2-Cys Peroxiredoxins (Prx) are homodimeric antioxidant proteins able to decompose H2O2, organic peroxides and peroxynitrites, using a highly reactive Cys residue present in its active sites, named peroxidatic cysteine (CysP). As a consequence of hydroperoxide reduction, a disulfide is formed with a second Cys residue (resolving cysteine - CysR) as a consequence of partial unfolding of CysP containing ±-helix. Therefore, during its catalytical cycle, 2-Cys Prx alternates between two states: fully folded (FF- reduced cysteines) and locally unfolded (LU-oxidized cysteines). After the hydroperoxide, the oxidized cysteines are reduced by thioredoxin (Trx). Structural studies reveals that the CysP is surrounded by three residues highly conserved among the Prx named catalytic triad which is composed by a proline, a threonine/serine and an arginine, Its assumed that Pro, Thr/Ser and Arg, residue acts in the stabilizing the deprotonated form of CysP ,lowering its pKa raising its activity over its substrates. Recently, was proposed that the conserved Thr/Ser residue also would be involved in the recognition and substrate, specificity, but several aspects of reactivity, structural alternations still elusive. Saccharomyces cerevisiae possesses two very related cytosolic isoforms of Prx (Tsa1 and Tsa2) (86% of identity and 96% of similarility). However, are significant differences concerning the pKa of CysP (Tsa1=5.4 and Tsa2= 6.3). Recently the crystallographic structure Tsa1 from S. cerevisiae was determined, and its analysis revealed that the only difference in the active site of these enzymes lies on Thr (44) in Tsa1, which is replaced by a Ser in Tsa2, what indicates that this residue may be implicated in CysP pKa. Additional analysis of Tsa1 quaternary structure pointed that a second residual of Arg (146) is involved in the spatial positioning of CysP, by a polar molecular interaction net with Glu50 and Arg123 that implicates in the CysP folding and reactivity. Besides, the Thr44 residual is found very close to the CysP located in the opposite side of this net, also able to be involved in the FF/LU transition status and the interactions with Trx. The aims of this project are to investigate the participation of Thr44 on the FF and LU structural transition, the involvement of CysP on pKa and its structural significance to the Tsa1 reduction by Trx. To achieved these goals Tsa1T44A, Tsa1T44S and Tsa1T44V will be generated by site direct mutagenesis and after expression and purification the peroxidatic acticity over H2O2 and organic hydroperoxides will be evaluated by the DTT oxidation assay. The cysteine reactivity will be determinated by competitive kinetics with HRP (Horseradish peroxidase) and Trx linked peroxidase activity. Additionally, quaternary structure alterations as results of mutations will be evaluated with size-exclusion chromatography (SEC). These projects already has preliminary results and at this time were obtained the mutants Tsa1T44A and Tsa1T44V and the standardization of their expressions is in progress. We believe that the results obtained in this project may give important contributions for a better comprehension of mechanisms which involves the Prx pKa, structural transitions and its reduction by Trx.
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