Partial activation of the pyruvate dehydrogenase kinase by the lipoyl domain region of E2 and interchange of the kinase between lipoyl domain regions.

The binding of the pyruvate dehydrogenase (E1) component and the E1-specific kinase to the core-forming dihydrolipoyl acetyltransferase (E2) component facilitates a severalfold enhancement in the rate at which the kinase phosphorylate E1 (i.e. versus free kinase phosphorylating free E1). The kinase and E1 associate with small exterior linker region-connected domains in the E2 structure. The kinase binds to one of two lipoyl domains, and the E1 component binds to a domain in E2"s structure between the lipoyl domain region and the inner domain. Sixty of the latter domains assemble to form a dodecahedron-shaped inner core. Binding of the kinase to a detached lipoyl domain region enhanced kinase activity. This bi-lipoyl domain fragment induced a 2-fold enhancement in the slow rate of phosphorylation of peptide substrate and intact E260 gave only a 50% higher rate. In contrast, the lipoyl domain fragment gave only a 40% enhancement in the faster rate of phosphorylation of E1; whereas the rate of phosphorylation of E1 was markedly increased (4-10-fold depending on conditions) by kinase interaction with the intact E2 core. Binding of E1 to an E2 structure lacking only the bi-lipoyl domain region did not enhance kinase activity. Thus, binding of the kinase to the lipoyl domain region elicits a structural change which enhances kinase activity; however, other processes are required to explain the very large enhancement in phosphorylation of E1 effected by intact E2 core. Among the latter is a need for a mechanism allowing one kinase molecule to phosphorylate many E1 tetramers, whereas both E1 and the kinase stay bound to the oligomeric E2 core (i.e. phosphorylation appears to be much faster than the dissociation of either the kinase or E1 tetramers from E260 core). Exposure of kinase bound to the lipoyl domain fragment to intact E2 core for 10 s allowed a transition to a maximal (7-fold) activation of the kinase. In the opposite direction, an increasing level of the free bi-lipoyl domain fragment rapidly reduced, in a concentration-dependent manner the activity of kinase bound initially to intact E2. The data strongly support kinase transfer between free lipoyl domains and the intact E2 core and fit about a 12-fold tighter binding of the kinase to intact E2 cores over binding to free lipoyl domains. Such an interchange of the kinase between these E2 structures was confirmed by sucrose gradient studies.(ABSTRACT TRUNCATED AT 400 WORDS)