Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex. Identification of important amino acid residues.
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.
Jackson
J C
JC
Metabolic and Cardiovascular Diseases Research, Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901-1398, USA.
Vinluan
C C
CC
Dragland
C J
CJ
Sundararajan
V
V
Yan
B
B
Gounarides
J S
JS
Nirmala
N R
NR
Topiol
S
S
Ramage
P
P
Blume
J E
JE
Aicher
T D
TD
Bell
P A
PA
Mann
W R
WR
D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine
D010455:Peptides; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase