Investigation of acid-base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry.
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, FeCl(2), 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. pK(a) showing a bell-shaped plot. Oxidative catechol cleavage by a pyridine-monosubstituted beta-cyclodextrin in the presence of TACN and FeCl(2) 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.
Brivio
Michela
M
Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
Schlosrich
Janne
J
Ahmad
Mark
M
Tolond
Caroline
C
Bugg
Timothy D H
TD
D002384:Catalysis; D050548:Catechol 1,2-Dioxygenase / Q000235:genetics / Q000378:metabolism*; D002396:Catechols / Q000138:chemical synthesis / Q000737:chemistry; D003198:Computer Simulation; D005296:Ferrous Compounds / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D009154:Mutation; D011725:Pyridines / Q000737:chemistry*
D002396:Catechols; D005296:Ferrous Compounds; D011725:Pyridines; D050548:Catechol 1,2-Dioxygenase; C034221:catechol; C023666:pyridine