The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component.
The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.
Hipps
D S
DS
Department of Biochemistry, University of Cambridge, U.K.
Packman
L C
LC
Allen
M D
MD
Fuller
C
C
Sakaguchi
K
K
Appella
E
E
Perham
R N
RN
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D008058:Dihydrolipoamide Dehydrogenase; D014357:Trypsin