Interaction of 3,4-dienoyl-CoA thioesters with medium chain acyl-CoA dehydrogenase: Stereochemistry of inactivation of a flavoenzyme

The medium chain acyl-CoA dehydrogenase is rapidly inhibited by racemic 3,4 -dienoyl-CoA derivatives with a stoichiometry of two molecules of racemate per enzyme flavin. Synthesis of R- and S-3,4-decadienoyl-CoA shows that the R-enantiomer is a potent, stoichiometric, inhibitor of the enzyme. a-Proto n abstraction yields an enolate to oxidized flavin charge-transfer intermed iate prior to adduct formation. The crystal structure of the reduced, inact ive enzyme shows a single covalent bond linking the C-4 carbon of the 2,4-d ienoyl-CoA moiety and the N5 locus of reduced flavin. The kinetics of rever sal of adduct formation by release of the conjugated 2,4-diene were evaluat ed as a function of both acyl chain length and truncation of the CoA moiety . The adduct is most stable with medium chain length allenic inhibitors. Ho wever, the adducts with R-3,4-decadienoyl-pantetheine and -N-acetylcysteami ne are some 9- and > 100-fold more kinetically stable than the full-length CoA thioester. Crystal structures of these reduced enzyme species, determin ed to 2.4 Angstrom, suggest that the placement of H-bonds to the inhibitor carbonyl oxygen and the positioning of the catalytic base are important det erminants of adduct stability. The S-3,4-decadienoyl-CoA is not a significa nt inhibitor of the medium chain dehydrogenase and does not form a detectab le flavin adduct. However, the S-isomer is rapidly isomerized to the trans- trans-2,4-conjugated diene. Protein modeling studies suggest that the S-ena ntiomer cannot approach close enough to the isoalloxazine ring to form a fl avin adduct, but can be facilely reprotonated by the catalytic base. These studies show that truncation of CoA thioesters may allow the design of unex pectedly potent lipophilic inhibitors of fatty acid oxidation.