A New Method for Assessing the In Vitro and In Vivo Enzyme Reaction Mechanisms Using Stable Isotope Methodology

当前研究的目标是一种方法，即体内酶机制的直接选择，即L-histidine to urocanic acid catalyzed by histidine ammonia-lyase in human by using stable isotope methodology。两个健康的波动者（subjects A and B） received a single 100-mg oral dose of L-[3，3-イイD12イイD1HイイD22イイD2， 1'， 3'-イD115イイD1NイイD22イイD2] histidine（L-His-[M+4]） or L-[3， 3， 5'-イD12イイD1HイD23イD2，1'，3'-イD115イD1NイD22イD2] histidine （L-His-[M+5]）。血液和尿液样本在管理员和分析后发现了超过24小时的血液和尿液样本，在等离子体和尿液样本后标记的L-组氨酸的存在中表示L-组氨酸的存在的L-组氨酸[M+3]，L-His-[M+4]和UA-[M+3]由C-3和/或L-组氨酸的C-5和C-5 '组成的C-5。在C-5 '中发现酶催化氢交换的C-5'中的两种L-组氨酸和urocanic酸提供了一种理性解释，通过一种可逆转的机制在铁原反应中的碳离子中间体。（y-截获值）在C-中的氢交换所发生5'的异源酸环可以反映出氢能增加的稳定性或碳离子中间体的生命周期。在体内发现了大量的氢交换物，以接近于9.0 （T.），即由组氨酸氨裂解酶（Pseudomonas fluorescens）催化的体外酶反应。Furuta等人（1992年），J. Biol. Chem.，267， 12600-12605）在被聚合的碳原子被证明是在研究中显示的氢交换的事实，该研究提供了一个重要的价值，并被标记为对被哺乳动物氨裂解酶系统催化的机构提炼的Elimination反应的机理，无论是在体外还是体内。

The objective of the present study is an approach to the direct elucidation of in vivo enzyme mechanism for the conversion of L-histidine to urocanic acid catalyzed by histidine ammonia-lyase in human by using stable isotope methodology.Two healthy volunteers (subjects A and B) received a single 100-mg oral dose of L-[3,3-ィイD12ィエD1HィイD22ィエD2, 1', 3'-ィイD115ィエD1NィイD22ィエD2] histidine (L-His-[M+4]) or L-[3, 3, 5'-ィイD12ィエD1HィイD23ィエD2, 1', 3'-ィイD115ィエD1NィイD22ィエD2] histidine (L-His-[M+5]). Blood and urine samples were obtained over 24 hr after the administraton and analyzed by stable isotope dilution mass spectrometry.The mass spectrometric analyses of L-histidine and urocanic acid in the plasma and urine samples after the administration of labeled L-histidines indicated the presence of L-His-[M+3], L-His-[M+4] and UA-[M+3] formed by the deuterium-hydrogen exchanges at C-3 and/or C-5' of L-histidine and at C-5' of urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction.The time course data (y-intercept value) of hydrogen exchange occurred at C-5' of the imidazole ring of urocanic acid may reflect the stability or the lifetime of a carbanion intermediate into which hydrogen can be incorporated. The extent of the hydrogen exchange in vivo was found to be close to that of the in vitro enzyme reaction catalyzed by histidine ammonia-lyase (Pseudomonas fluorescens) at 9.0 (T. Furuta et al. (1992) J. Biol. Chem., 267, 12600-12605). The fact of the hydrogen exchange occurred at the conjugated carbon atoms demonstrated in the study offers a significant value with regard to the mechanistic elucidation of elimination reactions catalyzed by mammalian ammonia-lyase systems, both in vitro and in vivo.