Learning from Nature hwo Two-metal Centres Operate: Studies of Purple Acid Phosphatases (PAP) and Polyphenol Oxidases (PPO)-Enzymes and Models

本项目的主要目标是通过对酶和模型复合物的结构和功能研究的结合，解决两种金属离子对两类酶：紫色酸性磷酸酶（PAP）和多酚氧化酶（PPO与儿茶酚氧化酶（CO）和酪氨酸酶（Tyr））的各自作用的问题。作为起点，我们将进一步推进对结构已知系统（植物PAP和CO）的理解。在分子水平上对作用机制的描述仍存在一些争议，有几个方面仍有待阐明。大量的数据是失踪的活性物种，结合模式的基板（各自的作用，每个金属中心）和H+的重要性和作用及其在催化过程中的转移（pH值的依赖性）。此外，我们将把我们的注意力集中在同一个家庭的高度重要的酶，迄今为止仍然很差的特点：人PAP和酪氨酸。基于我们小组的专业知识，我们将纯化并尝试获得这些酶的X射线结构，以评估与亲本酶的已知结构的差异，并通过整体比较获得信息。了解酶机制的分子方面是目前生物无机化学中最具挑战性的目标之一，使用协同的跨学科方法似乎是实现这一目标的最有前途的方式。目前的项目是建立在这样一个方法，将包括：1。酶的分离和纯化（高量用于众所周知的程序-植物PAP和CO-和纯化程序的最终确定-人PAP和Tyr）2.结构（X射线）和生物化学研究（抑制剂，动力学）3.仿生模型，以获得可靠的结构，光谱和反应特性4.研究金属中心的分子、电子和磁性结构的物理方法（模型和酶）

The main objective of this project is to address the question of the respective roles of the two metal ions of two classes of bimetallic enzymes: the purple acid phosphatases (PAP) and the polyphenol oxidases (PPO with catechol oxidases (CO) and tyrosinase (Tyr)) via combination of structural and functional studies on enzymes and on model complexes. As a starting point, we will further advance the understanding of structurally-known systems (plant PAP and CO). Some controversies persist in the description of the mechanism of action at the molecular level and several aspects remain to be elucidated. A great deal of data are missing on the active species, the binding mode of the substrate (respective role of each metallic centre) and the importance and role of H+ and their transfer in the catalytic processes (pH dependence). In addition, we will focus our attention on highly important enzymes of the same family which to date remain poorly characterized: human PAP and Tyr. Based on our groups expertises, we will purify and attempt to obtain X-ray structures of these enzymes to assess the differences with the known structures of parent enzymes and get information through an overall comparison. Understanding molecular aspects of enzyme mechanisms is currently among the most challenging objectives in bioinorganic chemistry and using a synergistic interdisciplinary approach appears to be the most promising way of reaching this goal. The present project is built on such an approach and will include: 1. Isolation and purification of enzymes (High amount for well-known procedure - plant PAP and CO- and finalization of purification procedure - human PAP and Tyr) 2. Structural (X-ray) and biochemical studies (inhibitors, kinetics) 3. Biomimetic models to get reliable structural, spectroscopic and reactivity characteristics 4. Physical methods to investigate the molecular, electronic and magnetic structures of metal centres (models and enzymes)