Mukerjee N, Pietruszko R
Center of Alcohol Studies, Rutgers University, Piscataway, New Jersey 08855-0969.
J Biol Chem. 1994 Aug 26;269(34):21664-9.
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 enzyme from inactivation. The inactivation was irreversible upon dialysis and occurred without incorporation of the 14C-labeled isosorbide dinitrate. Inactivation was associated with formation of products, isosorbide-2-mononitrate and isosorbide-5-mononitrate. At 25 degrees C there were two pathways of product formation: a fast pathway, sensitive to aldehyde dehydrogenase inhibitors, and a slower pathway insensitive to inhibitors. The fast product formation and inactivation occurred simultaneously, and both were inhibited by chloral and by the irreversible active site-directed inhibitor bromoacetophenone. At 0 degree C the slow product formation was abolished, allowing study of the enzyme catalyzed reaction. The inactivation of the E1 isozyme at 0 degree C occurred 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 k3 values for the E1 isozyme when compared with that of the other two nitrate esters of isosorbide. Reversibility of inactivation by 2-mercaptoethanol suggested involvement of enzyme sulfhydryls. The inactivation appears to be mechanism-based and involves the esterase function of aldehyde dehydrogenase.
硝酸异山梨酯使人类醛脱氢酶（EC 1.2.1.3）的E1和E2同工酶失活，同时消除脱氢酶和酯酶活性。NAD可促进该过程，而三氯乙醛和NAD则可保护该酶不被失活。透析后失活是不可逆的，且失活过程中未掺入14C标记的硝酸异山梨酯。失活与产物异山梨醇-2-单硝酸酯和异山梨醇-5-单硝酸酯的形成有关。在25℃时，产物形成有两条途径：一条快速途径，对醛脱氢酶抑制剂敏感；另一条较慢途径，对抑制剂不敏感。快速产物形成和失活同时发生，且两者均受三氯乙醛和不可逆的活性位点导向抑制剂溴代苯乙酮抑制。在0℃时，缓慢的产物形成被消除，从而可以研究酶催化反应。0℃时E1同工酶的失活以单周转方式发生，导致80%的催化活性丧失，异山梨醇-2-单硝酸酯为主要产物。未检测到硝酸盐；在25℃时，检测到亚硝酸盐，但量低于化学计量。单硝酸酯也是醛脱氢酶的底物和失活剂。与异山梨醇的其他两种硝酸酯相比，异山梨醇-2-单硝酸酯对E1同工酶的K(i)和k3值最低。2-巯基乙醇使失活可逆，提示酶的巯基参与其中。失活似乎是基于机制的，涉及醛脱氢酶的酯酶功能。
