Mechanism-based Inactivation of Coenzyme B₁₂-dependent Diol Dehydratase by 3-Unsaturated 1,2-Diols and Thioglycerol

• Published in: Journal of biochemistry (Tokyo) Vol. 144; no. 4; pp. 437 - 446
• Main Authors: Toraya, Tetsuo, Tamura, Naohisa, Watanabe, Takeshi, Yamanishi, Mamoru, Hieda, Naoki, Mori, Koichi
• Format: Journal Article
• Language: English
• Published: England Japanese Biochemical Society 01.10.2008; Oxford University Press
• Subjects: adenosylcobalamin; Butylene Glycols - pharmacology; Cobamides - chemistry; Cobamides - metabolism; coenzyme B12; diol dehydratase; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors - pharmacology; Escherichia coli - enzymology; Glycerol - analogs & derivatives; Glycerol - pharmacology; Glycols - pharmacology; Kinetics; mechanism-based inactivation; Models, Molecular; Propanediol Dehydratase - antagonists & inhibitors; Propanediol Dehydratase - chemistry; Propanediol Dehydratase - metabolism; radical enzyme; radical enzyme; mechanism-based inactivation; coenzyme B; diol dehydratase; adenosylcobalamin
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvn086

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.