title: Thioester enolate stabilization in the acyl-CoA dehydrogenases: the effect of 5-deaza-flavin substitution.
abstract: The redox-inactive thioester analog 3-thia-octanoyl-CoA blocks transfer of a hydride equivalent to the flavin prosthetic group of the medium-chain acyl-CoA dehydrogenase with the accumulation of a stable enolate intermediate not encountered with normal substrates. Substitution of the normal flavin with 5-deaza-FAD would thus be expected to lead to enolate formation with both normal and 3-thia-substrate analogs, because reduction of the 5-deaza-enzyme is thermodynamically highly unfavorable. However, spectrophotometric titrations show that neither ligand forms significant enolate species with the 5-deaza-FAD enzyme. Similarly, the substituted dehydrogenase catalyzes undetectable alpha-proton exchange with octanoyl-CoA and ca. 1% of the corresponding rate with 3-thia-octanoyl-CoA when compared to the native enzyme. This inability to stabilize enolate species is not simply due to impaired binding of CoA-thioester analogs, because binding of a range of ligands is weakened by only 2- to 10-fold with the 5-deaza-enzyme. 4-Thia-trans-2-enoyl-CoA product is polarized normally on binding to the substituted protein, showing that this critical aspect of catalysis is apparently normal. These data, together with studies with CoA-persulfide and acetoacetyl- and p-nitrophenylacetyl-CoA, suggest that 5-deaza-FAD substitution exerts subtle, unanticipated, effects on the reductive half-reaction of the medium-chain acyl-CoA dehydrogenase. The involvement of charge-transfer interactions in the acidification of weakly acidic acyl-CoA thioesters is discussed.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: I
authors.initials: I
authors.lastname: Rudik
authors.forename: C
authors.initials: C
authors.lastname: Thorpe
mesh_terms: D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry*; D000818:Animals; D004952:Esters / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011485:Protein Binding; D013053:Spectrophotometry; D013552:Swine; D013816:Thermodynamics; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D004952:Esters; D008024:Ligands; D005182:Flavin-Adenine Dinucleotide; C023406:1-deaza-FAD; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase