Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol

Tetsuo Toraya, Naohisa Tamura, Takeshi Watanabe, Mamoru Yamanishi, Naoki Hieda, Koichi Mori

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)


The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co-C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5′-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg2+ and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5′-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H2S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.

Original languageEnglish
Pages (from-to)437-446
Number of pages10
JournalJournal of biochemistry
Issue number4
Publication statusPublished - Oct 2008


  • Adenosylcobalamin
  • Coenzyme B
  • Diol dehydratase
  • Mechanism-based inactivation
  • Radical enzyme

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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