Pre-steady state kinetic characterization of human peroxiredoxin 5: taking advantage of Trp84 fluorescence increase upon oxidation.
Human peroxiredoxin 5 (PRDX5) catalyzes different peroxides reduction by enzymatic substitution mechanisms. Enzyme oxidation caused an increase in Trp84 fluorescence, allowing performing pre-steady state kinetic measurements. The technique was validated by comparing with data available from the literature or obtained herein by alternative approaches. PRDX5 reacted with organic hydroperoxides with rate constants in the 10(6)-10(7)M(-1)s(-1) range, similar to peroxynitrite-mediated PRDX5 oxidation, whereas its reaction with hydrogen peroxide was slower (10(5)M(-1)s(-1)). The method allowed determining the kinetics of intramolecular disulfide formation as well as thioredoxin 2-mediated reduction. The reactivities of PRDXs with peroxides were surprisingly high considering thiol pK(a), indicating that other protein determinants are involved in PRDXs specialization. The order of reactivities between PRDX5 towards oxidizing substrates differ from other PRDXs studied, pointing to a selective action of PRDXs with respect to peroxide detoxification, helping to rationalize the multiple enzyme isoforms present even in the same cellular compartment.
Trujillo
Madia
M
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800 Montevideo, Uruguay. madiat@fmed.edu.uy
Clippe
André
A
Manta
Bruno
B
Ferrer-Sueta
Gerardo
G
Smeets
Aude
A
Declercq
Jean-Paul
JP
Knoops
Bernard
B
Radi
Rafael
R
D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry; D054464:Peroxiredoxins / Q000737:chemistry*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds; D013879:Thioredoxins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
D020033:Protein Isoforms; D012997:Solvents; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D014364:Tryptophan; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; D010100:Oxygen