Microscopic pKa values of Escherichia coli thioredoxin.
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.
Chivers
P T
PT
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Prehoda
K E
KE
Volkman
B F
BF
Kim
B M
BM
Markley
J L
JL
Raines
R T
RT
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
D000143:Acids; D002247:Carbon Isotopes; D011994:Recombinant Proteins; D013879:Thioredoxins; D011490:Protein Disulfide Reductase (Glutathione)