| These studies concern three areas which are peripherally related: the identification of a previously unknown prosthetic group of electron-transferring flavoprotein (ETF), a site-directed mutagenesis study of the 166 position of human medium chain acyl-CoA dehydrogenase (MCAD), and an examination of the non-physiological oxidase activity of the medium chain dehydrogenase.;The additional prosthetic group of electron-transferring flavoprotein was discovered upon analysis of denatured extracts of the mammalian protein. Chromatographic and enzymatic analyses showed that the heterodimeric electron carrier, ETF, contains one molecule of tightly bound AMP, in addition to the FAD redox center. AMP was found in the same stoichiometry in an ETF from the methylotrophic bacterium W3A1, suggesting that this prosthetic group is widely distributed in the ETF family. Possible reasons for the presence of AMP in ETF are discussed.;TRP 166 is located on a loop in MCAD which partially covers the si-face of the isoalloxazine ring system, protecting it from solvent. An aromatic residue at this position is conserved in all members of the acyl-CoA dehydrogenase family. This locus has been suggested as important in facilitating interflavin electron transfer between the dehydrogenase and ETF. The purification of a number of site-directed mutants at position 166 and related loci was attempted to explore their effects on catalysis. These efforts were largely thwarted by the instability of the mutant enzymes and, in particular, the failure to purify protein containing an aliphatic substitution at position 166 of MCAD. The loop containing position 166 seems to play an important role in flavin binding.;The binding normal substrates to MCAD profoundly suppresses the reactivity of the reduced flavin towards molecular oxygen, while bulky substrates can mediate significant oxidase activity. This work tests recent suggestions that the oxidase activity of MCAD towards indolepropionyl-CoA (IP-CoA) is solely due to reoxidation of the free reduced enzyme. Stopped-flow and steady state kinetic experiments are incompatible with this simple model, and require that at least two ligated, reduced enzyme species react with oxygen. These data were confirmed and extended with a Glu376Gln mutant of MCAD. Finally, the oxygen reactivity of the short chain acyl-CoA dehydrogenase from Megasphaera elsdenii was also shown to proceed via oxidation of reduced enzyme-ligand complexes. |
