The conserved, buried aspartic acid in oxidized Escherichia coli thioredoxin has a pKa of 7.5. Its titration produces a related shift in global stability.
Aspartic acid 26 in Escherichia coli thioredoxin is located at the bottom of a hydrophobic cavity, near the redox-active disulfide of the active site. Asp 26 is embedded in the protein except for part of the surface of one carboxyl oxygen. The high degree of evolutionary conversion of Asp 26 suggests that it plays a critical role in thioredoxin function. We have determined the pKa of Asp 26 by a novel electrophoretic method based on the relative electrophoretic mobilities of wild-type thioredoxin and of D26A thioredoxin (with Asp 26 replaced by alanine). The pKa of Asp 26 determined by this technique is 7.5, more than 3 units above the pKa of a solvated carboxyl side chain. The titration of Asp 26 is thermodynamically linked to the stability of thioredoxin. As expected if thioredoxin stability depends on the ionization state of Asp 26, delta Go WT, the free energy of the cooperative denaturation reaction of wild-type thioredoxin by guanidine hydrochloride, varies with pH in a sigmoidal fashion in the vicinity of pH 7.5. Over the same pH range, the free energy for D26A folding, delta Go D26A, is pH independent and D26A is highly stabilized compared to wild type. From the thermodynamic cycle describing the linkage of Asp 26 titration to thioredoxin stability, the difference in free energy between wild-type thioredoxin with protonated Asp 26 and wild-type thioredoxin with deprotonated Asp 26, delta delta Go (COOH----COO-), is calculated to be 4.9 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)
Langsetmo
K
K
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Fuchs
J A
JA
Woodward
C
C
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002021:Buffers; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry*
D002021:Buffers; D004220:Disulfides; D013438:Sulfhydryl Compounds; D001224:Aspartic Acid; D013879:Thioredoxins; D011392:Proline; D003545:Cysteine; D000409:Alanine