CATALYTIC AND FAD-BINDING RESIDUES OF MITOCHONDRIAL VERY LONG-CHAIN ACYL-COENZYME-A DEHYDROGENASE

Very long-chain acyl-CoA dehydrogenase (VLCAD) is one of four flavopro teins which catalyze the initial step of the mitochondrial beta-oxidat ion spiral, By sequence comparison with other acyl-CoA dehydrogenases, Glu-422 of VLCAD has been presumed to be the catalytic residue that a bstracts the a-proton in the alpha beta-dehydrogenation reaction. Repl acing Glu-422 with glutamine (E422Q) caused a loss of enzyme activity by preventing the formation of a charge transfer complex between VLCAD and palmitoyl-CoA. This result provides further evidence for Glu-422 being part of the active site of VLCAD, F418L is a disease-causing mut ation in human VLCAD deficiency, Unlike wild-type VLCAD, F418L and F41 8V contained no bound FAD when expressed at extremely high levels in t he baculovirus expression system, Although F418T and F418Y bound FAD a t a level similar to that of wild type VLCAD, both showed reduced V-ma x,, values toward palmitoyl-CoA, most likely due to a decrease in the rate of enzyme-bound FAD reduction, These data suggest that Phe-418 is involved in the binding and subsequent reduction of FAD, FAD-deficien t VLCADs (F418L, F418V, and apo-VLCAD) showed increased sensitivity to trypsinization. Loss of FAD may change the folding of VLCAD subunit.