Characterization of human and pig kidney long-chain-acyl-CoA dehydrogenases and their role in beta-oxidation

Abstract

Long-chain-acyl-CoA dehydrogenase (LCADH) has been produced by recombinant techniques from the human cDNA and purified after expression in Escherichia coli. Pig kidney LCADH was purified using an optimized method which also produces apparently pure short-chain-acyl-CoA dehydrogenase (SCADH) and medium-chain-acyl-CoA dehydrogenase (MCADH) in good yields. LCADH from both sources has a maximal turnover rate (Vmax, of 650-700min−1 at pH 7.6) with the best substrates, which is approximately fivefold higher than reported previously. The human enzyme has an approximately fivefold higher Km, compared with the pig kidney enzyme with substrates of chain length from C10, to C18, and a significantly different dependence of Vmax on the chain length. Pig kidney LCADH has a similar Vmax/Km with C10, to C18, substrates as MCADH does with C6, to C10, substrates. Recombinant human LCADH, however, is significantly less efficient (approximately fourfold with C,J than purified pig kidney enzyme. We conclude that human LCADH is either quantitatively less important in β-oxidation than in the pig, or that post-translational modifications, not present in the recombinant human enzyme, are required to optimize human LCADH activity. Our results demonstrate that LCADH is as important as the other acyl-CoA dehydrogenases in fatty acid oxidation at physiological, mitochondrial pH with optimal substrates of chain length C10 C14. The extent of the LCADH-flavin cofactor reduction observed with most substrates and the rate of the subsequent reoxidation with oxygen are markedly different from those found with human medium chain acyl-CoA dehydrogenase. Both LCADH are inactivated by the substrate analogue 2-octynoyl-CoA, possibly via covalent modification of GIu261, the active-site residue involved in deprotonation of the substrate (α)C-H.