PT  - JOURNAL ARTICLE
AU  - M. Verena Wenzl
AU  - Matteo Beretta
AU  - Martina Griesberger
AU  - Michael Russwurm
AU  - Doris Koesling
AU  - Kurt Schmidt
AU  - Bernd Mayer
AU  - Antonius C. F. Gorren
TI  - SITE-DIRECTED MUTAGENESIS OF ALDEHYDE DEHYDROGENASE-2 SUGGESTS THREE DISTINCT PATHWAYS OF NITROGLYCERIN BIOTRANSFORMATION
AID  - 10.1124/mol.111.071704
DP  - 2011 Jan 01
TA  - Molecular Pharmacology
PG  - mol.111.071704
4099  - http://molpharm.aspetjournals.org/content/early/2011/05/02/mol.111.071704.short
4100  - http://molpharm.aspetjournals.org/content/early/2011/05/02/mol.111.071704.full
AB  - 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 Cys-302 (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 Glu-268 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 Cys-302 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 Glu-268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu-268 shifts the reaction towards formation of free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Though 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.