Assembly and Full Functionality of Recombinantly Expressed Dihydrolipoyl Acetyltransferase Component of the Human Pyruvate Dehydrogenase Complex

Abstract

The dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) consists of 60 COOH-terminal domains as an inner assemblage and sequentially via linker regions an exterior pyruvate dehydrogenase (E1) binding domain and two lipoyl domains. Mature human E2, expressed in a protease-deficient Escherichia coli strain at 27°, was prepared in a highly purified form. Purified E2 had a high acetyltransferase activity, was well lipoylated based on its acetylation, and bound a large complement of bovine E1. Electron micrographs demonstrated that the inner core was assembled in the expected pentagonal dodecahedron shape with E1 binding around the inner core periphery.With saturating E1 and excess dihydrolipoyl dehydrogenase (E3) but no E3-binding protein (E3BP), the recombinant E2 supported the overall PDC reaction at 4% of the rate of bovine E2⅐E3BP subcomplex. The lipoates of assembled human E2 or its free bilipoyl domain region were reduced by E3 at rates proportional to the lipoyl domain concentration, but those of the E2⅐E3BP were rapidly used in a concentration-independent manner consistent with bound E3 rapidly using a set of lipoyl domains localized nearby. Given this restriction and the need for E3BP for high PDC activity, directed channeling of reducing equivalents to bound E3 must be very efficient in the complex.The recombinant E2 oligomer increased E1 kinase activity by up to 4-fold and, in a Ca 2؉ -dependent process, increased phospho-E1 phosphatase activity more than 15-fold. Thus the E2 assemblage fully provides the molecular intervention whereby a single E2-bound kinase or phosphatase molecule rapidly phosphorylate or dephosphorylate, respectively, many E2-bound E1. Thus, we prepared properly assembled, fully functional human E2 that mediated enhanced regulatory enzyme activities but, lacking E3BP, supported low PDC activity.Pyruvate dehydrogenase complexes from various sources are among the largest enzyme systems that serve strategic roles in metabolism (1, 2). The mammalian complex has a highly organized structure in which the dihydrolipoyl transacetylase (E2) 1 component has a central role in the organization and integrated chemical reactions of the complex, and supports enhanced functioning of dedicated kinase and phosphatase components (3-8). The other components of the mammalian complex required for the overall reaction are: the pyruvate dehydrogenase (E1) component, an ␣ 2 ␤ 2 tetramer present at 20 -30 copies (1); the dihydrolipoyl dehydrogenase (E3), a homodimer present in about 6 copies (9); and the E3-binding protein (E3BP) estimated at 6 -12 copies (10, 11). Dedicated and highly regulated kinase and phosphatase components control the conversion of E1 between an active (nonphosphorylated) form, E1a, and an inactive (phosphorylated) form, E1b.Mammalian PDC-E2 subunits have four flexibly connected domains and form the core of the complex in which 60 of its COOH-terminal inner (I) domains assemble into a dodecahedron-shaped structure with its other three domains ...

The Dihydrolipoamide S-Acetyltransferase Subunit of the Mitochondrial Pyruvate Dehydrogenase Complex from Maize Contains a Single Lipoyl Domain

“…The nearly homogeneous E2 had a specific activity of 191 ⌬A 232 min Ϫ1 mg Ϫ1 , considerably higher than the specific activity observed with purified recombinant human E2 (19.4 ⌬A 232 min Ϫ1 mg Ϫ1 ; Ref. 34). The purified maize E2 consisted of 52-, 53-, and 54-kDa polypeptides at a ratio of approximately 2:2:1, almost identical to the polypeptide pattern for native maize E2.…”

“…The formation of acetyldihydrolipoamide was measured by the ⌬A 232 for 1 min at 25°C. Background rates, without E2 (Ͻ5% of the actual rate), were observed and stabilized after approximately 20 s. The extinction coefficient for the acetyldihydrolipoamide ester bond is 4400 M Ϫ1 cm Ϫ1 ; however, rates were expressed as ⌬A 232 due to the potential for formation of undetermined amounts of biacetylated dihydrolipoamide (34).…”

Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase

“…Materials-Recombinant human E2, K46AE2, and K173AE2 were prepared as described previously (37,46). Recombinant human E2-E3BP was prepared as described elsewhere.…”

Immunoglobulin gene usage and immunohistochemical characteristics of human monoclonal antibodies to the mitochondrial autoantigens of primary biliary cirrhosis induced in the XenoMouse

“…The purification of PDC-E2 from beef heart mitochondria was isolated by polyethylene glycol precipitation. 12 Recombinant (r) PDC-E2, full-length, and rPDC-E2-inner lipoyl domain (rPDC-E2-ILD), rOGDC-E2, rBCOADC-E2, rE3BP, and r-MIT3 (TRIPLE HYBRID) were prepared as described previously. [13][14][15] Keyhole limpet hemocyanin (KLH) was used as a control antigen.…”