Project nitroglycerin

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Funded by FWF grants P21693 and P24946

 

Nitroglycerin (glyceryl trinitrate; GTN), is the most prominent representative of the organic nitrates or nitrovasodilators, a class of compounds that have been used clinically since the late 19th century for the treatment of coronary artery disease (angina pectoris), congestive heart failure and myocardial infarction. The beneficial clinical effect of GTN is due to dilation of large coronary arteries, resulting in improved blood supply to the heart and venodilation, resulting in increased venous pooling and consequent reduction of venous return and cardiac preload. The combination of increased supply and decreased demand of oxygen provides unique therapeutic benefit in cardiac ischemia.

It is well established that GTN is a prodrug that is bioactivated to yield nitric oxide (NO) or a related species which activates soluble guanylate cyclase in vascular smooth muscle, resulting in cGMP-mediated vasodilation. Several enzymatic patways may contribute to GTN bioactivation, but aldehyde dehydrogenase-2 (ALDH2) appears to be the key enzyme mediating the therapeutic effect of GTN. We focus on the characterization of the ALDH2-catalyzed reaction, with special emphasis being placed on the poorly understood direct reduction of the organic nitrate to NO and the mechanisms invovled in mechanism-based ALH2 inactivation that may contribute to the development of nitrate tolerance.

Proposed reaction mechanism of GTN denitration

After nucleophilic attack of Cys-302 on the first nitrate group of GTN, a thionitrate, the central intermediate, is formed. The major reaction pathway (a), in which Cys-301 and Cys-303 are involved, results in the formation of nitrite and an oxidized enzyme, presumably a species with a disulfide bond in the active site, which can be regenerated by reductants like DTT. b, depicts the pathway yielding an irreversibly inactivated enzyme, possibly due to oxidation of Cys-302 to a sulfinic acid. For this pathway Glu-268 is essential. c, leads to a reversibly inhibited enzyme and the production of NO. (Lang et al., J. Biol. Chem. 287, 38124-38134, 2012)

Stereoscopic representation of the complex of the ALDH2 E268Q/C301S/C303S mutant with GTN

A, GTN (yellow) is shown in a ball and stick representation. Active site residues are shown as sticks. The resting conformation of Cys-302 has been omitted for clarity. Dashed lines indicate polar interactions.

B, superposition of the active site of the triple mutant (green) with that of the apo wild-type enzyme (blue, PDB 1o05). Labels refer to the wild-type. (Lang et al., J. Biol. Chem. 287, 38124-38134, 2012)

Effect of daidzin on the denitration activity of ALDH2

A, schematic representation of the GTN complexed triple mutant (light green) superimposed onto the daidzin bound wild-type enzyme (dark green) (PDB 2vle). GTN and daidzin are shown as sticks in blue and red, respectively.

B, the activity of GTN denitration (10 and 100 μm GTN) was determined for varying daidzin concentrations (10 μm to 10 mm). Data are mean ± S.E. of three independent experiments. A best fit of both curves to a model assuming competitive inhibition was obtained with a Kd for GTN of 8.7 μm. C, corresponding Dixon plots. (Lang et al., J. Biol. Chem. 287, 38124-38134, 2012

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