Wenzl MV, Beretta M, Gorren AC, Zeller A, Baral PK, Gruber K, Russwurm M, Koesling D, Schmidt K, Mayer B
Role of the general base GLU-268 in nitroglycerin bioactivation and superoxide formation by aldehyde dehydrogenase-2. [JOURNAL ARTICLE]
J Biol Chem 2009 Jun 8.
Mitochondrial aldehyde dehydrogenase (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of nitric oxide (NO) or a related activator of soluble guanylate cyclase (sGC). ALDH2 denitrates GTN to 1,2-glyceryl dinitrate (1,2-GDN) and nitrite but also catalyzes reduction of GTN to nitric oxide (NO). To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wildtype (WT) enzyme with mutants lacking either the reactive Cys302 (C302S) or the general base Glu268 (E268Q). While the C302S mutation led to >90 % loss of all enzyme activities, the E268Q mutant exhibited virtually unaffected rates of GTN denitration despite low dehydrogenase and esterase activities. The nucleotide cofactor NAD caused a pronounced increase in the rates of 1,2-GDN formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified sGC or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase (SOD), suggesting that bioavailability of GTN-derived NO is limited by co-generation of superoxide. Formation of superoxide was confirmed by determination of hydroethidine oxidation that was inhibited by SOD and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited about 50 % lower rates of superoxide formation than the WT enzyme. Our results suggest that E268 is involved in the structural organization of the NAD binding pocket but is not required for GTN denitration. ALDH2-catalyzed superoxide formation may essentially contribute to oxidative stress in GTN-exposed blood vessels.
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