The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (α2β2) of E1α and E1β polypeptide chains. The domain structure of the E1α and E1β chains, and the protein-protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1α in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1α, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (α2β2). 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 unchanged. 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 dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.
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