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

机译：三同位素标记和动力学同位素效应：暴露酶系统中的AH转移步骤

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摘要

Kinetic isotope effect (KIE) studies can provide insight into the mechanismand kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 14, 2644-2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years, but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method and that such experiments are limited only by synthetic techniques, availability of 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）研究可以深入了解复杂催化级联的特定化学步骤的机制和动力学。氢kie测量的最近结果检测了酶动力学和催化功能之间的相关性，导致该地区的研究浪涌。遗憾的是，大多数酶的H-转移反应不是速率限制，观察到的kie不可能反映在感兴趣的化学步骤上的内在kies。鉴于他们重视了解所研究的化学步骤，精确测定观察到的数据的内在kie是必不可少的。 1975年，诺罗普制造了一种优雅的方法，可以从观察到的值（北罗姆，D.B.（1975）逐态分析酶反应中的动力学同位素效应的稳态分析。生物化学14,2644-2651]。 Northrop方法涉及使用所有三种氢同位素的Kie测量，其中一种用作参考同位素。多年来，这种方法已成功地与观察到的kies的不同组合使用，但在实验确定之前，没有理性选择参考同位素的标准从未进行过。在这里，我们使用两种不同酶作为模型反应，使用二羟氢醇和二氢纤维的还原与模型反应的二氢化酯和二氢丙酮素的不同参考同位素（以及不同的实验设计进行比较。已应用许多同位素标记图案以促进参考同位素的比较研究。结果表明了北罗波方法的多功能性，并且这种实验仅受合成技术，起始材料的可用性和与使用同位素组合相关的实验误差。

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  • 来源
    《Biochemistry》 |2011年第29期|共7页
  • 作者

    Arundhuti Sen; Atsushi Yahashiri; Amnon Kohen;

  • 作者单位

    The Department of Chemistry The University of Iowa Iowa City Iowa 52242 United States;

    The Department of Chemistry The University of Iowa Iowa City Iowa 52242 United States;

    The Department of Chemistry The University of Iowa Iowa City Iowa 52242 United States;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    Labeling; Isotope; Effects;

    机译：标记;同位素;效果;

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