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J. Biol. Chem., Vol. 265, Issue 13, 7116-7119, May, 1990
P Bross, S Engst, AW Strauss, DP Kelly, I Rasched and S Ghisla
The cDNA of human medium chain acyl-CoA dehydrogenase (MCADH) was modified
by in vitro mutagenesis, and the sequence encoding the mature form of MCADH
was introduced into an inducible expression plasmid. We observed synthesis
of the protein in Escherichia coli cells transformed with this plasmid with
measurable MCADH enzyme activity in cell extracts. Glutamic acid 376, which
has been proposed by Powell and Thorpe (Powell, P. J., and Thorpe, J.
(1988) Biochemistry 27, 8022- 8028) as an essential residue and the
proton-abstracting base at the active site of the enzyme, was mutated to
glutamine. After expression in bacteria of this plasmid, the corresponding
extracts show no detectable MCADH activity, although mutant MCADH-protein
production was detected by protein immunoblots. The mature enzyme and the
Gln376 mutant were purified to apparent homogeneity. The wild-type enzyme
is a yellow protein due to the content of stoichiometric FAD and had a
specific activity which is 50% of MCADH purified from pig kidney. The
Gln376 mutant is devoid of activity (less than 0.02% that of wild type,
expressed enzyme) and is green because of bound CoA persulfide. Properties
of the mutant enzyme suggest that the Glu376----Gln change specifically
affects substrate binding. These results prove that Glu376 plays an
important role in the initial step of dehydrogenation catalysis.
Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
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