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Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61.
J Biol Chem. 2003 Mar 14; 278(11):9203-11.JB

Abstract

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, Cys(95), Cys(82), and Cys(61), 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 Cys(61) 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 Cys(61) 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 10(6) m(-1) s(-1), and the low K(m) (9.0 microm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H(2)O(2) (K(m) > 1.7 mm). Rapid inactivation of Tpx due to Cys(61) overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H(2)O(2). 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.

Authors+Show Affiliations

Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

12514184

Citation

Baker, Laura M S., and Leslie B. Poole. "Catalytic Mechanism of Thiol Peroxidase From Escherichia Coli. Sulfenic Acid Formation and Overoxidation of Essential CYS61." The Journal of Biological Chemistry, vol. 278, no. 11, 2003, pp. 9203-11.
Baker LM, Poole LB. Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61. J Biol Chem. 2003;278(11):9203-11.
Baker, L. M., & Poole, L. B. (2003). Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61. The Journal of Biological Chemistry, 278(11), 9203-11.
Baker LM, Poole LB. Catalytic Mechanism of Thiol Peroxidase From Escherichia Coli. Sulfenic Acid Formation and Overoxidation of Essential CYS61. J Biol Chem. 2003 Mar 14;278(11):9203-11. PubMed PMID: 12514184.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61. AU - Baker,Laura M S, AU - Poole,Leslie B, Y1 - 2003/01/03/ PY - 2003/1/7/pubmed PY - 2003/5/15/medline PY - 2003/1/7/entrez SP - 9203 EP - 11 JF - The Journal of biological chemistry JO - J Biol Chem VL - 278 IS - 11 N2 - 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, Cys(95), Cys(82), and Cys(61), 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 Cys(61) 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 Cys(61) 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 10(6) m(-1) s(-1), and the low K(m) (9.0 microm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H(2)O(2) (K(m) > 1.7 mm). Rapid inactivation of Tpx due to Cys(61) overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H(2)O(2). 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/12514184/Catalytic_mechanism_of_thiol_peroxidase_from_Escherichia_coli__Sulfenic_acid_formation_and_overoxidation_of_essential_CYS61_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)71291-5 DB - PRIME DP - Unbound Medicine ER -