BIOCHEMICAL-CHARACTERIZATION OF PURIFIED, HUMAN RECOMBINANT LYS304-]GLU MEDIUM-CHAIN ACYL-COA DEHYDROGENASE CONTAINING THE COMMON DISEASE-CAUSING MUTATION AND COMPARISON WITH THE NORMAL ENZYME

Recombinant, normal human medium-chain acyl-CoA dehydrogenase (MCADH) and the common, human disease-causing K304E mutant ([Glu304]MCADH) pro tein 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 expr ession in E. coli cells at reduced temperature (28 degrees C). Express ion in the same system at 37 degrees C results in very low amounts of active mutant protein. Several catalytic and physicochemical parameter s of these two proteins have been determined and were compared to thos e of purified pig kidney MCADH. Although [Glu304]MCADH has approximate ly the same rate of substrate reduction with dodecanoyl-CoA and the sa me V-max as human MCADH with the best substrate for the latter, octano yl-CoA, the K-m in the mutant MCADH is fourfold higher, which generate s a correspondingly lower catalytic efficiency. Importantly, V-max obt ained using the natural acceptor, electron transfer flavoprotein, is o nly a third that for human MCADH. The V-max/K-m versus chain-length pr ofile of the mutant shows a maximum with dodecanoyl-CoA which differs markedly from that of human MCADH, which has maximal efficiency with o ctanoyl-CoA. The substrate specificity of the mutant is broader with a less pronounced activity peak resembling long-chain acyl-CoA dehydrog enase. The purified mutant enzyme exhibits a reduced thermal stability compared to human wild-type MCADH. The major difference between the t wo proteins expressed in E. coli is the more pronounced lability of th e K304E mutant in crude extracts, which suggests a higher susceptibili ty to attack by endogenous proteases. Differences between tetrameric [ Glu304]MCADH which survives the first step(s) of purification and corr esponding MCADH are minor. The overall differences in properties of [G lu304]MCADH together with its impaired folding and tetramer assembly m ay contribute to the generation of the abnormalities observed in patie nts homozygous for the K304E mutation.