title: Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly.
abstract: The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex.
authors.affiliation: Department of Biochemistry, University of Cambridge, U.K.
authors.forename: I A
authors.initials: IA
authors.lastname: Lessard
authors.forename: R N
authors.initials: RN
authors.lastname: Perham
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000737:chemistry*; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins