Abstract:
:The pH dependence of the 13C chemical shifts of the side-chain carboxyl carbons of all Asp and Glu residues in the reduced and oxidized states of human thioredoxin and in a mixed disulfide complex of human thioredoxin with a target peptide from the transcription factor NF kappa B has been investigated by multidimensional triple-resonance NMR spectroscopy. While the titration curves for most of the side-chain carboxyl resonances exhibit simple Henderson-Hasselbalch behavior with pKa values not far from those found for model compounds, several side chains give rise to two- or three-step titration curves, indicative of the influence of multiple ionizations. In particular, the triad formed by Asp58, Asp60, and Asp61 forms such a complex network of titrating groups. The ionization behavior of Asp26 shows an abnormally high pKa value for an aspartate residue in all states of human thioredoxin, with pKa values of 9.9 in the reduced state, 8.1 in the oxidized state, 8.9 in the mixed disulfide complex, and 8.6 in an active site mutant in which Cys35 was replaced by Ala. The unambiguous determination of the pKa values of Asp26 for a variety of states of human thioredoxin presented in this paper is highly significant in view of two recent reports on Escherichia coli thioredoxin which presented contradicting pKa values for Asp26 and Cys35 [Wilson et al. (1995) Biochemistry 34, 8931-8939; Jeng et al. (1995) Biochemistry 34, 10101-10105]. The stabilization of the protonated side chain of Asp26 in human thioredoxin is achieved via a hydrogen-bonding network involving the hydroxyl group of the neighboring Ser28 which is then connected to the active site region (comprising Cys32 and Cys35) via bound water molecules. The coupling of the buried Asp26 to the active site is responsible for the influence of the Asp26 ionization behavior on the titration shifts of active site residues.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Qin J,Clore GM,Gronenborn AMdoi
10.1021/bi952299hsubject
Has Abstractpub_date
1996-01-09 00:00:00pages
7-13issue
1eissn
0006-2960issn
1520-4995pii
bi952299hjournal_volume
35pub_type
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