Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilusand their role in catalysis

The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase com plex of Bacillus stearothermophilus is a heterotetramer (alpha (2)beta (2)) of E1 alpha. and E1 beta polypeptide chains. The domain structure of the E 1 alpha and E1 beta chains, and the protein-protein interactions involved i n assembly, have been studied by means of limited proteolysis. It appears t hat there may be two conformers of E1 alpha in the E1 heterotetramer, one b eing more susceptible to proteolysis than the other. A highly conserved reg ion in E1 alpha, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (alpha (2)beta (2)) As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchang ed. The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1. However, a pyruvate dehydrogen ase complex assembled in vitro with cleaved E1p exhibits a markedly lower o verall catalytic activity than that assembled with untreated E1. This impli es that active site coupling between the E1 and E2 components has been impa ired. This has important implications for the way in which a tethered lipoy l domain can interact with E1 in the assembled complex.