Reactivity of the active site base in the acyl-CoA dehydrogenase family

The importance of the active site glutamate residue in the acyl-CoA dehydrogenase family has been investigated in two different ways.;Mechanistic studies showed that 3-thia-octanoyl-CoA in conjunction with the facile one electron oxidant ferricenium hexafluorophosphate irreversibly and stoichiometrically inactivated medium chain acyl-CoA dehydrogenase from porcine kidney. From radioactive labelling and peptide analysis, the target was shown to be GLU 376. This amino acid residue has previously been determined to function as the catalytic base in the reductive half reaction of the enzyme.;The importance of the position of the active site base in the reactivity of certain ligands such as 2-pentynoyl-CoA and 3-methyl-3-butenoyl-CoA with the acyl-CoA dehydrogenases has been assessed by probing three flavoenzymes, short chain acyl-CoA dehydrogenase, a mutant of isovaleryl-CoA dehydrogenase and the wild type enzyme with a series of acyl-CoA thioesters. The short chain enzyme and the mutant protein, which both have a glutamate residue in the same position as the medium chain enzyme. show a similar sensitivity towards inactivation by 2-pentynoyl-CoA and the ability to catalyze isomerization reactions. In contrast to these two enzymes, wild type isovaleryl-CoA dehydrogenase, which has the active glutamate residue in a different position within its amino acid sequence, reacts very differently with these thioesters. Evidence has been obtained which suggests that the variation in reactivity towards these ligands is due to the different orientation of the active site base within the substrate binding site of these three enzymes.