Activated function of the pyruvate dehydrogenase phosphatase through Ca2+-facilitated binding to the inner lipoyl domain of the dihydrolipoyl acetyltransferase.
Micromolar Ca2+ facilitates approximately 10-fold enhancement of pyruvate dehydrogenase phosphatase (PDP) activity by aiding the association of PDP with the dihydrolipoyl acetyltransferase (E2) component. Connected by linker regions, E2 consists of two lipoyl domains, the NH2-lipoyl domain (L1) and the interior lipoyl domain (L2), and a pyruvate dehydrogenase component binding domain surrounding a 60-mer inner core. Using recombinant constructs of L1 or L2, E2-enhanced PDP activity was markedly decreased by L2 but not by L1, effectively competing with intact E2 in Ca2+-dependent binding of PDP (half-maximal reduction at 2.0 microM L2 versus 6.7 microM E2 subunit). Using L2 fused to glutathione S-transferase resulted in direct Ca2+-dependent binding of PDP to L2 (Kd, approximately 1.7 microM L2). Affinity-bound glutathione S-transferase-L2 was used to purify PDP to homogeneity by selective binding and elution by Ca2+ chelation. The large activity enhancement of PDP by E2 was eliminated by enzymatic removal of lipoates from E2 and restored by their enzymatic reintroduction. The critical role of the L2 lipoate is not in binding of PDP to E2, since PDP was still bound by delipoylated L2, and delipoylated L2 inhibited E2-enhanced PDP activity, although lipoylated L2 was more effective in each of these tests. Thus, pyruvate dehydrogenase complex activity is increased by enhanced availability of PDP to its E2-bound, phosphorylated pyruvate dehydrogenase substrate as a consequence of the Ca2+-facilitated interchange of PDP among the mobile L2 domains and an essential (undetermined) step engaging the L2 lipoate.
Chen
G
G
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Wang
L
L
Liu
S
S
Chuang
C
C
Roche
T E
TE
D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D007668:Kidney / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex; D013329:Structure-Activity Relationship
D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase; D008274:Magnesium; D002118:Calcium