Oxidative modification of citrate synthase by peroxyl radicals and protection with novel antioxidants
In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 126.96.36.199), the first enzyme of the citric acid cycle. We used 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 824h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC50 values of 9, 14, 34, 37, 150, and 160μM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.
|Keywords||AAPH, Aconitase, Antioxidants, Citrate synthase, Enzyme inactivation, Epigallocatechin gallate, Naphthalenediols, Oxidative stress|
|Journal||Journal of Enzyme Inhibition and Medicinal Chemistry|
Chepelev, N.L. (Nikolai L.), Bennitz, J.D. (Joshua D.), Wright, J.S, Smith, J. C, & Willmore, W. (2009). Oxidative modification of citrate synthase by peroxyl radicals and protection with novel antioxidants. Journal of Enzyme Inhibition and Medicinal Chemistry, 24(6), 1319–1331. doi:10.3109/14756360902852586