、光谱和 一个独特的、独一无二的氢酶模型平台上的计算方法。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或不稳定来提供基本的某些反应性 引导细胞中的能量和物质。该工作将以以下形式提供额外的基础资源 综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.