Damage caused to Saccharomyces cerevisiae SY4 plasma membrane H(+)-ATPase by Fe- and Cu-Fenton reagents was determined in secretory vesicles containing enzyme in which Cys residues were replaced singly or in pairs by Ala. Cys-221 situated in a beta-sheet domain between M2 and M3 segments, phosphorylation domain-located Cys-409 and Cys-532 situated at the ATP-binding site play a role in the inactivation. In the presence of all three residues the enzyme exhibited a certain basic inactivation, which did not change when Cys-532 was replaced with Ala. In mutants having intact Cys-532 but lacking one or both other cysteines, replacement of Cys-221 with Ala led to lower inactivation, suggesting that Cys-221 may serve as a target for metal-catalyzed oxidation and intact Cys-532 promotes this target role of Cys-221. In contrast, the absence of Cys-409 caused higher inactivation by Fe-Fenton. Cys-532 thus seems to serve as a target for Fe-Fenton, intact Cys-409 causing a conformational change that makes Cys-532 less accessible to oxidation. The mutant lacking both Cys-221 and Cys-409 is more sensitive to Fe-Fenton than to Cu-Fenton and the absence of both Cys residues thus seems to expose presumable extra Fe-binding sites. These data and those on protection by ATP, ADP, 1,4-dithiothreitol and deferrioxamine B point to complex interactions between individual parts of the enzyme molecule that determine its sensitivity towards Fenton reagents. ATPase fragmentation caused by the two reagents differed in that the Fe-Fenton reagent produced in Western blot "smears" whereas the Cu-Fenton reagent produced defined fragments.

• MeSH
• buněčná membrána účinky léků   enzymologie   MeSH
• cystein metabolismus   MeSH
• měď MeSH
• mutace MeSH
• oxidační stres účinky léků   MeSH
• peroxid vodíku farmakologie   MeSH
• protonové ATPasy chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae chemie   účinky léků   enzymologie   MeSH
• substituce aminokyselin MeSH
• volné radikály MeSH
• vztahy mezi strukturou a aktivitou MeSH
• železo farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• Fenton's reagent MeSH Prohlížeč
• měď MeSH
• peroxid vodíku MeSH
• protonové ATPasy MeSH
• volné radikály MeSH
• železo MeSH