Reconstitution of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes: analysis of protein X involvement and interaction of homologous and heterologous dihydrolipoamide dehydrogenases.
Optimal conditions for rapid and efficient reconstitution of pyruvate dehydrogenase complex (PDC) activity are demonstrated by using an improved method for the dissociation of the multienzyme complex into its constituent E1 (substrate-specific 2-oxoacid decarboxylase) and E3 (dihydrolipoamide dehydrogenase) components and isolated E2/X (where E2 is dihydrolipoamide acyltransferase) core assembly. Selective cleavage of the protein X component of the purified E2/X core with the proteinase arg C decreases the activity of the reconstituted complex to residual levels (i.e. 8-12%); however, significant recovery of reconstitution is achieved on addition of a large excess (i.e. 50-fold) of parent E3. N-terminal sequence analysis of the truncated 35,000-M(r) protein X fragment locates the site of cleavage by arg C at the extreme N-terminal boundary of a putative E3-binding domain and corresponds to the release of a 15,000-M(r) N-terminal fragment comprising both the lipoyl and linker sequences. In native PDC this region of protein X is shown to be partly protected from proteolytic attack by the presence of E3. Recovery of complex activity in the presence of excess E3 after arg C treatment is thought to result from low-affinity interactions with the partly disrupted subunit-binding domain on X and/or the intact analogous subunit binding domain on E2. Contrasting recoveries for arg C-modified E2/X/E1 core, and untreated E2/E1 core of the 2-oxoglutarate dehydrogenase complex, reconstituted with excess bovine heart E3, pig heart E3 or yeast E3 point to subtle differences in subunit interactions with heterologous E3s and offer an explanation for the inability of previous investigators to achieve restoration of PDC function after selective proteolysis of the protein X component.
Sanderson
S J
SJ
Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
Khan
S S
SS
McCartney
R G
RG
Miller
C
C
Lindsay
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D009994:Osmolar Concentration; D010455:Peptides / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism
D010455:Peptides; D011768:Pyruvate Dehydrogenase Complex; C489061:pyruvate dehydrogenase complex component X, Bos taurus; D007655:Ketoglutarate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase; C044499:submandibular proteinase A; D012697:Serine Endopeptidases