Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.
The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube.
D000123:Acetyltransferases / Q000032:analysis; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D044943:Fatty Acid Desaturases / Q000032:analysis; D005227:Fatty Acids; D005420:Flavoproteins / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008970:Molecular Weight; D011485:Protein Binding; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis*
D005227:Fatty Acids; D005420:Flavoproteins; D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D044943:Fatty Acid Desaturases; D000123:Acetyltransferases