Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2

Abstract

Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2.