Enzymatic Activation of Water

生命系统利用来自环境的化学能来合成各种各样的生物聚合物；核酸、蛋白质、多糖和复杂的甘油脂。这些聚合物不是静态的，而是处于恒定的流动状态，不断地被合成、修复和/或降解。这些生物聚合物的降解都是热力学上有利的水解过程。因此，活生物体为了维持其聚合结构而进行了一场西西弗斯式的斗争。正如我们所知，生命之所以可能存在，只是因为大量的水是一种无效的反应物。大量利用水来降解生物分子的酶的一个主要化学作用是在受控的方式下增强水的反应性。结构生物学和诱变研究已经提出了水活化的潜在化学机制，包括脱溶、碱催化和金属离子配位，但这些机制的贡献基本上尚未得到验证。动力学同位素效应是提供最直接的过渡态结构的实验测量。由于技术上的困难，以前无法确定动力学同位素对水活化的影响，在本研究中，将开发三种研究磷酸酯水解的方法，这些方法可以很容易地适用于其他酶，包括蛋白酶和脂肪酶。这三种方法依赖于质谱领域取得的不同进展。前两种方法利用了热转化（热解）与同位素比值质谱仪的耦合。第三种方法采用全分子质谱法，利用新一代四极飞行时间质谱仪的能力，利用串联质谱法精确测定分子的同位素组成。动力学同位素效应将增强我们对酶促水活化机制的理解，并可能导致改进蛋白质和其他仿生催化剂的设计。当确定KIEs的方法得到充分开发和测试后，将积极寻求与其他酶学家进一步合作，使用GC/TC/IRMS设备的特殊功能。磷酸盐的分析不仅与酶学有关，而且与其他各种跨学科领域有关。同位素组成的测定在环境、气候变化和考古研究中具有重要意义；除了用作代谢示踪剂和表征化学反应机制的工具外。生物化学本科专业活跃，每年约有30名大四学生从事实验室研究项目，并参加妇女和少数民族暑期研究项目。这个项目将提高几个在我的实验室和合作者参与研究的本科生的意识。这些人，连同支持的研究生和博士后助理，将介绍现代质谱的能力，以及如何通过稳定同位素的应用来增强这些能力。这些机会应该为他们的科学事业带来强大和广泛适用的分析技能。

Living systems use chemical energy derived from the environment to synthesize a large variety of biological polymers; nucleic acids, proteins, polysaccharides and complex glycerolipids. These polymers are not static, but in a constant state of flux, continuously being synthesized, repaired and/or degraded. The degradations of each of these biological polymers are thermodynamically favorable hydrolytic processes. Thus living organisms engage in a Sisyphean struggle to maintain their polymeric structures. Life, as we know it, is possible only because bulk water is an ineffective reactant. A major chemical role of the large number of enzymes that use water to degrade biological molecules is to enhance the reactivity of water in a controlled fashion. Potential chemical mechanisms of water activation, include desolvation, base catalysis and metal ion coordination have been suggested by structural biology and mutagenesis studies, but the contribution of each of these mechanisms remains essentially untested. Kinetic isotope effects are the experimental measurements that provide the most direct insight into transition state structures. Technical difficulties have previously prevented the determination of kinetic isotope effects on water activation, in this research three methods for studying phosphoester hydrolysis that could be readily adapted to other enzymes, including proteases and lipases will be developed. These three methods rely on different advances that have been made in the realm of mass spectrometry. The first two methods take advantage of the coupling of thermal conversion (pyrolysis) to an isotope ratio mass spectrometer. The third method employs whole molecule mass spectrometry and takes advantage of the capabilities of the new generation of quadrupole-time of flight mass spectrometers to precisely determine the isotopic composition of molecules utilizing tandem mass spectrometry. The kinetic isotope effects will enhance our understanding of the mechanism(s) of enzymatic water activation and may lead to improved design of protein and other biomimetic catalysts. When the methodology for determining KIEs is well-developed and tested, further collaborations with other enzymologists will be actively sought to use the special capabilities of the GC/TC/IRMS facility. Analysis of phosphate is relevant not only to enzymology but to a variety of other interdisciplinary areas. Determination of isotopic composition is important in the study of the environment, climate change and archaeology; in addition to their uses as metabolic tracers and tools in the characterization of chemical reaction mechanisms. CWRU has an active biochemistry undergraduate program with ~30 seniors each year engaging in laboratory research projects and participation in summer research programs for women and disadvantaged minorities. This project will increase the awareness of several undergraduates participating in the research in my lab and with collaborators. These individuals, along with the supported graduate student and post-doctoral associate, will be introduced to the capabilities of modern mass spectrometry and how they may be enhanced by the application of stable isotopes. These opportunities should bring a strong and widely applicable analytical skill set to their scientific careers.