ENZYME-ACTIVATED INHIBITORS, ALTERNATE SUBSTRATES, AND A DEAD END INHIBITOR OF THE GENERAL ACYL-COA DEHYDROGENASE

Abstract

Aspects of the binding and dehydrogenation of acyl-CoA thiol esters by the general acyl-CoA dehydrogenase from pig liver were investigated using a dead-end inhibitor, S-octyl-CoA, several alternate substrates, and 3 active site-directed inhibitors. Experiments with S-octyl-CoA indicate that the carbonyl group of acyl-CoA thiol esters is not absolutely required for binding to the enzyme. The mode of binding of the 8-carbon thiol ether can be distinguished from the mode of binding of the enoyl-CoA product, octenoyl-CoA. Octanoyl pantetheine, octanoyl-etheno-CoA and octanoyl-3''-dephospho-CoA are alternate substrates of the dehydrogenase. Steady state kinetic constants obtained with these alternate substrates indicate that the adenosine 5''-diphosphate, but not the 3''-phosphate, of the nucleotide moiety of acyl-CoA substrates contribute to the tight binding of the substrates. The substrate analogs 3-butynoyl-CoA and 3-octynoyl-CoA are active site-directed, mechanism-based irreversible inhibitors of the dehydrogenase. These inhibitors covalently modify the apoprotein rather than the flavin. This finding and the fact that 2,3-octadienoyl-CoA also completely and irreversibly inhibits the enzyme indicate that the 3-acetylenic thiol esters inhibit the enzyme by a mechanism involving: base-catalyzed abstraction of a protein at C-2 followed by isomerization to the allene carbanion, protonation of the carbanion and attack of a nucleophile in the enzyme-active site on C-3 of the 2,3-dienoyl-CoA. The data show that the alkynoyl-CoA are activated and bound at the active site of the enzyme. Apparently, abstraction of a proton at C-2 of acyl-CoA substrates is the initial step in the catalytic pathway of dehydrogenation of substrates by the enzyme.