Role of the General Base Glu-268 in Nitroglycerin Bioactivation and Superoxide Formation by Aldehyde Dehydrogenase-2

Abstract

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of NO or a related activator of soluble guanylate cyclase. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate and nitrite but also catalyzes reduction of GTN to NO. To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wild type (WT) enzyme with mutants lacking either the reactive Cys-302 (C302S) or the general base Glu-268 (E268Q). Although 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 co-factor NAD caused a pronounced increase in the rates of 1,2-glyceryl dinitrate formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified soluble guanylate cyclase or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase, 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 superoxide dismutase and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited ∼50% lower rates of superoxide formation than the WT enzyme. Our results suggest that Glu-268 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.

Site-Directed Mutagenesis of Aldehyde Dehydrogenase-2 Suggests Three Distinct Pathways of Nitroglycerin Biotransformation

“…In 2002, Stamler and coworkers (Chen et al, 2002) showed that ALDH2 denitrates GTN to 1,2-glyceryl dinitrate (1,2-GDN) and nitrite and proposed that reduction of nitrite by components of the mitochondrial electron transfer chain could provide the link between ALDH2-catalyzed GTN metabolism and vasorelaxation. However, we found that 1) GTN activates sGC in the presence of purified ALDH2 , 2) ALDH2 catalyzes the reduction of GTN to NO, accounting for 5 to 10% of total GTN turnover (Beretta et al, 2008a;Wenzl et al, 2009), and 3) mitochondrial biotransformation of nitroglycerin is not affected by respiratory substrates or inhibitors (Kollau et al, 2009), suggesting that ALDH2 catalysis is sufficient for GTN bioactivation in blood vessels.…”

“…The mechanism underlying ALDH2-catalyzed GTN reduction is not known, but the reaction seems to occur within the active site pocket with Cys302 serving as the common essential nucleophile (Larson et al, 2007;Wenzl et al, 2009). It was proposed by Stamler and coworkers (Chen et al, 2002) that GTN denitration resembles ester hydrolysis by ALDH2 (Mann and Weiner, 1999) (i.e., nucleophilic attack of a nitro group of GTN by Cys302, resulting in formation of a thionitrate intermediate and release of the corresponding alcohol, preferentially 1,2-GDN).…”

Nitrate Therapy

“…58 Recent data obtained with purified ALDH-2 provide evidence that ALDH-2 could be a source of GTN-triggered ROS formation. 59 The pathways leading to GTN tolerance in this hypothesis are summarized in Figure 6.…”

Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor, improves wound healing through cGMP/cGMP-dependent protein kinase

“…We compared NO-Cbi to direct NO donors, rather than to indirect NO donors such as nitroglycerin or other organic nitrates, because the latter require cellular biotransformation and can increase production of reactive oxygen species [33,34]. The increased potency of NO-Cbi compared to DetaNONOate was particularly surprising, because Deta-NONOate releases two nitric oxide molecules per mole of compound.…”