Interaction of acyl coenzyme A substrates and analogs with pig kidney medium-chain acyl-CoA dehydrogenase

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Abstract
Several alkylthio coenzyme A (CoA) derivatives (from ethyl- to hexadecyl-SCOA) have been synthesized to probe the substrate binding site in the flavoprotein medium-chain acyl-CoA dehydrogenase from pig kidney. All bind to apparently equivalent sites with a stoichiometry of four per tetramer. A plot of log Kd vs. hydrocarbon chain length is linear from 2 to 16 carbons with a free energy of binding of 390 cal/methylene group. These data suggest an acyl-binding site of moderate hydrophobicity and imply that the observed substrate specificity of the medium-chain dehydrogenase is not achieved simply by the length of the hydrocarbon binding pocket. Extrapolation of the graph to zero chain length predicts a Kd of 1 mM for the CoA moiety. The difference between this value and the experimentally determined value of 206 .mu.M may be attributed to a contribution from the ionization of the sulfhydryl group in CoASH. The interaction of several eight-carbon intermediates of .beta.-oxidation (trans-2- and trans-3-octenoyl-CoA and L-3-hydroxy- and 3-ketooctanoyl-CoA) with the dehydrogenase has also been studied. All but the L-3-OH derivative bind tightly to the enzyme (with Kd values in the 50-90 nM range) and are very effective inhibitors of the dehydrogenation of octanoyl-CoA. The trans-3-enoyl analogue produces an immediate, intense, long-wavelength band (.lambda.max = 820 nm), which probably represents a charge-transfer interaction between the delocalized .alpha.-carbanion donor and oxidized flavin as the acceptor. The L-3-OH analogue is a reductant of the flavin, yielding 3-ketooctanoyl-CoA. The results of these studies suggest that all of the 28 intermediates of palmitoyl-CoA oxidation plus those derived from the degradation of unsaturated fatty acid chains could bind to the medium chain length dehydrogenase from pig kidney. Possible metabolic consequences of this are discussed.