INACTIVATION OF HUMAN ALDEHYDE DEHYDROGENASE BY ISOSORBIDE DINITRATE

Isosorbide dinitrate inactivated E1 and E2 isozymes of human aldehyde dehydrogenase (EC 1.2.1.3), abolishing both dehydrogenase and esterase activities. NAD promoted, whereas chloral and NAD protected the enzym e from inactivation. The inactivation was irreversible upon dialysis a nd occurred without incorporation of the C-14-labeled isosorbide dinit rate. Inactivation was associated with formation of products, isosorbi de-2-mononitrate and isosorbide-5-mononitrate, At 25 degrees C there w ere two pathways of product formation: a fast pathway, sensitive to al dehyde dehydrogenase inhibitors, and a slower pathway insensitive to i nhibitors. The fast product formation and inactivation occurred simult aneously, and both were inhibited by chloral and by the irreversible a ctive site-directed inhibitor bromoacetophenone. At 0 degrees C the sl ow product formation was abolished, allowing study of the enzyme catal yzed reaction. The inactivation of the E1 isozyme at 0 degrees C occur red in a single turnover that accounted for 80% of catalytic activity loss with isosorbide-2-mononitrate being the major product. No nitrate was ever detected; at 25 degrees C, nitrite was detected but in less than stoichiometric amounts. The mononitrates were also substrates and inactivators of aldehyde dehydrogenase. Isosorbide-2-mononitrate had the lowest K-i and k(3), values for the E1 isozyme when compared with that of the other two nitrate esters of isosorbide. Reversibility of i nactivation by 2-mercaptoethanol suggested involvement of enzyme sulfh ydryls. The inactivation appears to be mechanism-based and involves th e esterase function of aldehyde dehydrogenase.