1999 Fiscal Year Final Research Report Summary
A New Method for Assessing the In Vitro and In Vivo Enzyme Reaction Mechanisms Using Stable Isotope Methodology
| Project/Area Number |
09672199
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Tokyo University of Pharmacy and Life Science |
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Principal Investigator |
FURUTA Takashi Tokyo University of Pharmacy and Life Science, Pharmacy, Associate Professor, 薬学部, 助教授 (70120152)
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| 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)
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| Project Period (FY) |
1997 – 1999
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| Keywords | L-Histidine / Urocanic Acid / Histidine Ammonia-Lyase / In Vivo Enzyme Mechanism / In Vivo Enzyme Activities / Metabolism / Stable Isotopes / GC-MS / MS |
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| 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.
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