Department of Chemistry, University of Michigan, Ann Arbor, MI, United States; Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, United States.
Methods Enzymol. 2022;669:151-172. doi: 10.1016/bs.mie.2021.12.018. Epub 2022 Jan 28.
Adenosylcobalamin- (AdoCbl) dependent enzyme reactions involved the transfer of hydrogen atoms between the 5'-carbon of the coenzyme and the substrates and products of the reaction. Tritium and deuterium kinetic isotope effect measurements are, therefore, a valuable tool to probe the mechanisms of AdoCbl-dependent enzymes, as they can provide information about the reaction pathway and the rate-determining step. Furthermore, if the intrinsic kinetic isotope effect can be isolated, information on the nature of the transition state associated with hydrogen transfer can be obtained. In this chapter I present methods for the preparation of isotopically-labeled AdoCbl and their use in rapid chemical quench experiments that allow isotope effects on specific steps in the reaction to be isolated. These techniques are illustrated with examples from my laboratory's studies on the AdoCbl dependent enzyme, glutamate mutase.
腺钴胺素（AdoCbl）依赖性酶反应涉及辅酶 5'-碳与反应的底物和产物之间氢原子的转移。因此，氚和氘动力学同位素效应测量是研究 AdoCbl 依赖性酶机制的一种有价值的工具，因为它们可以提供有关反应途径和速率决定步骤的信息。此外，如果可以分离出固有动力学同位素效应，则可以获得与氢转移相关的过渡态的性质的信息。在本章中，我介绍了制备同位素标记的腺钴胺素的方法及其在快速化学猝灭实验中的应用，这些实验可以分离反应中特定步骤的同位素效应。这些技术通过我实验室对腺钴胺素依赖性酶谷氨酸变位酶的研究示例进行了说明。
