DIRECT MEASUREMENT OF THE ASPARTIC-ACID-26 PK(A) FOR REDUCED ESCHERICHIA-COLI THIOREDOXIN BY C-13 NMR

Because of interference from the pH-dependent behavior of nearby group s in the active site of Escherichia coil thioredoxin, the pK(a) of the buried carboxyl group of the aspartic acid at position 26 has been di fficult to quantitate. We report a direct measurement of this pK(a) us ing an NMR method utilizing the correlation between the (CH)-H-beta pr oton resonances and the (CO)-C-13 of the titrating carboxyl group. The experiments show unequivocally that the pK(a) is 7.3-7.5, rather than the value of 9 or greater recently proposed by Wilson, N. A., et al. [(1995) Biochemistry 34, 8931-8939]. The assignment of the titrating r esonances to Asp 26 is unambiguous: the values of the (CH)-H-beta chem ical shifts correspond exactly to those of Asp 26, and their titration in the pH range 5.7-10.0 is the same as that observed previously for the proton resonances alone. In addition, the chemical shift of the ca rboxyl C-13 resonance at pH 5.7 is upfield of those of the other carbo xyl and carboxamide resonances, diagnostic for a protonated carboxyl g roup. The resonances assigned to Asp 26 are the only ones that titrate in the pH range 5.7-10.5. None of the other aspartate and glutamate r esidues in the molecule are titrated in this pH range, consistent with their positions on the surface of the molecule. The pK(a) measured fo r Asp 26 in reduced thioredoxin is identical within experimental error to that measured in the oxidized form of the protein. This is signifi cant for the reductive mechanism of thioredoxin: the buried salt bridg ed/hydrogen-bonded side chains of Asp 26 and Lys 57 are likely to cont ribute to the facility of the reaction by providing a convenient sourc e and sink for protons in the hydrophobic environment of the complex b etween thioredoxin and its substrates.