title: Mechanism-based inactivation of coenzyme B12-dependent diol dehydratase by 3-unsaturated 1,2-diols and thioglycerol.
abstract: 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, Mg(2+) 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). H(2)S 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.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Okayama, Japan. toraya@cc.okayama-u.ac.jp
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Toraya
authors.forename: Naohisa
authors.initials: N
authors.lastname: Tamura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Watanabe
authors.forename: Mamoru
authors.initials: M
authors.lastname: Yamanishi
authors.forename: Naoki
authors.initials: N
authors.lastname: Hieda
authors.forename: Koichi
authors.initials: K
authors.lastname: Mori
mesh_terms: D002072:Butylene Glycols / Q000494:pharmacology*; D003038:Cobamides / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005990:Glycerol / Q000031:analogs & derivatives* / Q000494:pharmacology; D006018:Glycols / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D011408:Propanediol Dehydratase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D002072:Butylene Glycols; D003038:Cobamides; D004791:Enzyme Inhibitors; D006018:Glycols; C009465:thioglycerol; D011408:Propanediol Dehydratase; C000913:cobamamide; D005990:Glycerol; C034598:3-butene-1,2-diol