NDLI: Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

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Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Vohník, S. Tuma, R. Woodward, C. Hanson, C. Thomas, G. J. Fuchs, J. A.
Description	Country affiliation: United States Author Affiliation: Vohník S ( Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 64110, USA.)
Abstract	The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.
ISSN	09618368
e-ISSN	1469896X
Journal	Protein Science
Issue Number	1
Volume Number	7
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	1998-01-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Escherichia Coli Chemistry Thioredoxins Binding Sites Cysteine Hydrogen-ion Concentration Models, Molecular Mutation Genetics Oxidation-reduction Protein Conformation Spectrum Analysis, Raman Sulfhydryl Compounds Titrimetry Research Support, U.s. Gov't, P.h.s. Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

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