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

Abstract

To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.

Aldehyde Dehydrogenase Inhibitors: a Comprehensive Review of the Pharmacology, Mechanism of Action, Substrate Specificity, and Clinical Application

“…This suggests that ALDH3 may be subject to a unique mode of inhibition by GTN that differs from the inhibition of ALDH2. A recent series of studies involving sitedirected mutagenesis experiments led Wenzl et al (2011) to propose three distinct pathways for GTN biotransformation by ALDH2. The dominant pathway involves the formation of nitrite and active site Cys (301 and 303) disulfide formation (Fig.…”

Cytochrome P450 and Non-Cytochrome P450 Oxidative Metabolism: Contributions to the Pharmacokinetics, Safety, and Efficacy of Xenobiotics

“…From an endogenous standpoint, two key roles for ALDHs are the conversion of retinaldehyde to retinoic acid, a metabolic conversion that is involved in the regulation of a host of homeostatic functions, as well as the metabolism of acetaldehyde, a by-product of ethanol metabolism (Stagos et al, 2010;Singh et al, 2013). One of the key xenobiotic roles for ALDH is the bioactivation of nitroglycerin to nitric oxide, resulting in the drug's vasodilatory effects (Chen et al, 2002;Wenzl et al, 2011).…”

Potent Inhibition of Aldehyde Dehydrogenase-2 by Diphenyleneiodonium: Focus on Nitroglycerin Bioactivation

“…This was later confirmed with ALDH2-deficient blood vessels in which the high-affinity pathway of GTN-induced relaxation is absent (Chen et al, 2005). The main route of ALDH2-catalyzed denitration of GTN yields 1,2-glycerol dinitrate (1,2-GDN) and inorganic nitrite, but a minor reaction results in direct formation of NO (Wenzl et al, 2011). Although direct NO formation by ALDH2 observed with the purified enzyme is likely to explain vascular GTN bioactivity, it remains to be shown that this reaction does indeed mediate GTN-induced relaxation of blood vessels.…”

“…Esterase activity of ALDH2 (6.6 mg/ml) was measured by monitoring the formation of p-nitrophenolate from p-nitrophenyl acetate (pNPA; 0.1 ALDH2-Catalyzed Bioactivation of GTN Measured as Activation of Purified sGC. Activation of purified sGC (50 ng) by increasing concentrations of GTN (1 nM-440 mM) was determined by incubation of 4 mg of purified ALDH2 at 37°C for 10 minutes in the presence of 0.5 mM [a-32 P]GTP (∼250,000 cpm), 1 mM NAD 1 and 1000 U/ml superoxide dismutase as described previously elsewhere (Wenzl et al, 2009a(Wenzl et al, , 2011Beretta et al, 2010). DPI was present at the indicated concentrations (0.01-100 mM), and DEA/NO (1 mM) served as control for maximal sGC activity.…”