Hexadienoyl -CoA as an active site probe of acyl -CoA dehydrogenase and enoyl -CoA

Medium-chain acyl-CoA dehydrogenase (MCAD) and enoyl-CoA hydratase (ECH) are the first two enzymes in the fatty acid beta-oxidation pathway. Substrate polarization by these two enzymes has been investigated using hexadienoyl-CoA (HD-CoA), a product analog of MCAD and a substrate analog for ECH. Raman difference spectroscopy has revealed that MCAD and ECH polarize HD-CoA to similar extents. Binding of HD-CoA to each enzyme results in distortion of the HD enone fragment towards an enolate-like structure and uncoupling of the terminal ethylenic bond from the dienoyl skeleton.;13C NMR experiments using labeled HD-CoAs bound to ECH are in close agreement with the Raman studies and reveal a decrease in electron density at the HD C1 and C3 carbons and an increase in electron density at C2. In contrast, 13C NMR spectra of HD-CoA bound to MCAD are characterized by large (12--13 ppm) upheld shifts in the HD C1 and C3 resonances. The changes in chemical shift are ascribed to stabilization of an enolate-like structure by the MCAD active site and also to the effect of the ring current from the enzyme's flavin cofactor.