Kinetics of peroxiredoxins and their role in the decomposition of peroxynitrite.
Methodologies and results of studies on the kinetics of peroxiredoxins (Prx) are reviewed. Peroxiredoxins are broad-spectrum peroxidases that catalyze the reduction of H2O2, organic hydroperoxides and peroxynitrite by thiols. Their catalytic cycle starts with the oxidation of a particularly reactive cysteine residue (C(P)) to a sulfenic acid derivative by the peroxide substrate, the sulfenic acid then reacts with a thiol to form a disulfide, and the cycle is completed by thiol/disulfide exchange reactions that regenerate the ground-state enzyme. Depending on the subtype of peroxiredoxin, the thiol reacting with the primary oxidation product (E-SOH) may be a cysteine residue of a second subunit (typical 2-Cys Prx), a cysteine residue of the same subunit (atypical 2-Cys Prx) or reducing substrate (1-Cys Prx and at least one example of an atypical 2-Cys Prx). In a typical 2-Cys Prx the intra-subunit disulfide formation with the second "resolving" cysteine (C(R)) is mandatory for the reduction by the specific substrate, which is a protein characterized by a CXXC motif such as thioredoxin, tryparedoxin or AhpF. These consecutive redox reactions define the catalysis as an enzyme substitution mechanism, which is corroborated by a ping-pong pattern that is commonly observed in steady-state analyses, chemical identification of catalytic intermediates and stopped-flow analyses of partial reactions. More complex kinetic patterns are discussed in terms of cooperativity between the subunits of the oligomeric enzymes, generation of different oxidized intermediates or partial over-oxidation of C(P) to a sulfinic acid. Saturation kinetics is often not observed indicating that a typical complex between reduced enzyme and hydroperoxide is not formed and that, in these cases, formation of the complex between the oxidized enzyme and its reducing substrate is slower than the reaction within this complex. Working with sulphur catalysis, Prxs are usually less efficient than the heme- or selenium-containing peroxidases, but in some cases the k(+1) values (bimolecular rate constant for oxidation of reduced E by ROOH) are comparable, the overall range being 2 x 10(3)-4 x 10(7) M(-1)s(-1) depending on the hydroperoxide and the individual Prx. For the reduction of peroxynitrite k(+1) values of 1 x 10(6) up to 7 x 10(7) M(-1)s(-1) have been measured. The net forward rate constants k'(+2) for the reductive part of the cycle range between 2 x 10(4)-1 x 10(7) M(-1)s(-1). These kinetic characteristics qualify the peroxiredoxins as moderately efficient devices to detoxify hydroperoxides, which is pivotal to organisms devoid of more efficient peroxidases, and as most relevant to the detoxification of peroxynitrite. In higher organisms, their specific role is seen in the regulation of signalling cascades that are modulated by H2O2, lipid hydroperoxides or peroxynitrite.
Trujillo
Madia
M
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay. madiat@fmed.edu.uy
Ferrer-Sueta
Gerardo
G
Thomson
Leonor
L
Flohé
Leopold
L
Radi
Rafael
R
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain / Q000502:physiology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010545:Peroxides / Q000737:chemistry* / Q000378:metabolism*; D054464:Peroxiredoxins / Q000737:chemistry* / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000737:chemistry* / Q000378:metabolism*; D020836:Protein Structure, Quaternary / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology; D013434:Sulfenic Acids / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D008667:Metalloproteins; D010545:Peroxides; D013434:Sulfenic Acids; D030421:Peroxynitrous Acid; D054464:Peroxiredoxins; D003545:Cysteine