Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and their role in catalysis.
The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (alpha2beta2) of E1alpha and E1beta polypeptide chains. The domain structure of the E1alpha and E1beta chains, and the protein-protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1alpha in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1alpha, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (alpha2beta2). As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged. The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1. However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.
Chauhan
H J
HJ
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK.
Domingo
G J
GJ
Jung
H I
HI
Perham
R N
RN
D000595:Amino Acid Sequence; D002384:Catalysis; D002918:Chymotrypsin / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014357:Trypsin / Q000378:metabolism
D011768:Pyruvate Dehydrogenase Complex; D002918:Chymotrypsin; D014357:Trypsin; D011767:Pyruvate Decarboxylase