CHARACTERIZATION OF HUMAN AND PIG-KIDNEY LONG-CHAIN-ACYL-COA DEHYDROGENASES AND THEIR ROLE IN BETA-OXIDATION

Long-chain-acyl-CoA dehydrogenase (LCADH) has been produced by recombi nant techniques from the human cDNA and purified after expression in E scherichia coli. Pig kidney LCADH was purified using an optimized meth od which also produces apparently pure short-chain-acyl-CoA dehydrogen ase (SCADH) and medium-chain-acyl-CoA dehydrogenase (MCADH) in good yi elds. LCADH from both sources has a maximal turnover rate (V-max of 65 0-700 min(-1) at pH 7.6) with the best substrates, which is approximat ely fivefold higher than reported previously. The human enzyme has an approximately fivefold higher K-m compared with the pig kidney enzyme with substrates of chain length from C-10 to C-18 and a significantly different dependence of V-max on the chain length. Pig kidney LCADH ha s a similar V-max/K-m with C-10 to C-14 substrates as MCADH does with C-6 to C-10 substrates. Recombinant human LCADH, however, is significa ntly less efficient (approximately fourfold with C-12) than purified p ig kidney enzyme. We conclude that human LCADH is either quantitativel y less important in beta-oxidation than in the pig, or that post-trans lational modifications, not present in the recombinant human enzyme, a re required to optimize human LCADH activity. Our results demonstrate that LCADH is as important as the other acyl-CoA dehydrogenases in fat ty acid oxidation at physiological, mitochondrial pH with optimal subs trates of chain length C-10-C-14. The extent of the LCADH-flavin cofac tor reduction observed with most substrates and the rate of the subseq uent reoxidation with oxygen are markedly different from those found w ith human medium chain acyl-CoA dehydrogenase. Both LCADH are inactiva ted by the substrate analogue 2-octynoyl-CoA, possibly via covalent mo dification of Glu261, the active-site residue involved in deprotonatio n of the substrate (alpha)C-H.