β-Hydroxydecanoyl Thioester Dehydrase. Complete Characterization ot the Fate of the "Suicide" Substrate 3-Decynoyl-NAC

Article abstract of DOI:10.1021/ja00277a041

β-Hydroxydecanoyl thioester dehydrase, the pivotal enzyme in the biosynthesis of unsaturated fatty acids under anaerobic conditions, catalyzes the interconversion of thioesters of (R)-3-hydroxydecanoic acid (1), (E)-2-decenoic acid (2), and (Z)-3-decenoic acid (3).Dehydrase is irreversibly inactivated by the N-acetylcysteamine thioester of 3-decynoic acid (3-decynoyl-NAC), via dehydrase-catalyzed conversion of the acetylenic thioester to 2,3-decadienoyl-NAC.This is the classic example of "suicide" or "mechanism-based" enzyme inactivation.NMR-based experiments have been carried out in order to define the mechanistic relationship between "normal" catalysis and suicide inactivation of dehydrase, by providing detailed structural information on the enzyme-bound inactivator moiety. 3-<2-(13)C>Decynoyl-NAC was synthesized and incubated with homogeneous dehydrase. (13)C NMR spectroscopy at 100.6 MHz showed that when 2,3-decadienoyl-NAC is attacked by the active-site histidine, the product is (3-imidazolyl-3-decenoyl)-NAC.This adduct is slowly isomerized to (3-imidazolyl-2-decenoyl)-NAC.One molecule of inactivator is bound per subunit of the dimeric enzyme.Model histidine-allene adducts have been made and characterized.Comparisons of NMR data reveal that the double-bond configuration of the decenoyl moiety of the enzyme-bound inactivator is E.Analysis of these findings strongly suggests that the histidine residue that is alkylated by 2,3-decadienoyl-NAC is the active-site base.The structure of the product formed by inactivation of dehydrase by 3-decynoyl-NAC and the mechanism of the inactivation are readily explained in terms of the mechanisms of the normal dehydrase-catalyzed reactions as well as the stereochemical relationships between enzyme and substrates in those normal reactions.