HETEROLOGOUSLY EXPRESSED INNER LIPOYL DOMAIN OF DIHYDROLIPOYL ACETYLTRANSFERASE INHIBITS ATP-DEPENDENT INACTIVATION OF PYRUVATE-DEHYDROGENASE COMPLEX - IDENTIFICATION OF IMPORTANT AMINO-ACID-RESIDUES

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mit ochondrion, is diminished in animal models of diabetes, Studies with p urified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydro lipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdom ain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inac tivation of the complex. By combining molecular modelling, site-direct ed mutagenesis and biophysical characterizations, we have also identif ied two amino acid residues in this subdomain (IIe(229) and Phe(231)) that largely determine the magnitude of this effect.