高效智能人工硒酶的理性设计与功能研究

How to remove reactive oxygen species (ROS) with high efficiency and intelligence is a challenge in the field of biomimetic selenoenzyme, the resolution of which will help to maintain redox balance in a dynamic state and protect organism against oxidative damage. Here, we proposed a novel idea “Rational design of artificial enzymes based on multiple synergistic catalytic factors such as catalysis, recognition, and microenvironment” to create highly efficient artificial selenoenzymes. In order to achieve this goal, protein simulation, genetic engineering, selective modification, and supramolecular complexation methods were used to incorporate a glutathione peroxidase (GPx)-like active center into natural proteins. Meanwhile, the biological function of GPx mimics can be reversibly controlled by light, temperature, or Ca2+ at the catalytic site, recognition site, and entire active center using the strategies, including the conformational transformation of environmentally responsive molecules, the reversibility of supramolecular complexation, and the allosteric effect of proteins. Taking the advantages of interdisciplinary, we will further make a thorough study of the structural and functional relationship of artificial selenoenzyme. This includes the crystallization and structure determination of artificial selenoenzymes, and the clarification of their physicochemical properties, enzymatic kinetics and thermodynamic processes, as well as antioxidant activity at cellular and subcellular level. It is intended that the future design and construction of smart artificial selenoenzyme with high efficiency will be favored by exploring the microscopic reaction mechanism and revealing the essence of catalysis.

如何高效智能清除体内过量的活性氧自由基（ROS），调控细胞内氧化还原微环境的动态平衡，以保护机体免受氧化损伤是当前仿硒酶研究中的难点问题。本项目提出“催化、识别与微环境多因素协同仿酶”的新思路，以天然蛋白质为骨架，综合运用蛋白质模拟、基因工程、选择性化学修饰和超分子复合等方法，精确构筑类谷胱甘肽过氧化物酶（GPx）催化活性中心，获得高效的GPx酶模型。同时，利用环境响应分子变构、超分子可逆复合和蛋白质别构效应等，在GPx模拟物的催化部位、识别部位和整个活性中心不同层面上，实现光、温度或Ca2+对其抗氧化功能的可逆调控。借助多学科交叉的优势，阐明人工硒酶的理化性质、酶促动力学与热力学过程、细胞和亚细胞水平的抗氧化活性，并解析其晶体结构，深入研究它们的结构与功能的关系。进一步通过探索酶的微观催化反应机理，揭示其高效催化的本质，为未来设计与构筑高效智能的人工硒酶提供指导。

酶是自然界长期进化而产生的一种生物催化剂，是一切生命代谢活动的基础。在单分子水平上开展仿酶研究，不仅可为酶的结构与功能探索提供新的思路和方法，还可发展各种新型人工酶制剂，弥补天然酶来源有限以及体内/体外实际应用中的各种不利因素。本项目执行后，基于蛋白质结构计算与分子模拟，提出了催化、识别与微环境多因素协同仿酶设计的新思路。运用基因定点突变、半胱氨酸缺陷型表达和选择性化学修饰，精确构筑了类酶活性中心，发展了蛋白质别构调控和超分子调控两类酶活性调控新方法，成功制备了钙离子、光、pH调控的多种高效智能的人工酶模型。相较于天然谷胱甘肽过氧化物酶，上述人工酶的催化效率和底物转一性均达到了天然酶相同数量级水平，并研究了其酶动力学与热力学过程，测定了其饱和动力学和酶-底物结合的热力学参数，以及亚细胞水平的抗氧化能力，明确了人工酶的催化最适pH值和温度。最终，通过纳米孔单分子检测技术，还成功捕获人工酶催化循环过程中不稳定的中间体，包括硒醇（ESeH）、硒硫（ESeSG）以及用传统手段难以捕捉的次硒酸（ESeOH），为深入探索其结构与功能关系奠定了基础。