Enzymatic Activation of Water

生命系统使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。这些生物聚合物中的每一种的降解都是化学上有利的水解过程。因此，活的有机体为了维持它们的聚合物结构而进行着一场西西弗斯式的斗争。正如我们所知，生命之所以可能，只是因为大量的水是一种无效的反应物。利用水降解生物分子的大量酶的主要化学作用是以受控的方式增强水的反应性。水活化的潜在化学机制，包括去溶剂化，碱催化和金属离子配位已被结构生物学和诱变研究所提出，但这些机制中的每一个的贡献仍然基本上未经测试。动力学同位素效应是提供对过渡态结构最直接洞察的实验测量。技术上的困难，以前阻止了水活化的动力学同位素效应的测定，在这项研究中，可以很容易地适应于其他酶，包括蛋白酶和脂肪酶的研究磷酸酯水解的三种方法将被开发。这三种方法依赖于质谱领域取得的不同进展。前两种方法利用热转化（热解）与同位素比质谱仪的耦合。第三种方法采用全分子质谱法，并利用新一代四极杆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，并可能导致蛋白质和其他仿生催化剂的改进设计。 当确定KIE的方法得到充分开发和测试后，将积极寻求与其他酶学家的进一步合作，以利用GC/TC/IRMS设施的特殊能力。 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、气候变化和考古学研究中非常重要;此外，它们还可用作代谢示踪剂和表征化学反应机制的工具。 CWRU有一个活跃的生物化学本科课程，每年约有30名高年级学生从事实验室研究项目，并参加针对妇女和弱势少数群体的夏季研究项目。这个项目将提高参与我实验室研究的几个本科生和合作者的意识。 这些人，沿着与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增强。这些机会应该为他们的科学生涯带来强大且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.