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.
Mukerjee
N
N
Center of Alcohol Studies, Rutgers University, Piscataway, New Jersey 08855-0969, USA.
Dryjanski
M
M
Dai
W
W
Katzenellenbogen
J A
JA
Pietruszko
R
R
D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004950:Esterases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D007783:Lactones / Q000378:metabolism* / Q000494:pharmacology*; D015394:Molecular Structure; D011753:Pyrones / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
C104710:3-isopropyl-6-bromomethylenetetrahydropyran-2-one; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D007783:Lactones; D011753:Pyrones; C104711:3-phenyl-6-bromomethylenetetrahydropyran-2-one; D000444:Aldehyde Dehydrogenase; D004950:Esterases; D003545:Cysteine