|M.Sc Student||Shekhter Talia|
|Subject||Synthetic Proteins as a Tool for Understanding Redox|
Properties of Grx3
|Department||Department of Chemistry||Supervisor||PROFESSOR EMERITUS Ehud Keinan|
Glutaredoxin 3 from E.coli (Grx3) is an important redox enzyme consisting of 82 residues. These include the redox active motif Cys-Pro-Tyr-Cys, typical of the glutaredoxin (Grx) family. This family of enzymes that belong to the thioredoxin (Trx) super-family, catalyze thiol-disulfide exchange reactions.
In reduced Grxs, the pKa of the N-terminal active-site nucleophilic cysteine residue (Cys11 in Grx3) is lowered significantly. The stabilization of the corresponding thiolate is expected to influence the redox potential of these enzymes.
Although Grx1 and Grx3 share the same active site sequence (CPYC), they exhibit different redox potentials (-233 and -198 mV, respectively). Residue 8 in both Grx3 and Grx1 has attracted significant attention due to its proximity to the nucleophilic thiolate of Cys11 in the reduced form of the enzymes. Arg occupies position 8 in Grx1 and Lys occupies this position in Grx3.
In order to better assess the role of residue 8 on the catalytic efficiency of the glutaredoxins, it was essential to study both the redox potential and the kinetics of equilibration. Several analogs of Grx3 were chemically synthesized with either lysine, arginine, citrulline, ornithine or norvaline at position 8 (Scheme 1A).
Studying the redox equilibration reaction between each of the above mutants and Trx (Scheme 1B), we found no significant change in the thermodynamic parameters. These results suggest that in the wildtype Lys8 stabilizes both reduced and oxidized forms of Grx3 to approximately the same extent. Still, one analog, Grx3(Lys8Arg), showed a 10 mV lowering the redox potential in comparison with the wild-type Grx3(Lys8). These findings provide partial explanations of the increased reductive efficiency of Grx1.
In contrast to the thermodynamic parameters, the kinetics of equilibration reaction between Grx3 and Trx showed significant differences among the analogs. We found that, even though residue 8 is not directly involved with the catalytic mechanism, it affects the nucleophilic thiolate through electrostatic and hydrogen bonding interactions. When these interactions are perturbed, the reaction proceeds faster. Finally, when these interactions are absent, as in the case of Nva, the water molecules solvate the thiolate, resulting in faster reaction ? 8-fold faster than Lys and 40-fold faster than Arg. These observations strongly suggests that breaking the electrostatic interactions with residue 8 in the reduced and the oxidized state contributes approximately 1.4 kcal/mol to the activation barrier of the catalytic reaction. These interactions directly affect the rate determining step and serves to slow down the turnover of the enzyme.