Identification of the catalytic residue of human short/branched chain acyl-CoA dehydrogenase by in vitro mutagenesis.

ABSTRACT

The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes which catalyze the alpha,beta-dehydrogenation of acyl-CoA esters, transferring electrons to electron transferring flavoprotein. We have recently cloned and characterized the cDNA for human short/branched chain acyl-CoA dehydrogenase (SBCAD). Based on homology with the other ACDs we hypothesized that E381 is the catalytic residue for this enzyme. Alteration of this amino acid to glutamine, glycine or arginine resulted in an inactive enzyme. Substitution of aspartate at this position led to an enzyme with reduced activity compared to the wild type. An E381G/G260E double mutation (which places a glutamate in a position homologous to the catalytic residue identified in other members of this gene family) restored enzyme activity. These data confirm the crucial nature of E381 to the activity of this enzyme and strongly support its role as the alpha-proton abstracting base.