PARTIAL ACTIVATION OF THE PYRUVATE-DEHYDROGENASE KINASE BY THE LIPOYLDOMAIN REGION OF E2 AND INTERCHANGE OF THE KINASE BETWEEN LIPOYL DOMAIN REGIONS

The binding of the pyruvate dehydrogenase (E1) component and the E1-sp ecific kinase to the core-forming dihydrolipoyl acetyltransferase (E2) component facilitates a severalfold enhancement in the rate at which the kinase phosphorylates E1 (i.e. versus free kinase phosphorylating free E1). The kinase and E1 associate with small exterior linker regio n-connected domains in the E2 structure. The kinase binds to one of tw o lipoyl domains, and the E1 component binds to a domain in E2''s stru cture 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 kina se activity. This bi-lipoyl domain fragment induced a 2-fold enhanceme nt in the slow rate of phosphorylation of peptide substrate and intact E260 gave only a 50% higher rate. In contrast, the lipoyl domain frag ment gave only a 40% enhancement in the faster rate of phosphorylation of E1; whereas the rate of phosphorylation of E1 was markedly increas ed (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-l ipoyl domain region did not enhance kinase activity. Thus, binding of the kinase to the lipoyl domain region elicits a structural change whi ch enhances kinase activity; however, other processes are required to explain the very large enhancement in phosphorylation of E1 effected b y intact E2 core. Among the latter is a need for a mechanism allowing one kinase molecule to phosphorylate many E1 tetramers, whereas both E 1 and the kinase stay bound to the oligomeric E2 core (i.e. phosphoryl ation appears to be much faster than the dissociation of either the ki nase 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 direc tion, an increasing level of the free bi-lipoyl domain fragment rapidl y reduced, in a concentration-dependent manner, the activity of kinase bound initially to intact E2. The data strongly support kinase transf er 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 thes e E2 structures was confirmed by sucrose gradient studies. Our results are consistent with movement of the tightly bound kinase between lipo yl domains and suggest the possibility of rapid movement by direct kin ase transfer (i.e. without an intermediate step of kinase dissociation ).