scholarly journals Activated Function of the Pyruvate Dehydrogenase Phosphatase through Ca2+-facilitated Binding to the Inner Lipoyl Domain of the Dihydrolipoyl Acetyltransferase

1996 ◽  
Vol 271 (45) ◽  
pp. 28064-28070 ◽  
Author(s):  
Guilan Chen ◽  
Lijuan Wang ◽  
Shengjiang Liu ◽  
Christina Chuang ◽  
Thomas E. Roche
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Dihydrolipoyl Acetyltransferase ◽  
Pyruvate Dehydrogenase Phosphatase ◽  
Inner Lipoyl Domain ◽  
Lipoyl Domain

scholarly journals Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex

1998 ◽  
Vol 334 (3) ◽  
pp. 703-711 ◽  
Author(s):  
John C. JACKSON ◽  
Christine C. VINLUAN ◽  
Carol J. DRAGLAND ◽  
Vijayayakumar SUNDARARAJAN ◽  
Bing YAN ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Catalytic Subunits ◽  
Dependent Inactivation ◽  
Dihydrolipoyl Acetyltransferase ◽  
Animal Models Of Diabetes ◽  
Inner Lipoyl Domain ◽  
Lipoyl Domain

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mitochondrion, is diminished in animal models of diabetes. Studies with purified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydrolipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdomain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inactivation of the complex. By combining molecular modelling, site-directed mutagenesis and biophysical characterizations, we have also identified two amino acid residues in this subdomain (Ile229 and Phe231) that largely determine the magnitude of this effect.

scholarly journals Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex

1993 ◽  
Vol 289 (1) ◽  
pp. 81-85 ◽  
Author(s):  
J Quinn ◽  
A G Diamond ◽  
A K Masters ◽  
D E Brookfield ◽  
N G Wallis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Structural Studies ◽  
Gene Encoding ◽  
E Coli ◽  
Inner Lipoyl Domain ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.

scholarly journals 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

1994 ◽  
Vol 297 (1) ◽  
pp. 137-143 ◽  
Author(s):  
D S Hipps ◽  
L C Packman ◽  
M D Allen ◽  
C Fuller ◽  
K Sakaguchi ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Tight Binding ◽  
Limited Proteolysis ◽  
Binding Domain ◽  
Dihydrolipoyl Acetyltransferase ◽  
Dihydrolipoyl Dehydrogenase ◽  
Lipoyl Domain

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.

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