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Papers In Press, published online ahead of print January 3, 2003
Department of Biochemistry, Wake Forest University, Winston-Salem, NC 27157
Corresponding Author: lbpoole{at}wfubmc.edu
Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase (TrxR) to reduce alkyl hydroperoxides. Tpx contains three Cys residues, C95, C82 and C61, and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin (Prx) family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S and C82,95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity and the C82S mutation only slightly affects reaction rates. Furthermore, a sulfenic acid intermediate at C61 generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole- (NBD-)labeled C82,95S, confirming the identity of C61 as the peroxidatic center. In stopped flow kinetic studies, Tpx and Trx1 form a Michaelis complex during turnover with a catalytic efficiency of 3.0 x 10E6 M-1 s-1, and the low Km (9.0 µM) of Tpx for CHP demonstrates substrate specificity towards alkyl hydroperoxides over H2O2 (Km > 1.7 mM). Rapid inactivation of Tpx due to C61 overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxy-eicosatetranoic acid (15-HPETE), but not H2O2. Unlike most other 2-Cys Prxs, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond, yet is homodimeric in solution.
J. Biol. Chem, 10.1074/jbc.M209888200
Submitted on September 26, 2002
Revised on December 30, 2002
Accepted on January 3, 2003
Catalytic mechanism of Thiol peroxidase from Escherichia coli: Sulfenic acid formation and overoxidation of essential Cys61
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