Peroxynitrite-mediated oxidation of the C85S/C152E mutant of dihydrofolate reductase from Escherichia coli: functional and structural effects.
Peroxynitrite is a potent reactive oxygen species that is believed to mediate deleterious protein modifications in a wide variety of neurodegenerative disorders. In this study, we have analysed the effects of oxidative damage induced by peroxynitrite on a cysteine-free mutant of dihydrofolate reductase (SE-DHFR), from a functional and a structural point of view. The peroxynitrite-mediated oxidation results in the inhibition, concentration-dependent, of the catalytic activity. This effect is strongly influenced by the HCO(3)(-)/CO(2) buffering system, that we observed to significantly affect the yield of protein oxidation by modulating the peroxynitrite-induced modification of aromatic residues. Because of this effect, in presence of bicarbonate system, we have observed a protection of enzymatic activity of SE-DHFR with regard to peroxynitrite. The thermodynamic stability of the oxidized protein has been studied in comparison with the non-oxidized protein by differential scanning calorimetry. The thermodynamic parameters obtained showed a decrease of stability of SE-DHFR upon oxidation, evaluated in terms of Gibbs free energy of about 1.25 kcal/mol at 25 degrees C, with respect to the non-oxidized protein. Together, these data indicate that structural and functional alterations induced by peroxynitrite may play a direct role in compromising DHFR function in multiple pathological conditions.
Pucciarelli
Stefania
S
Post-Graduate School in Clinical Biochemistry, Department of Molecular, Cellular and Animal Biology, University of Camerino, 62032, Camerino (MC), Italy. stefania.pucciarelli@unicam.it
Spina
Michele
M
Montecchia
Francesca
F
Lupidi
Giulio
G
Eleuteri
Anna Maria
AM
Fioretti
Evandro
E
Angeletti
Mauro
M
D015153:Blotting, Western; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015151:Immunoblotting; D009154:Mutation; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D017382:Reactive Oxygen Species; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D003553:Cystine; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D003545:Cysteine; D010100:Oxygen