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Characterization of the East Asian variant of aldehyde dehydrogenase-2: bioactivation of nitroglycerin and effects of Alda-1.

Beretta M, Gorren AC, Wenzl MV, Weis R, Russwurm M, Koesling D, Schmidt K, Mayer B - J. Biol. Chem. (2009)

Bottom Line: In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1.The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant.In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, 8010 Graz, Austria

ABSTRACT
The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at > or = 5 mM. In the presence of 1 mM NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

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Effect of Alda-1 and NAD on the esterase activities of ALDH2*1 and ALDH2*2. Esterase activity was measured photometrically with 0.1 mm p-NPA as described in “Experimental Procedures” in the absence or presence of 1 mm (A) or increasing concentrations (B) NAD and 10 μm Alda-1 or 0.5% DMSO (vehicle control). Activities were calculated as fold increase over controls (mean values ± S.E.) obtained in three independent determinations. Note that the control value of ALDH2*2 is only 0.11% of the ALDH2*1 control.
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Figure 4: Effect of Alda-1 and NAD on the esterase activities of ALDH2*1 and ALDH2*2. Esterase activity was measured photometrically with 0.1 mm p-NPA as described in “Experimental Procedures” in the absence or presence of 1 mm (A) or increasing concentrations (B) NAD and 10 μm Alda-1 or 0.5% DMSO (vehicle control). Activities were calculated as fold increase over controls (mean values ± S.E.) obtained in three independent determinations. Note that the control value of ALDH2*2 is only 0.11% of the ALDH2*1 control.

Mentions: Esterase activities were measured photometrically as p-NPA hydrolysis in the absence and presence of 1 mm NAD, 10 μm Alda-1, or a combination of both. As shown in Fig. 4A, the activities of ALDH2*1 and ALDH2*2 (190 ± 5.5 and 0.21 ± 0.01 nmol × min−1 × mg−1, respectively) were increased by NAD and Alda-1 to similar extent (6- and 9-fold, respectively). In the combined presence of NAD and Alda-1, however, the effect of either compound on wild type ALDH2 was almost abolished (1.7-fold stimulation), whereas the esterase activity of ALDH2*2 was stimulated 73-fold (note that the esterase activity of ALDH2*2 was still 20-fold lower than that of ALDH2*1 under these conditions). The peculiar discrepancy between the effects of the Alda-1/NAD combination on the two ALDH2 variants is apparently due to a biphasic effect of NAD on the esterase activity of ALDH2. As shown in Fig. 4B, the esterase activities of ALDH2*1 and ALDH2*2 were maximally stimulated by 1 and 5 mm NAD, respectively, whereas higher concentrations of the coenzyme led to marked inhibition of both ALDH2 variants. Alda-1 stimulated the esterase activities of both enzymes in the absence of NAD and potentiated the effect of the coenzyme, in particular its effect on ALDH2*2. Intriguingly, Alda-1 additionally caused a pronounced ∼20-fold left shift of NAD-induced inhibition of wild type ALDH2, which was half-maximal at ∼10 and 0.5 mm in the absence and presence of Alda-1.


Characterization of the East Asian variant of aldehyde dehydrogenase-2: bioactivation of nitroglycerin and effects of Alda-1.

Beretta M, Gorren AC, Wenzl MV, Weis R, Russwurm M, Koesling D, Schmidt K, Mayer B - J. Biol. Chem. (2009)

Effect of Alda-1 and NAD on the esterase activities of ALDH2*1 and ALDH2*2. Esterase activity was measured photometrically with 0.1 mm p-NPA as described in “Experimental Procedures” in the absence or presence of 1 mm (A) or increasing concentrations (B) NAD and 10 μm Alda-1 or 0.5% DMSO (vehicle control). Activities were calculated as fold increase over controls (mean values ± S.E.) obtained in three independent determinations. Note that the control value of ALDH2*2 is only 0.11% of the ALDH2*1 control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2801295&req=5

Figure 4: Effect of Alda-1 and NAD on the esterase activities of ALDH2*1 and ALDH2*2. Esterase activity was measured photometrically with 0.1 mm p-NPA as described in “Experimental Procedures” in the absence or presence of 1 mm (A) or increasing concentrations (B) NAD and 10 μm Alda-1 or 0.5% DMSO (vehicle control). Activities were calculated as fold increase over controls (mean values ± S.E.) obtained in three independent determinations. Note that the control value of ALDH2*2 is only 0.11% of the ALDH2*1 control.
Mentions: Esterase activities were measured photometrically as p-NPA hydrolysis in the absence and presence of 1 mm NAD, 10 μm Alda-1, or a combination of both. As shown in Fig. 4A, the activities of ALDH2*1 and ALDH2*2 (190 ± 5.5 and 0.21 ± 0.01 nmol × min−1 × mg−1, respectively) were increased by NAD and Alda-1 to similar extent (6- and 9-fold, respectively). In the combined presence of NAD and Alda-1, however, the effect of either compound on wild type ALDH2 was almost abolished (1.7-fold stimulation), whereas the esterase activity of ALDH2*2 was stimulated 73-fold (note that the esterase activity of ALDH2*2 was still 20-fold lower than that of ALDH2*1 under these conditions). The peculiar discrepancy between the effects of the Alda-1/NAD combination on the two ALDH2 variants is apparently due to a biphasic effect of NAD on the esterase activity of ALDH2. As shown in Fig. 4B, the esterase activities of ALDH2*1 and ALDH2*2 were maximally stimulated by 1 and 5 mm NAD, respectively, whereas higher concentrations of the coenzyme led to marked inhibition of both ALDH2 variants. Alda-1 stimulated the esterase activities of both enzymes in the absence of NAD and potentiated the effect of the coenzyme, in particular its effect on ALDH2*2. Intriguingly, Alda-1 additionally caused a pronounced ∼20-fold left shift of NAD-induced inhibition of wild type ALDH2, which was half-maximal at ∼10 and 0.5 mm in the absence and presence of Alda-1.

Bottom Line: In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1.The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant.In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, 8010 Graz, Austria

ABSTRACT
The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at > or = 5 mM. In the presence of 1 mM NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

Show MeSH
Related in: MedlinePlus