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
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.
Available from: Astrid Schrammel
- "The evidence is based on inhibition of GTN-induced relaxation by ALDH2 inhibitors  and the absence of the high affinity pathway of GTN vasodilation in ALDH2-deficient mice . 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 . "
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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.
Available from: Christian L Heine
- "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 , 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.
Available from: molpharm.aspetjournals.org
- "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. "
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ABSTRACT: Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI), a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2. DPI (0.3 μM) inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. Denitration and bioactivation of 1 - 2 μM GTN, assayed as 1,2-glycerol dinitrate formation and activation of purified sGC, respectively, were inhibited by DPI with a half-maximally active concentration of about 0.2 μM in a GTN-competitive manner. Molecular modeling indicated that DPI binds to the catalytic site of ALDH2, and this was confirmed by experiments showing substrate-competitive inhibition of the dehydrogenase and esterase activities of the enzyme. Our data identify ALDH2 as highly sensitive target of DPI and explain inhibition of GTN-induced relaxation by this drug observed previously. In addition, the data provide new evidence for the essential role of ALDH2 in GTN bioactivation and may have implications to other fields of ALDH2 research, such as hepatic ethanol metabolism and cardiac ischemia/reperfusion injury.
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