CAREER: Determination of enzyme mechanisms using new kinetic isotope effect approaches

“生命过程化学”项目将通过这一职业奖，支持纽约州立大学布法罗分校的安德鲁·默金博士开发研究酶促反应机制的新方法。本研究的目的是确定两种酶的过渡态结构，催化反应与未解决的机制。为了实现这一目标，Murkin博士开发了一种基于二维核磁共振光谱的测量动力学同位素效应的方法，该方法比以前的方法具有更高的灵敏度和精度。将计算得到的理论动力学同位素效应与实验测量结果相匹配，建立过渡态模型。当一个分子发生反应并达到过渡态时，反应或经历几何或静电变化的原子在被较重的同位素取代时，会产生不同的速率。这种动力学同位素效应提供了一种实验上可获得的机制中速率限制阶段底物的图像。该奖项开发的技术应该引起广大科学家的兴趣，他们依赖于动力学同位素效应来研究各种过程，包括设计新疗法。该计划还旨在鼓励年轻人接受生物化学和相关科学方面的培训。为了激发年轻人的教育兴趣，将通过在中学和大学实验室进行的信息丰富的实践、探究式实验，向大布法罗学校的K-12学生（包括代表性不足的年轻人）介绍酶。在大学层面，过渡态抑制剂的设计和同位素动力学效应的测量将被纳入本科和研究生的化学课程。

With this CAREER award, the Chemistry of Life Processes Program is supporting Dr. Andrew Murkin of the University at Buffalo, The State University of New York, in the development of new methods for studying the mechanisms of enzymatic reactions. The objective of this research is to determine the transition state structures of two enzymes that catalyze reactions with unresolved mechanisms. To reach this goal, Dr. Murkin developed a method for measuring kinetic isotope effects based on two-dimentional NMR spectroscopy that delivers better sensitivity and precision than previous methods. Transition state models will be constructed by matching the theoretical kinetic isotope effects obtained computationally to those measured experimentally.When a molecule undergoes a reaction and reaches the transition state, the atoms that react or experience geometric or electrostatic changes exert differences in rates when substituted with heavier isotopes. Such kinetic isotope effects provide an experimentally accessible picture of the substrate during the rate-limiting step in a mechanism. The techniques developed from this award should be of interest to a wide audience of scientists who depend on kinetic isotope effects to study a variety of processes, including the design of new therapies. This program is also designed to encourage youth to pursue training in biochemistry and related sciences. To stimulate educational interest among youth, an introduction to enzymes will be provided to K-12 students in greater-Buffalo schools, including underrepresented youths, through informative hands-on, inquiry-based experiments conducted in middle schools and in a university laboratory. At the university level, the design of transition state inhibitors and measurement of kinetic isotope effects will be integrated into undergraduate and graduate chemistry courses.