title: Microscopic pKa values of Escherichia coli thioredoxin.
abstract: Thiol:disulfide oxidoreductases have a CXXC motif within their active sites. To initiate the reduction of a substrate disulfide bond, the thiolate form of the N-terminal cysteine residue (CXXC) of this motif performs a nucleophilic attack. Escherichia coli thioredoxin [Trx (CGPC)] is the best characterized thiol:disulfide oxidoreductase. Previous determinations of the active-site pKa values of Trx have led to conflicting interpretations. Here, 13C-NMR spectroscopy, site-specific isotopic labeling, and site-directed mutagenesis were used to demonstrate that analysis of the titration behavior of wild-type Trx requires the invocation of microscopic pKa values for two interacting active-site residues: Asp26 (7.5 and 9.2) and Cys32 (CXXC; 7.5 and 9.2). By contrast, in two Trx variants, D26N Trx and D26L Trx, Cys32 exhibits a pKa near 7.5 and has a well-defined, single-pKa titration curve. Similarly, in oxidized wild-type Trx, Asp26 has a pKa near 7.5. In CVWC and CWGC Trx, Cys32 exhibits a single pKa near 6.2. In all five enzymes studied here, there is no evidence for a Cys35 (CXXC) pKa of < 11. This study demonstrates that a comprehensive approach must be used to unravel complex titration behavior of the functional groups in a protein.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
authors.forename: P T
authors.initials: PT
authors.lastname: Chivers
authors.forename: K E
authors.initials: KE
authors.lastname: Prehoda
authors.forename: B F
authors.initials: BF
authors.lastname: Volkman
authors.forename: B M
authors.initials: BM
authors.lastname: Kim
authors.forename: J L
authors.initials: JL
authors.lastname: Markley
authors.forename: R T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D000143:Acids / Q000737:chemistry; D001665:Binding Sites; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology*; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013223:Statistics as Topic; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics; D017186:Titrimetry
chemical_list: D000143:Acids; D002247:Carbon Isotopes; D011994:Recombinant Proteins; D013879:Thioredoxins; D011490:Protein Disulfide Reductase (Glutathione)