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

使用稳定同位素方法评估体外和体内酶反应机制的新方法

• 批准号: 09672199
• 负责人: FURUTA Takashi
• 金额: $ 2.05万
• 依托单位: Tokyo University of Pharmacy and Life Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672199/
• 关键词: L-Histidine; Urocanic Acid; Histidine Ammonia-Lyase; In Vivo Enzyme Mechanism; In Vivo Enzyme Activities; Metabolism; Stable Isotopes; GC-MS; MS; 代謝異常症; 体内動態; 酵素反応機構; 安定同位体

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.

本研究的目的是用稳定同位素方法直接阐明在人体内组氨酸解氨酶催化L-组氨酸转化为尿囊酸的酶机制。两名健康志愿者(受试者A和B)单剂量口服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]组氨酸(L-His-[M+4]或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]或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]组氨酸(L-His-[M+5])。给药后24小时取血、尿标本，用稳定同位素稀释质谱仪进行分析，经血尿液L-组氨酸和尿犬酸的质谱分析，发现有L-组氨酸[M+3]、L-组氨酸[M+4]和尿犬酸C-5‘位的氢-氢交换形成的[M+3]、L-His-[M+4]和UA-[M+3]。L组氨酸和尿刀酸酶催化C-5‘位氢交换的发现，为消除反应中通过碳负离子中间体逐步可逆的机理提供了合理的解释。尿刀酸咪唑环C-5’位氢交换的时间历程数据(y截距值)可能反映了碳负离子中间体的稳定性或寿命。体内氢交换的程度与组氨酸解氨酶(荧光假单胞菌)在9.0℃时催化的体外酶反应的程度接近(T.Furuta et al.(1992)J.Biol.化学，267,12600-12605)。本研究证明了共轭碳原子上发生氢交换的事实对于解释哺乳动物氨解酶系统在体外和体内催化消除反应的机理具有重要价值。