title: The catalytic site of glutathione peroxidases.
abstract: In GPxs, the redox-active Se or S, is at hydrogen bonding distance from Gln and Trp residues that contribute to catalysis. From sequence homology of >400 sequences and modeling of the DmGPx as a paradigm, Asn136 emerged as a fourth essential component of the active site. Mutational substitution of Asn136 by His, Ala, or Asp results in a dramatic decline of specific activity. Kinetic analysis indicates that k(+1), the rate constant for the oxidation of the enzyme, decreases by two to three orders of magnitude, whereas the reductive steps characterized by k'(+2) are less affected. Accordingly, MS/MS analysis shows that in Asn136 mutants, the peroxidatic Cys45 stays largely reduced also in the presence of a hydroperoxide, whereas in the wild-type enzyme, it is oxidized, forming a disulfide with the resolving Cys. Computational calculation of pK(a) values indicates 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. These data disclose that the catalytic site of GPxs has to be redrawn as a tetrad, including Asn136, and suggest a mechanism accounting for the extraordinary catalytic efficiency of GPxs.
authors.affiliation: Department of Biology and CRIBI Biotechnology Centre, Padova, Italy.
authors.forename: Silvio C E
authors.initials: SC
authors.lastname: Tosatto
authors.forename: Valentina
authors.initials: V
authors.lastname: Bosello
authors.forename: Federico
authors.initials: F
authors.lastname: Fogolari
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Mauri
authors.forename: Antonella
authors.initials: A
authors.lastname: Roveri
authors.forename: Stefano
authors.initials: S
authors.lastname: Toppo
authors.forename: Leopold
authors.initials: L
authors.lastname: Flohé
authors.forename: Fulvio
authors.initials: F
authors.lastname: Ursini
authors.forename: Matilde
authors.initials: M
authors.lastname: Maiorino
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology; D005979:Glutathione Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D005979:Glutathione Peroxidase; D013880:Thioredoxin-Disulfide Reductase