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Recombinant, normal human medium-chain acyl-CoA dehydrogenase (MCADH) and the common, human disease-causing K304E mutant ([Glu304]MCADH) protein were expressed in Escherichia coli using an optimized system, and the enzymes were purified to apparent homogeneity. The crucial factor leading to the production of active [Glu304]MCADH protein is the expression in E. coli cells at reduced temperature (28 degrees C). Expression in the same system at 37 degrees C results in very low amounts of active mutant protein. Several catalytic and physicochemical parameters of these two proteins have been determined and were compared to those of purified pig kidney MCADH. Although [Glu304]MCADH has approximately the same rate of substrate reduction with dodecanoyl-CoA and the same V(max) as human MCADH with the best substrate for the latter, octanoyl-CoA, the K(m) in the mutant MCADH is fourfold higher, which generates a correspondingly lower catalytic efficiency. Importantly, V(max) obtained using the natural acceptor, electron transfer flavoprotein, is only a third that for human MCADH. The V(max)/K(m) versus chain-length profile of the mutant shows a maximum with dodecanoyl-CoA which differs markedly from that of human MCADH, which has maximal efficiency with octanoyl-CoA. The substrate specificity of the mutant is broader with a less pronounced activity peak resembling long-chain acyl-CoA dehydrogenase. The purified mutant enzyme exhibits a reduced thermal stability compared to human wild-type MCADH. The major difference between the two proteins expressed in E. coli is the more pronounced lability of the K304E mutant in crude extracts, which suggests a higher susceptibility to attack by endogenous proteases. Differences between tetrameric [Glu304]MCADH which survives the first step(s) of purification and corresponding MCADH are minor. The overall differences in properties of [Glu304]MCADH together with its impaired folding and tetramer assembly may contribute to the generation of the abnormalities observed in patients homozygous for the K304E mutation.

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 650-700 min(-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 C14 substrates as MCADH does with C6 to C10 substrates. Recombinant human LCADH, however, is significantly less efficient (approximately fourfold with C12) than purified pig kidney enzyme. We conclude that human LCADH is either quantitatively less important in beta-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 Glu261, the active-site residue involved in deprotonation of the substrate (alpha)C-H.