TAILIEUCHUNG - Báo cáo khóa học: Escherichia coli thioredoxin inhibition by cadmium Two mutually exclusive binding sites involving Cys32 and Asp26

Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd 2+ led us to investigate the thioredoxin–cadmium interaction used calorimetric and spectroscopicmethods at different pHvalues to explore the relative contribution of putative binding residues (Cys32, Cys35, Trp28, Trp31 and Asp26) within or near the active site. At pH 8 or two binding sites were identified by isothermal titration calorimetry with affinity constants of 10·10 6 M )1 and 1·10 6 M )1 . . | Eur. J. Biochem. 271 1299-1309 2004 FEBS 2004 doi Escherichia coli thioredoxin inhibition by cadmium Two mutually exclusive binding sites involving Cys32 and Asp26 Fran oise Rollin-Genetet1 Catherine Berthomieu2 Anne-Hélène Davin1 and Eric Quemeneur1 1CEA Valrho DSV-DIEP Service de Biochimie postgénomique et Toxicologie Nucleaire Bagnols-sur-Ceze 2CEA Cadarache DSV-DEVM Laboratoire de Bioenergetique Cellulaire UMR 6191 CEA-CNRS Aix-Marseille II Saint-Paul-lez-Durance France Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd2 led us to investigate the thioredoxin-cadmium interaction properties. We used calorimetric and spectroscopic methods at different pH values to explore the relative contribution of putative binding residues Cys32 Cys35 Trp28 Trp31 and Asp26 within or near the active site. At pH 8 or two binding sites were identified by isothermal titration calorimetry with affinity constants of 10 X 106 M 1 and 1 X 106 M 1. For both sites a proton was released upon Cd2 binding. One mole of Cd2 per mole of reduced thioredoxin was measured by mass spectrometry at these pH values demonstrating that the two binding sites were partially occupied and mutually exclusive. Cd2 binding at either site totally inhibited the thiol-disulfide transferase activity of Trx. The absence of Cd2 interaction detected for oxidized or alkylated Trx and the inhibition of the enzymatic activity of thioredoxin by Cd2 supported the role of Cys32 at the first site. The fluorescence profile of Cd2 -bound thioredoxin differed however from that of oxidized thioredoxin indicating that Cd2 was not coordinated with Cys32 and Cys35. From FTIR spectroscopy we inferred that the second site might involve Asp26 a buried residue that deprotonates at a rather high and unusual pKa for a carboxylate . The pKa of the two residues Cys32 and Asp26 have been shown to be interdependent Chivers T. P. 1997 .

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