Open AccessBiochemical Characterization of Purified, Human Recombinant Lys304→Glu Medium‐Chain Acyl‐Coa Dehydrogenase Containing the Common Disease‐Causing Mutation and Comparison with the Normal Enzyme
Author(s) -
Kieweg Volker,
Krautle FranzGeorg,
Nandy Andreas,
Engst Stefan,
Vock Petra,
AbdelGhany AbdelGhany,
Bross Peter,
Gregersen Niels,
Rasched Ihab,
Strauss Arnold,
Ghisla Sandro
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00548.x
Subject(s) - mutant , enzyme , biochemistry , recombinant dna , escherichia coli , dehydrogenase , microbiology and biotechnology , chemistry , mutant protein , biology , gene
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 °C). Expression in the same system at 37 °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.