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

Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010, Austria.
Molecular pharmacology (Impact Factor: 4.13). 05/2011; 80(2):258-66. DOI: 10.1124/mol.111.071704
Source: PubMed


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.

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    • "The evidence is based on inhibition of GTN-induced relaxation by ALDH2 inhibitors [13] and the absence of the high affinity pathway of GTN vasodilation in ALDH2-deficient mice [15]. The main route of ALDH2-catalyzed denitration of GTN yields 1,2-glycerol dinitrate (1,2-GDN) and inorganic nitrite, but our data obtained with several ALDH2 mutants suggest that GTN bioactivity is mediated by a minor pathway resulting in the direct formation of NO [16]. "
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    ABSTRACT: The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterizTed as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., Eur. J. Pharmacol. 314:347-350, 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes an hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2.
    Biochemical Pharmacology 01/2015; 218(4). DOI:10.1016/j.bcp.2014.12.021 · 5.01 Impact Factor
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    • "To determine the amount of NO released by the NO donors under the applied experimental conditions, we measured the conversion of oxyhemoglobin to methemoglobin spectrophotometrically from the absorbance difference between 420 and 401 nm as published [25], but with 50 mM triethanolamine (TEA; pH 7.4) instead of KP i as the buffer. "
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    ABSTRACT: Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg(2+). In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constant of 8.9±0.4 and 0.55±0.06M(-1)•s(-1) in the presence and absence of Mg(2+). The maximum yield of GSNO was close to 100% in the presence of Mg(2+) but only about half as high in its absence. From the latter observation we conclude that, in the absence of Mg(2+), nitrosation starts by formation of a complex between NO and O2, which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg(2+), demonstrating O2(-) formation. The reaction in the presence of Mg(2+) appears to involve formation of a Mg(2+)-GSH complex. SOD did not affect Mg(2+)-stimulated nitrosation, suggesting that no O2(-) is formed in that reaction. Replacing GSH by other thiols revealed that reaction rates increased with the pKa of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg(2+)-stimulated NO/O2-induced nitrosothiol formation may be a physiologically relevant reaction.
    Free Radical Biology and Medicine 09/2014; 76. DOI:10.1016/j.freeradbiomed.2014.08.024 · 5.74 Impact Factor
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    • "To determine how much NO is released by DEA/NO under the present experimental conditions, we measured the conversion of oxyhemoglobin (oxy-Hb) to methemoglobin (met-Hb) spectrophotometrically from the absorbance difference between 420 and 401 nm according to a published procedure [31], but with 50 mM TEA (pH 7.4) instead of KP i as the buffer. "
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    ABSTRACT: Nitrosothiols are increasingly regarded as important participants in a range of physiological processes, yet little is known about their biological generation. Nitrosothiols can be formed from the corresponding thiols by nitric oxide in a reaction that requires the presence of oxygen and is mediated by reactive intermediates (NO2 or N2O3) formed in the course of NO autoxidation. Since the autoxidation of NO is second order in NO, it is extremely slow at submicromolar NO concentrations, casting doubt on its physiological relevance. In this paper we present evidence that at submicromolar NO concentrations the aerobic nitrosation of glutathione does not involve NO autoxidation but a reaction that is first order in NO. We show that this reaction produces nitrosoglutathione efficiently in a reaction that is strongly stimulated by physiological concentrations of Mg(2+). These observations suggest that direct aerobic nitrosation may represent a physiologically relevant pathway of nitrosothiol formation.
    Free Radical Biology and Medicine 05/2013; 63. DOI:10.1016/j.freeradbiomed.2013.04.034 · 5.74 Impact Factor
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