Study of the fully folded-locally unfolded transition and the resolution step in human peroxiredoxin 5

STEPHANIE PORTILLO; DIEGO SEBASTIAN VAZQUEZ; JAVIER SANTOS; E. LAURA COITIÑO; FERRER-SUETA, GERARDO

Foz do Iguaçu, Brazil

Congreso; 23rd Congress of the IUMBM and 44th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology; 2015

International Union for Biochemistry and Molecular Biology and Brazilian Society for Biochemistry and Molecular Biology

Human peroxiredoxin 5 (PRDX5) is an antioxidant enzyme that reduces different peroxides in a very effective way. It uses two cysteine residues in catalysis: the peroxidatic cysteine ?Cp? that reacts with the peroxide to form a cysteine sulfenic acid ?Cp-SOH? and the resolving cysteine ?Cr? that reacts with Cp-SOH, forming a disulfide bond. Since in the reduced enzyme Cp and Cr are 14 Å apart, a conformational change must occur to approach them, designated as the fully folded (FF)-locally unfolded (LU)transition. The aim of this work is to elucidate whether the FF-LU equilibrium is already present in the reduced enzyme or the transition is triggered by forming Cp-SOH. To assess the impact of the transition on the resolution step, we also evaluated the rate constant of the reaction and the pKa?s of the species involved. The FF-LU transition was mapped using molecular dynamics (MD) simulations. PRDX5 was simulated for 1 μs of plane MD and 500 ns of accelerated molecular dynamics (aMD) both in the thiol and sulfenic states using the Amber14 suite. The rate constant for the disulfide bond formation was measured using a pseudo-first order approach, with H2O2 in excess. At 200 μM of H 2O2 the rate-limiting step corresponds to the disulfide bond formation, allowing the measurement of a k app.The MD analysis supports the idea of a FF-LU transition not triggered by p-SOH, since in the reduced protein (Cp-SH) the α-helix bearing the active site starts to unfold in the microsecond scale. Regarding the resolution reaction a maximum rate constant of 26 s -1 is obtained, corresponding to the milisecond time-scale. From this evidence we conclude that the rate of the resolution reaction would not be limited by the conformational change and that the reduced enzyme would display both FF/LU conformations.