ASSEMBLY AND FULL FUNCTIONALITY OF RECOMBINANTLY EXPRESSED DIHYDROLIPOYL ACETYLTRANSFERASE COMPONENT OF THE HUMAN PYRUVATE-DEHYDROGENASE COMPLEX

The dihydrolipoyl acetyltransferase (E2) component of mammalian pyruva te dehydrogenase complex (PDC) consists of 60 COOH-terminal domains as an inner assemblage and sequentially via linker regions an exterior p yruvate dehydrogenase (E1) binding domain and two lipoyl domains, Matu re human E2, expressed in a protease-deficient Escherichia coli strain at 27 degrees, was prepared in a highly purified form, Purified E2 ha d a high acetyltransferase activity, was well lipoylated based on its acetylation, and bound a large complement of bovine E1. Electron micro graphs demonstrated that the inner core was assembled in the expected pentagonal dodecahedron shape with El binding around the inner core pe riphery, With saturating E1 and excess dihydrolipoyl dehydrogenase (E3 ) but no E3-binding protein (E3BP), the recombinant E2 supported the o verall PDC reaction at 4% of the rate of bovine E2 E3BP subcomplex, Th e lipoates of assembled human E2 or its free bilipoyl domain region we re reduced by E3 at rates proportional to the lipoyl domain concentrat ion, but those of the E2 E3BP were rapidly used in a concentration-ind ependent manner consistent with bound E3 rapidly using a set of lipoyl domains localized nearby, Given this restriction and the need for E3B P for high PDC activity, directed channeling of reducing equivalents t o bound E3 must be very efficient in the complex, The recombinant E2 o ligomer increased E1 kinase activity by up to 4-fold and, in a Ca2+-de pendent process, increased phospho-E1 phosphatase activity more than 1 5-fold. Thus the E2 assemblage fully provides the molecular interventi on whereby a single E2-bound kinase or phosphatase molecule rapidly ph osphorylate or dephosphorylate, respectively, many E2-bound E1. Thus, we prepared properly assembled, fully functional human E2 that mediate d enhanced regulatory enzyme activities but, lacking E3BP, supported l ow PDC activity.