title: Expression and characterisation of the homodimeric E1 component of the Azotobacter vinelandii pyruvate dehydrogenase complex.
abstract: We have cloned and sequenced the gene encoding the homodimeric pyruvate dehydrogenase component (E1p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and expressed and purified the E1p component in Escherichia coli. Cloned E1p can be used to fully reconstitute complex activity. The enzyme was stable in high ionic strength buffers, but was irreversibly inactivated when incubated at high pH, which presumably was caused by its inability to redimerize correctly. This explains the previously found low stability of the wild-type E1p component after resolution from the complex at high pH. Cloned E1p showed a kinetic behaviour exactly like the wild-type complex-bound enzyme with respect to its substrate (pyruvate), its allosteric properties, and its effectors. These experiments show that acetyl coenzyme A acts as a feedback inhibitor by binding to the E1p component. Limited proteolysis experiments showed that the N-terminal region of E1p was easily removed. The resulting protein fragment was still active with artificial electron acceptors but had lost its ability to bind to the core component (E2p) and thus reconstitute complex activity. E1p was protected against proteolysis by E2p. The allosteric effector pyruvate changed E1p into a conformation that is more resistant to proteolysis.
authors.affiliation: Department of Biomolecular Sciences, Wageningen Agricultural University, The Netherlands.
authors.forename: A F
authors.initials: AF
authors.lastname: Hengeveld
authors.forename: A H
authors.initials: AH
authors.lastname: Westphal
authors.forename: A
authors.initials: A
authors.lastname: de Kok
mesh_terms: D000595:Amino Acid Sequence; D001395:Azotobacter / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D011768:Pyruvate Dehydrogenase Complex