Haloenol lactones as inactivators and substrates of aldehyde dehydrogenase.

Human aldehyde dehydrogenase (EC 1.2.1.3) isozymes E1 and E2 were irreversibly inactivated by stoichiometric concentrations of the haloenol lactones 3-isopropyl-6(E)-bromomethylene tetrahydro-pyran-2-one and 3-phenyl-6(E)-bromomethylene tetrahydropyran-2-one. No inactivation occurred with the corresponding nonhalogenated enol lactones. Both the dehydrogenase and esterase activities were abolished. Activity was not regained on dialysis or treatment with 2-mercaptoethanol. The inactivation was subject to substrate protection: NAD afforded protection which increased in the presence of the aldehyde-substrate competitive inhibitor chloral. Saturation kinetics gave positive gamma-axis intercepts, allowing the determination of binding constants. Inactivation stiochiometry determined with 14C-labeled 3-(1-naphthyl)-6(E)-iodomethylene tetrahydropyran-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 within a similar time frame. Opening of the lactone ring to form enzyme-acyl intermediate with active site cysteine appears to be a necessary prerequisite to inactivation, since halogen in the lactone ring is nonreactive. Thus, the inactivation of aldehyde dehydrogenase by haloenol lactones is mechanism-based. Inactivation by haloenol lactones occurs in a manner analogous to that of chymotrypsin with which aldehyde dehydrogenase shares esterase activity and binding of haloenol lactones at the active site.