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Originally published In Press as doi:10.1074/jbc.M209888200 on January 3, 2003

J. Biol. Chem., Vol. 278, Issue 11, 9203-9211, March 14, 2003
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Catalytic Mechanism of Thiol Peroxidase from Escherichia coli
SULFENIC ACID FORMATION AND OVEROXIDATION OF ESSENTIAL CYS61*

Laura M. S. Baker and Leslie B. PooleDagger

From the Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

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 to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys95, Cys82, and Cys61, and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). 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 Cys61 generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole-labeled C82S,C95S, confirming the identity of Cys61 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 × 106 M-1 s-1, and the low Km (9.0 µM) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H2O2 (Km > 1.7 mM). Rapid inactivation of Tpx due to Cys61 overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H2O2. Unlike most other 2-Cys peroxiredoxins, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond yet is homodimeric in solution.

* Project support was provided by National Institutes of Health Grant R01 GM50389 and by an Established Investigatorship from the American Heart Association (to L. B. P).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-6711; Fax: 336-716-7671; E-mail: lbpoole@wfubmc.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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