HALOENOL LACTONES AS INACTIVATORS AND SUBSTRATES OF ALDEHYDE DEHYDROGENASE

Human aldehyde dehydrogenase (EC 1.2.1.3) isozymes E1 and E2 were irre versibly inactivated by stoichiometric concentrations of the haloenol lactones 3-isopropyl-(E)-bromomethylene tetrahydro-pyran-2-one and 3-p henyl-6(E)-bromomethylene tetrahydropyran-2-one. No inactivation occur red with the corresponding nonhalogenated enol lactones. Both the dehy drogenase and esterase activities were abolished. Activity was not reg ained on dialysis or treatment with 2-mercaptoethanol. The inactivatio n was subject to substrate protection: NAD afforded protection which i ncreased in the presence of the aldehyde-substrate competitive inhibit or chloral. Saturation kinetics gave positive y-axis intercepts, allow ing the determination of binding constants. Inactivation stiochiometry determined with C-14-labeled 3-(1-naphthyl)-6(E)-iodomethylene tetrah ydropyran-2-one was found to correspond to the active-site number. The nonhalogenated lactone, 3-(1-naphthyl)-6(E)-methylene tetrahydropyran -1-one was shown to be a substrate for aldehyde dehydrogenase via its esterase function. Inactivation and enzymatic hydrolysis occurred with in a similar time frame. Opening of the lactone ring to form enzyme-ac yl intermediate with active site cysteine appears to be a necessary pr erequisite to inactivation, since halogen in the lactone ring is nonre active. Thus, the inactivation of aldehyde dehydrogenase by haloenol l actones is mechanism-based. Inactivation by haloenol lactones occurs i n a manner analogous to that of chymotrypsin with which aldehyde dehyd rogenase shares esterase activity and binding of haloenol lactones at the active site.