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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

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

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KIEWEG, Volker, Franz-Georg KRÄUTLE, Andreas NANDY, Stefan ENGST, Petra VOCK, Abdel Ghany ABDEL GHANY, Peter BROSS, Niels GREGERSEN, Ihab RASCHED, Arnold W. STRAUSS, Sandro GHISLA, 1997. 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. In: European Journal of Biochemistry. 246(2), pp. 548-556. ISSN 0014-2956. eISSN 1432-1033. Available under: doi: 10.1111/j.1432-1033.1997.00548.x

@article{Kieweg1997Bioch-8241, title={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}, year={1997}, doi={10.1111/j.1432-1033.1997.00548.x}, number={2}, volume={246}, issn={0014-2956}, journal={European Journal of Biochemistry}, pages={548--556}, author={Kieweg, Volker and Kräutle, Franz-Georg and Nandy, Andreas and Engst, Stefan and Vock, Petra and Abdel Ghany, Abdel Ghany and Bross, Peter and Gregersen, Niels and Rasched, Ihab and Strauss, Arnold W. and Ghisla, Sandro} }

Abdel Ghany, Abdel Ghany Rasched, Ihab First publ. in: European Journal of Biochemistry 246 (1997), 2, pp. 548-556 Bross, Peter 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 Vmax as human MCADH with the best substrate for the latter, octanoyl-CoA, the Km in the mutant MCADH is fourfold higher, which generates a correspondingly lower catalytic efficiency. Importantly, Vmax obtained using the natural acceptor, electron transfer flavoprotein, is only a third that for human MCADH. The Vmax/Km 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. Kräutle, Franz-Georg 1997 Bross, Peter Gregersen, Niels Kieweg, Volker Ghisla, Sandro 2011-03-24T17:41:42Z Strauss, Arnold W. 2011-03-24T17:41:42Z Strauss, Arnold W. application/pdf Nandy, Andreas eng Engst, Stefan Vock, Petra Abdel Ghany, Abdel Ghany 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 Nandy, Andreas Kieweg, Volker Vock, Petra Engst, Stefan Kräutle, Franz-Georg Gregersen, Niels Rasched, Ihab Ghisla, Sandro deposit-license

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