Site-directed mutagenesis and 1H NMR spectroscopy of an interdomain segment in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.
Deletion of two of the three homologous lipoyl domains that form the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain of the Escherichia coli pyruvate dehydrogenase complex can be achieved by in vitro deletion in the structural gene aceF. A site-directed mutagenesis of this shortened aceF gene was carried out to replace the glutamine residue at position 291 (wild-type numbering) with a histidine residue. Residue 291 is near the middle of a long segment (about 30 amino acid residues) of polypeptide chain, rich in alanine, proline, and charged amino acids, that links the remaining lipoyl domain to the dihydrolipoamide dehydrogenase (E3) binding domain in the E2p chain. A fully active enzyme complex was still assembled, and despite the enormous size of the particle (Mr approximately 4 x 10(6)), sharp resonances attributable to the single new histidine residue per E2p chain could be detected in the 400-MHz 1H NMR spectrum of the complex. The sharpness of these resonances, their chemical shifts (7.94 and 7.05 ppm), and the apparent pKa (6.4) of the side chain were all consistent with this histidine residue being exposed to solvent in a conformationally flexible region of the E2p polypeptide chain. These experiments provide direct proof for the conformational flexibility of this region of polypeptide chain, which is thought to play an important part in the movement of the lipoyl domain required for active site coupling in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Texter
F L
FL
Department of Biochemistry, University of Cambridge, England.
Radford
S E
SE
Laue
E D
ED
Perham
R N
RN
Miles
J S
JS
Guest
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005973:Glutamine; D006639:Histidine; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000378:metabolism
D011768:Pyruvate Dehydrogenase Complex; D005973:Glutamine; D006639:Histidine; D006859:Hydrogen