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Issue 7, 2009
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Investigation of acid–base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry

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Abstract

The extradiol and intradiol catechol dioxygenase reaction mechanisms proceed via a common proximal hydroperoxide intermediate, which is processed via different Criegee 1,2-rearrangements. An R215W mutant of extradiol dioxygenase MhpB, able to produce a mixture of extradiol and intradiol cleavage products, was analysed at pH 5.2–8.6, and the yield of extradiol product was found to be highly pH-dependent, whereas the yield of intradiol product was pH-independent. The acid–base chemistry of a biomimetic reaction for extradiol oxidative catechol cleavage was also investigated, using 1,4,7-triazacyclononane, FeCl2, and pyridine in methanol, in which pyridine is proposed to act as both a general base and (in protonated form) a general acid. Kinetic experiments using a range of meta- and para-substituted pyridines gave a Brønsted plot of log(v) vs. pKa showing a bell-shaped plot. Oxidative catechol cleavage by a pyridine-monosubstituted β-cyclodextrin in the presence of TACN and FeCl2 in methanol yielded only intradiol cleavage products. It is therefore proposed that bifunctional acid–base catalysis is required for iron (II)-dependent extradiol catechol cleavage, whereas the rate-determining step for intradiol catechol cleavage does not involve acid–base catalysis.

Graphical abstract: Investigation of acid–base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry

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Article information


Submitted
02 Dec 2008
Accepted
18 Dec 2008
First published
09 Feb 2009

Org. Biomol. Chem., 2009,7, 1368-1373
Article type
Paper

Investigation of acid–base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry

M. Brivio, J. Schlosrich, M. Ahmad, C. Tolond and T. D. H. Bugg, Org. Biomol. Chem., 2009, 7, 1368
DOI: 10.1039/B821559F

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