RECOMBINANT HUMAN LIVER MEDIUM-CHAIN ACYL-COA DEHYDROGENASE - PURIFICATION, CHARACTERIZATION, AND THE MECHANISM OF INTERACTIONS WITH FUNCTIONALLY DIVERSE C-8-COA MOLECULES

We offer a large scale purification procedure for the recombinant huma n liver medium-chain acyl-CoA. dehydrogenase (HMCAD). This procedure r outinely yields 100-150 mg of homogeneous preparation of the enzyme fr om 80 L of the Escherichia coli host cells. A comparative investigatio n of kinetic properties of the human liver and pig kidney enzymes reve aled that, except for a few minor differences, both of these enzymes a re nearly identical. We undertook detailed kinetic and thermodynamic i nvestigations for the interaction of HMCAD-FAD with three C-8-CoA mole cules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation at the alpha-beta ca rbon centers; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate a nd product of the enzyme, respectively, whereas 2-octynoyl-CoA is know n to inactivate the enzyme. Our experimental results demonstrate that all three C-8-CoA molecules first interact with HMCAD-FAD to form corr esponding Michaelis complexes, followed by two subsequent isomerizatio n reactions. The latter accompany either subtle changes in the electro nic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzy me-bound flavin (in case of octanoyl-CoA). The rate and equilibrium co nstants intrinsic to the above microscopic steps exhibit marked simila rity with different C-8-CoA molecules. However, the electronic structu ral changes accompanying the 2-octynoyl-CoA-dependent inactivation of the enzyme is 3-4 orders of magnitude slower than the above isomerizat ion re actions. Hence, the octanoyl-CoA-dependent reductive half-react ion and the 2-octynoyl-CoA-dependent covalent modification of the enzy me occur during entirely different microscopic steps. Arguments are pr esented that the origin of the above difference lies in the protein co nformation-dependent orientation of Glu-376 in the vicinity of the C-8 -CoA binding site.