Roles of ligand structure and active-site mutations in medium chain acyl -CoA dehydrogenase

In order to obtain human liver medium-chain acyl-CoA dehydrogenase (MCAD) on a large-scale basis, a plasmid containing the MCAD gene was cloned into E. coli competent cells and expressed. The wild-type MCAD yield was 130 mg from 32 L of cultured cells.;Interactions of functionally diverse C8-CoA's, octanoyl-CoA, octenoyl-CoA, and octenoyl-CoA with the wild-type enzyme were examined via UV/Vis rapid-scanning and single-wavelength stopped-flow, and all exhibited similar biphasic rate constants upon binding, indicating the formation of similar enzyme-ligand complexes. Inactivation by octenoyl-CoA was found to occur subsequent to the formation of the enzyme-ligand complex.;The thermodynamic contribution upon binding/reaction of octanoyl-CoA or octenoyl-CoA was examined and yielded the following conclusions: (1) enthalpic changes (DeltaH0) upon interaction, were more favorable with octanoyl-CoA than octenoyl-CoA; (2) the heat capacity changes (DeltaC p0) for the reaction of octanoyl-CoA were more favorable than for octenoyl-CoA; and (3) the free energy change (DeltaG 0) is equal for the binding/reaction of octanoyl-CoA or octenoyl-CoA.;To examine the role of the 3'-phosphate of coenzyme-A in enzyme catalysis, studies of normal and 3'-phosphate truncated ligands were performed. The dephosphorylated C 4-CoA substrate demonstrated a fivefold higher Km and a tenfold lower kcat than the phosphorylated substrate. The dephosphorylated C8-CoA had an eightfold higher Km, but the kcat increased by fivefold, indicating a reduced binding affinity for the dephosphorylated substrates with different overall catalytic effects.;Isothermal titration calorimetry of octenoyl-CoA or dephospho-octenoyl-CoA with the human liver enzyme demonstrated that the free energy and enthalpy changes were less favorable without the 3'-phosphate, indicating that removal of the 3'-phosphate destabilizes the ground and transition states of the enzyme-ligand complex.;Site-directed mutagenesis of amino acids, Asn-191 → Ala and Glu-376 → Asp, showed the following effects on ligand binding and/or catalysis: (1) the mutation of Asn-191 → Ala had little effect on catalysis of short and medium chain substrates; (2) the Glu-376 → Asp mutation decreases the dehydrogenase activity by twentyfold with IPCoA and octanoyl-CoA substrates; and (3) the Glu-376 → Asp mutation leads to pronounced and opposite effects upon binding acetoacetyl-CoA and IACoA as compared to wild-type enzyme.