Structure and funktion of human Acyl-CoA dehydrogenases. Iinvestigations on the Sudden Infant Death Syndrome

The aim of this work was to establish an isolation method for recombinantly expressed human Acyl-CoA dehydrogenases form E. coli. The main interest was supposed to be the mutant K304E of medium-chain specific Acyl-CoA dehydrogenase. This enzyme may play an important role in the Sudden Infant Death.An amino acid exchange at position 304 of the native enzyme, leads to a charge reversal an probably to an instability at this very position. Previous attempts to isolate this enzyme failed due to minute amounts or aggregation and degradation phenomena. A new method to detect and quantify recombinant Acyl-CoA dehydrogenase by the use of monospecific antiobodies was set up to unambiguosly measure inactive enzyme in crude extracts.A chromatographic separation scheme based on maximum three different steps was developed to purify different forms of human Acyl-CoA Dehydrogenase. The mutant K304E of medim-chain specific acyl-CoA dehydrogenase could for the first time be separated in a highly pure form. Also, a transient binding of molecular chaperone GroEL could be shown for the mutant enzyme.The biological activity of mutant K304E was against all expectations comparable to the native enzyme although the substrate specificity differed markedly towards long-chain fatty acid substrates. This could also be demonstrated by different extents of bleaching upon binding to substrates of various chain-lengths. The natural electron acceptor ETF also showed different binding behaviour to the mutant enzyme. All together, this lead to the assumption of slightly different shaped conformation of the mutant enzyme.In analogy of the medium-chain specific enzyme, the recombinant long-chain specific enzyme could also be isolated using the same separation scheme. Long-chain specific Acyl-CoA dehydrogenase could crystalized and ist structure could be resolved