title: Differential metabolism of organic nitrates by aldehyde dehydrogenase 1a1 and 2: substrate selectivity, enzyme inactivation, and active cysteine sites.
abstract: Organic nitrate vasodilators (ORN) exert their pharmacologic effects through the metabolic release of nitric oxide (NO). Mitochondrial aldehyde dehydrogenase (ALDH2) is the principal enzyme responsible for NO liberation from nitroglycerin (NTG), but lacks activity towards other ORN. Cytosolic aldehyde dehydrogenase (ALDH1a1) can produce NO from NTG, but its activity towards other ORN is unknown. Using purified enzymes, we showed that both isoforms could liberate NO from NTG, isosorbide dinitrate (ISDN), and nicrorandil, while only ALDH1a1 metabolized isosorbide-2-mononitrate and isosorbide-5-mononitrate (IS-5-MN). Following a 10-min incubation with purified enzyme, 0.1 mM NTG and 1 mM ISDN potently inactivated ALDH1a1 (to 21.9% ± 11.1% and 0.44% ± 1.04% of control activity, respectively) and ALDH2 (no activity remaining and 4.57% ± 7.92% of control activity, respectively), while 1 mM IS-5-MN exerted only modest inactivation of ALDH1a1 (reduced to 89% ± 4.3% of control). Cytosolic ALDH in hepatic homogenates incubated at the vascular EC(50) concentrations of ORN was inactivated by NTG (to 45.1% ± 8.1% of control activity) while mitochondrial ALDH was inactivated by NTG and nicorandil (to 68.2% ± 10.0% and 78.7% ± 19.8% of control, respectively). Via site-directed mutagenesis, the active sites of ORN metabolism of ALDH2 (Cys-319) and ALDH1a1 (Cys-303) were found to be identical to those responsible for their dehydrogenase activity. Cysteine-302 of ALDH1a1 and glutamate-504 of ALDH2 were found to modulate the rate of ORN metabolism. These studies provide further characterization of the substrate selectivity, inactivation, and active sites of ALDH2 and ALDH1a1 toward ORN.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, New York 14260-1200, USA.
authors.forename: Pei-Suen
authors.initials: PS
authors.lastname: Tsou
authors.forename: Nathaniel A
authors.initials: NA
authors.lastname: Page
authors.forename: Sean G
authors.initials: SG
authors.lastname: Lee
authors.forename: Sun Mi
authors.initials: SM
authors.lastname: Fung
authors.forename: Wing Ming
authors.initials: WM
authors.lastname: Keung
authors.forename: Ho-Leung
authors.initials: HL
authors.lastname: Fung
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000080924:Aldehyde Dehydrogenase 1 Family; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009566:Nitrates / Q000378:metabolism*; D009930:Organic Chemicals / Q000378:metabolism; D050697:Retinal Dehydrogenase; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D009566:Nitrates; D009930:Organic Chemicals; D000080924:Aldehyde Dehydrogenase 1 Family; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase; D003545:Cysteine