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Volume 271, Number 45, Issue of November 8, 1996 pp. 28064-28070
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, June 28, 1996, and in revised form, August 28, 1996)

From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

Micromolar Ca²⁺ facilitates ~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 µM L2 versus 6.7 µM E2 subunit). Using L2 fused to glutathione S-transferase resulted in direct Ca²⁺-dependent binding of PDP to L2 (K_d, ~1.7 µM 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.

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