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Microscopic pKa values of Escherichia coli thioredoxin. Biochemistry 1997 Dec 02;36(48):14985-91

Date

12/16/1997

Pubmed ID

9398223

DOI

10.1021/bi970071j

Scopus ID

2-s2.0-0030608830   152 Citations

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.

Author List

Chivers PT, Prehoda KE, Volkman BF, Kim BM, Markley JL, Raines RT

Author

Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Acids
Binding Sites
Carbon Isotopes
Escherichia coli
Genetic Variation
Hydrogen-Ion Concentration
Models, Chemical
Mutagenesis, Site-Directed
Nuclear Magnetic Resonance, Biomolecular
Protein Disulfide Reductase (Glutathione)
Recombinant Proteins
Statistics as Topic
Thioredoxins
Titrimetry
jenkins-FCD Prod-444 eb4ebd1a08581aba961d3befd3b851a3c3ec6b46