Proton-transfer effects in the active-site region of Escherichia coli thioredoxin using two-dimensional sup 1 H NMR

Journal Article · Tue Apr 30 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00231a023· OSTI ID:5556326

Dyson, H J; Tennant, L L ^[1]; Holmgren, A ^[2]

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1. Research Institute of Scripps Clinic, La Jolla CA (USA)
2. Karolinska Institutet, Stockholm (Sweden)

A series of two-dimensional (2D) correlated {sup 1}H NMR spectra of reduced and oxidized Escherichia coli thioredoxin have been used to probe the effects of pH in the vicinity of the active site, -Cys{sub 32}-Gly-Pro-Cys{sub 35}-, using the complete proton resonance assignments available for thioredoxin. In either oxidation state, the majority of residues of the thioredoxin molecule remain unchanged between pH 5.7 and pH 10, as indicated by the identical chemical shifts of the C{sup {alpha}}H, C{sup {beta}}H, and other protons. In reduced thioredoxin, a fairly widespread region around the active-site dithiol is affected by the titration of a group or groups with pK{sub a} approximately 7.1-7.4 in {sup 2}H{sub 2}O. Another titration, with pK{sub a} approximately 8.4, affects a smaller region of the protein. Oxidized thioredoxin contains a disulfide and no free thiol groups; nevertheless, the proton resonances of many groups in the active-site region were observed to titrate with a pK{sub a} of 7.5, probably as a result of an abnormally high pK{sub a} value for the carboxyl group of the buried Asp-26 residue. For reduced thioredoxin, the results indicate that Asp-26 is titrating in this pH range, as well as both thiol groups. The new results are strongly suggestive that the mechanism of thioredoxin-catalyzed protein disulfide reduction may be critically dependent on proton transfer as well as electron transfer within the active site.

• OSTI ID: 5556326
• Journal Information: Biochemistry; (United States), Vol. 30:17; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
DITHIOLS
NUCLEAR MAGNETIC RESONANCE
ELECTRON TRANSFER
BIOCHEMICAL REACTION KINETICS
CHEMICAL SHIFT
ESCHERICHIA COLI
HEAVY WATER
PROTONS
REDOX POTENTIAL
BACTERIA
BARYONS
ELEMENTARY PARTICLES
FERMIONS
HADRONS
HYDROGEN COMPOUNDS
KINETICS
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEONS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXYGEN COMPOUNDS
REACTION KINETICS
REAGENTS
RESONANCE
THIOLS
WATER
550601* - Medicine- Unsealed Radionuclides in Diagnostics