title: Real-time monitoring of intermediates reveals the reaction pathway in the thiol-disulfide exchange between disulfide bond formation protein A (DsbA) and B (DsbB) on a membrane-immobilized quartz crystal microbalance (QCM) system.
abstract: Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.
authors.affiliation: From the Innovative Flex Course for Frontier Organic Material Systems (iFront), Yamagata University, Yamagata 992-8510, Japan and Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Yazawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Furusawa
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Okahata
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D058525:Quartz Crystal Microbalance Techniques; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013501:Surface-Active Agents / Q000737:chemistry; D014451:Ubiquinone / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D013501:Surface-Active Agents; D014451:Ubiquinone; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli
keywords: Disulfide Bond Formation Protein; Enzyme Kinetics; Kinetics; Membrane Lipids; Membrane Proteins; Protein Chemistry; Quartz Crystal Microbalance; Supported Lipid Bilayer; Transient Kinetic Analysis