Cysteine homologues of glutathione peroxidases (GPxs) have more recently surprised with rate constants for the oxidation of the peroxidatic cysteine (CP), k ́+1 of > 106 M-1s-1. This observation conflicts with the common view that the pronounced reactivity just results from dissociation of CP enforced by hydrogen bonding within the catalytic triad composed of Cys/Sec, Gln and Trp, be- cause even a fully dissociated cysteine (or any other low MW thiol) does not react with H2O2 with a rate constant > 5 x 103 M-1s-1. Sequence homology of > 400 sequences, modeling of the Droso- phila melanogaster GPx (DmGPx) as a paradigm, and site-directed mutagenesis revealed a pivotal role of Asn136 as a fourth essential component of the active site. Mutational substitution of Asn136 by His, Ala or Asp results in a decline of specific activity by 97, 98 and 100%, respectively. In steady-state kinetic analysis of the muteins k ́+1 is decreased by two- to three orders of magnitude, while the reductive steps characterized by k ́+2 are less affected. Accordingly, MS/MS analysis shows that in Asn136 mutants the peroxidatic Cys45 (CP) stays largely reduced also in the presence of a hydroperoxide, while in the wild-type enzyme it is oxidized forming a disulfide with the resolving Cys (CR). Computational calculation of pKa values from wild-type and mutant Dm GPxs in- dicates that the residues facing the catalytic thiol, Asn136, Gln80 and, to a lesser extent Trp135, contribute to the dissociation of the thiol group, Asn136 being most relevant. It has, however, to be postulated that Asn 136 and Gln 80 activate CP not only by enforc- ing thiol dissociation but also by proton shuttling leading to polari- zation of the substrate’s peroxo bond, protonation of leaving groups and product stabilization. In conclusion, the catalytic site of GPxs has to be redrawn as a tetrad, wherein “carboxamide cataly- sis” is most relevant to the extraordinary catalytic efficiency of GPxs.

What makes the peroxidatic cysteine in CysPHGPx reactive?

TOSATTO, SILVIO;BOSELLO TRAVAIN, VALENTINA;ROVERI, ANTONELLA;TOPPO, STEFANO;URSINI, FULVIO;MAIORINO, MATILDE
2008

Abstract

Cysteine homologues of glutathione peroxidases (GPxs) have more recently surprised with rate constants for the oxidation of the peroxidatic cysteine (CP), k ́+1 of > 106 M-1s-1. This observation conflicts with the common view that the pronounced reactivity just results from dissociation of CP enforced by hydrogen bonding within the catalytic triad composed of Cys/Sec, Gln and Trp, be- cause even a fully dissociated cysteine (or any other low MW thiol) does not react with H2O2 with a rate constant > 5 x 103 M-1s-1. Sequence homology of > 400 sequences, modeling of the Droso- phila melanogaster GPx (DmGPx) as a paradigm, and site-directed mutagenesis revealed a pivotal role of Asn136 as a fourth essential component of the active site. Mutational substitution of Asn136 by His, Ala or Asp results in a decline of specific activity by 97, 98 and 100%, respectively. In steady-state kinetic analysis of the muteins k ́+1 is decreased by two- to three orders of magnitude, while the reductive steps characterized by k ́+2 are less affected. Accordingly, MS/MS analysis shows that in Asn136 mutants the peroxidatic Cys45 (CP) stays largely reduced also in the presence of a hydroperoxide, while in the wild-type enzyme it is oxidized forming a disulfide with the resolving Cys (CR). Computational calculation of pKa values from wild-type and mutant Dm GPxs in- dicates that the residues facing the catalytic thiol, Asn136, Gln80 and, to a lesser extent Trp135, contribute to the dissociation of the thiol group, Asn136 being most relevant. It has, however, to be postulated that Asn 136 and Gln 80 activate CP not only by enforc- ing thiol dissociation but also by proton shuttling leading to polari- zation of the substrate’s peroxo bond, protonation of leaving groups and product stabilization. In conclusion, the catalytic site of GPxs has to be redrawn as a tetrad, wherein “carboxamide cataly- sis” is most relevant to the extraordinary catalytic efficiency of GPxs.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2273859
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