PMID:9398223
| Citation |
Chivers, PT, Prehoda, KE, Volkman, BF, Kim, BM, Markley, JL and Raines, RT (1997) Microscopic pKa values of Escherichia coli thioredoxin. Biochemistry 36:14985-91 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi970071j |
| Keywords |
Acids/chemistry; Binding Sites; Carbon Isotopes; Escherichia coli/enzymology; Genetic Variation; Hydrogen-Ion Concentration; Models, Chemical; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Protein Disulfide Reductase (Glutathione)/chemistry; Protein Disulfide Reductase (Glutathione)/genetics; Recombinant Proteins/chemistry; Statistics as Topic; Thioredoxins/chemistry; Thioredoxins/genetics; Titrimetry |
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