"Dehydrogenase" and "oxidase" reactions of medium-chain fatty acyl-CoA dehydrogenase utilizing chromogenic substrates: role of the 3',5'-adenosine diphosphate moiety of the coenzyme A thioester in catalysis.

We undertook a comparative investigation of the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction utilizing indole-, furyl-, and 4-(dimethylamino)phenyl-substituted propionyl- and acryloyl-CoAs as potential substrate/product pairs. All these propionyl-CoA derivatives undergo MCAD-catalyzed conversion into their corresponding acryloyl-CoAs via both "dehydrogenase" (in the presence of "organic" electron acceptors) and "oxidase" (buffer-dissolved oxygen serving as the electron acceptor) pathways [Johnson, J. K., Wang, Z. X., & Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. The steady-state kinetic parameters for the enzyme utilizing these substrates reveal that the KmS (for the CoA substrates) and kcatS for the dehydrogenase reaction are at least an order of magnitude higher than those for the oxidase reaction. As with the CoA substrates, the enzyme catalyzes the conversion of indolepropionyl pantetheine phosphate (IPPP) into indoleacryloyl pantetheine phosphate (IAPP) via these two pathways. However, with IPPP as substrate, the Km (for IPPP) and kcat values of the dehydrogenase and oxidase reactions are the same. These, coupled with the spectral changes of the enzyme-product complexes as well as the binding affinities of the enzyme-substrate/product complexes, lead to the following conclusions: (1) The aromatic/heterocyclic group-containing substrates are converted into their corresponding products via both the dehydrogenase and the oxidase pathways. (2) The 3',5'-ADP moiety of the CoA thioester provides a significant fraction of the total binding energy in stabilizing the enzyme-substrate/product complexes.(ABSTRACT TRUNCATED AT 250 WORDS)