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[摘要]：Tyrosine nitration is a covalent post-translational protein modification associated with various diseases related to oxidative/nitrative stress. A role for nitration of tyrosine in protein inactivation has been proposed; however, few studies have established a direct link between this modification and loss of protein function. In the present study, we determined the effect of nitration of Tyr(345) and Tyr(368) in the beta-subunit of the F-1-ATPase using site-directed mutagenesis. Nitration of the beta-subunit, achieved by using TNM (tetranitromethane), resulted in 66% ATPase activity loss. This treatment resulted in the modification of several asparagine, methionine and tyrosine residues. However, nitrated tyrosine and ATPase inactivation were decreased in reconstituted F-1 with Y368F (54%), Y345F (28%) and Y345,368F (1 %) beta-subunits, indicating a clear link between nitration at these positions and activity loss, regardless of the presence of other modifications. Kinetic studies indicated that an F-1 with one nitrated tyrosine residue (Tyr(345) or Tyr(368)) or two Tyr(368) residues was sufficient to grant inactivation. Tyr(368) was four times more reactive to nitration due to its lower pK(a). Inactivation was attributed mainly to steric hindrance caused by adding a bulky residue more than the presence of a charged group or change in the phenolic pKa clue to the introduction of a nitro group. Nitration at this residue would be more relevant under conditions of low nitrative stress. Conversely, at high nitrative stress conditions, both tyrosine residues would contribute equally to ATPase inactivation. |
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