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

Research Project

Project/Area Number 09672199
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionTokyo University of Pharmacy and Life Science

Principal Investigator

FURUTA Takashi  Tokyo University of Pharmacy and Life Science, Pharmacy, Associate Professor, 薬学部, 助教授 (70120152)

Co-Investigator(Kenkyū-buntansha) SHIBASAKI Hiromi  Tokyo University of Pharmacy and Life Science,Pharmacy, Assistant, 薬学部, 助手 (20206121)
KASUYA Yasuji  Tokyo University of Pharmacy and Life Science,Pharmacy, Professor, 薬学部, 教授 (90096686)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥1,300,000 (Direct Cost: ¥1,300,000)
KeywordsL-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
Research Abstract

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.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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