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首页> 外文期刊>Biochemistry >Triple Isotopic Labeling and Kinetic Isotope Effects: Exposing H-Transfer Steps in Enzymatic Systems
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Triple Isotopic Labeling and Kinetic Isotope Effects: Exposing H-Transfer Steps in Enzymatic Systems

机译：三重同位素标记和动力学同位素效应：暴露酶系统中的H转移步骤

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Kinetic isotope effect (KIE) studies can provide insight intonthe mechanismand kinetics of specific chemical steps in complex catalyticncascades. Recent results from hydrogen KIE measurements have examinedncorrelations between enzyme dynamics and catalytic function,nleading to a surge of studies in this area. Unfortunately, most enzymaticnH-transfer reactions are not rate limiting, and the observed KIEs do notnreliably reflect the intrinsic KIEs on the chemical step of interest. Givenntheir importance to understanding the chemical step under study,naccurate determination of the intrinsic KIE from the observed data isnessential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B.n(1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 14, 2644u00012651]. The Northrop methodninvolves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method hasnbeen successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of referencenisotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinctnexperimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions.nA number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The resultsndemonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availabilitynof starting materials, and the experimental error associated with the use of distinct combinations of isotopologues.
机译：动力学同位素效应（KIE）研究可为复杂催化级联反应中特定化学步骤的机理和动力学提供洞见。氢KIE测量的最新结果已经检验了酶动力学与催化功能之间的相关性，从而导致该领域的研究激增。不幸的是，大多数酶促H转移反应没有速率限制，并且观察到的KIE不能可靠地反映目标化学步骤中的内在KIE。考虑到了解所研究的化学步骤的重要性，从观察到的数据中准确测定内在KIE至关重要。 1975年，诺斯罗普（诺斯罗普）开发了一种优雅的方法，可从其观测值评估内在的KIE [诺斯罗普（Northrop），D。B.n（1975）酶反应中动力学同位素效应的稳态分析。生物化学14，2644u00012651]。诺斯罗普方法涉及使用所有三种氢同位素进行的KIE测量，其中三种氢同位素之一用作参考同位素。多年来，该方法已成功地与观察到的KIE的不同组合一起使用，但是，从未通过实验确定合理选择参考同位素的标准。在这里，我们使用两种不同的酶将二氢叶酸和二氢生物蝶呤还原为模型反应，比较了不同的参考同位素（因此进行了不同的实验设计）。n已使用多种同位素标记方式来促进参考同位素的比较研究。结果证明了诺斯罗普方法的多功能性，并且此类实验仅受合成技术，起始材料的可用性以及与使用不同同位素异构体组合相关的实验误差的限制。

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