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But-3-ene-1,2-diol: A Mechanism-Based Active Site Inhibitor for Coenzyme B-12-Dependent Glycerol Dehydratase

Lookup NU author(s): Antonius Pierik, Emeritus Professor Bernard Golding


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Coenzyme B-12-dependent glycerol dehydratase is a radical enzyme that catalyses the conversion of glycerol into 3-hydroxypropanal and propane-1,2-diol into propanal via enzyme-bound intermediate radicals. The substrate analogue but-3-ene-1,2-diol was studied in the expectation that it would lead to the 4,4-dihydroxylbut-2-en-1-yl radical, which is stabilised (allylic) and not reactive enough to retrieve a hydrogen atom from 5'-deoxyadenosine, thereby interrupting the catalytic cycle. Racemic and enantiomerically pure but-3-ene-1,2-diols and their [1,1-H-2(2)], [2-H-2] and [4,4-H-2(2)] isotopomers were synthesised and characterised by NMR spectroscopy. (S)-[4-C-14]but-3-ene-1,2-diol was also prepared. Kinetic measurements showed but-3-ene-1,2-diol to be a competitive inhibitor of glycerol dehydratase (K-i=0.21 mM, k(i)= 5.0 x 10(-2)s(-1)). With [4-C-14]but-3-ene-1,2-diol it was demonstrated that species derived from the dial become tightly bound to the enzyme's active site, but not covalently bound, because the radioactivity could be removed upon denaturation of the enzyme. EPR measurements with propane-1,2-diol as substrate generated sharp signals after 10 s that disappeared after about 1 min. In contrast, EPR resonances appeared and disappeared more slowly when but-3-ene-1,2-diol was incubated with the enzyme. Among the deuteroted isotopomers, only [1,1-H-2(2)]but-3-ene-1,2-diol showed a significantly different EPR spectrum from that of the unlabelled diol; this indicated that coupling between the unpaired electron and a deuterium at C-1 was stronger than with deuterium at C-2 or C-4. The experiments suggest the formation of the 1,2-dihydroxybut-3-en-1-yl radical, which decomposes to unidentified product(s).

Publication metadata

Author(s): Pierik AJ, Graf T, Pemberton L, Golding BT, Retey J

Publication type: Article

Publication status: Published

Journal: ChemBioChem

Year: 2008

Volume: 9

Issue: 14

Pages: 2268-2275

ISSN (print): 1439-4227

ISSN (electronic): 1439-7633

Publisher: Wiley - VCH Verlag GmbH & Co. KGaA


DOI: 10.1002/cbic.200800213


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Funder referenceFunder name
HPRN-CT-2002-00195European Commission