A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli

• Published in: The Journal of biological chemistry Vol. 275; no. 46; pp. 35908 - 35913
• Main Authors: Boschi-Muller, S, Azza, S, Sanglier-Cianferani, S, Talfournier, F, Van Dorsselear, A, Branlant, G
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 17-11-2000
• Subjects: Binding Sites; Biochemistry; Biochemistry, Molecular Biology; Catalysis; Cysteine - chemistry; Cysteine - metabolism; Disulfides - chemistry; Disulfides - metabolism; Dithionitrobenzoic Acid; Dithiothreitol - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Life Sciences; Methionine - analogs & derivatives; Methionine - metabolism; methionine sulfoxide reductase; Methionine Sulfoxide Reductases; Models, Chemical; Molecular Weight; Mutation; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Peptides - chemistry; Peptides - metabolism; Reducing Agents - analysis; Spectrometry, Mass, Electrospray Ionization; sulfenic acid; Sulfenic Acids - chemistry; Sulfenic Acids - metabolism; Sulfhydryl Compounds - analysis; Thioredoxins - metabolism; MsrA; methionine sulfoxide reductase; DTNB; DTT; thionitrobenzoate; dimedone; methionine sulfoxide
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M006137200

Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. The proposed chemical mechanism of Escherichia coli MsrA involves three Cys residues (positions 51, 198, and 206). A fourth Cys (position 86) is not important for catalysis. In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 --> Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated. Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate. This intermediate has been characterized by chemical probes and mass spectrometry analyses. Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process.