| Abstract
| - The medium chain acyl-CoA dehydrogenase is rapidly inhibited by racemic 3,4-dienoyl-CoAderivatives with a stoichiometry of two molecules of racemate per enzyme flavin. Synthesis of R- andS-3,4-decadienoyl-CoA shows that the R-enantiomer is a potent, stoichiometric, inhibitor of the enzyme.α-Proton abstraction yields an enolate to oxidized flavin charge-transfer intermediate prior to adductformation. The crystal structure of the reduced, inactive enzyme shows a single covalent bond linking theC-4 carbon of the 2,4-dienoyl-CoA moiety and the N5 locus of reduced flavin. The kinetics of reversalof adduct formation by release of the conjugated 2,4-diene were evaluated as a function of both acylchain length and truncation of the CoA moiety. The adduct is most stable with medium chain lengthallenic inhibitors. However, the adducts with R-3,4-decadienoyl-pantetheine and -N-acetylcysteamine aresome 9- and >100-fold more kinetically stable than the full-length CoA thioester. Crystal structures ofthese reduced enzyme species, determined to 2.4 Å, suggest that the placement of H-bonds to the inhibitorcarbonyl oxygen and the positioning of the catalytic base are important determinants of adduct stability.The S-3,4-decadienoyl-CoA is not a significant inhibitor of the medium chain dehydrogenase and doesnot form a detectable flavin adduct. However, the S-isomer is rapidly isomerized to the trans-trans-2,4-conjugated diene. Protein modeling studies suggest that the S-enantiomer cannot approach close enoughto the isoalloxazine ring to form a flavin adduct, but can be facilely reprotonated by the catalytic base.These studies show that truncation of CoA thioesters may allow the design of unexpectedly potent lipophilicinhibitors of fatty acid oxidation.
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