PMID:15639221
| Citation |
Pucciarelli, S, Spina, M, Montecchia, F, Lupidi, G, Eleuteri, AM, Fioretti, E and Angeletti, M (2005) Peroxynitrite-mediated oxidation of the C85S/C152E mutant of dihydrofolate reductase from Escherichia coli: functional and structural effects. Arch. Biochem. Biophys. 434:221-31 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1016/j.abb.2004.11.008 |
| Keywords |
Blotting, Western; Calorimetry, Differential Scanning; Chromatography, Gel; Chromatography, High Pressure Liquid; Cysteine/chemistry; Cystine/chemistry; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Immunoblotting; Mutation; Oxidative Stress; Oxygen/chemistry; Oxygen/metabolism; Peroxynitrous Acid/pharmacology; Reactive Oxygen Species; Spectrometry, Fluorescence; Temperature; Tetrahydrofolate Dehydrogenase/genetics; Thermodynamics; Time Factors; Tryptophan/chemistry; Tyrosine/analogs & derivatives; Tyrosine/chemistry |
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