Requirements for the Adaptor Protein Role of Dihydrolipoyl Acetyltransferase in the Up-regulated Function of the Pyruvate Dehydrogenase Kinase and Pyruvate Dehydrogenase Phosphatase
1998; Elsevier BV; Volume: 273; Issue: 23 Linguagem: Inglês
10.1074/jbc.273.23.14130
ISSN1083-351X
AutoresDa‐Qing Yang, Xiaoming Gong, Alexander V. Yakhnin, Thomas E. Roche,
Tópico(s)Glycogen Storage Diseases and Myoclonus
ResumoThe dihydrolipoyl acetyltransferase (E2 component) is a 60-mer assembled via its COOH-terminal domain with exterior E1-binding domain and two lipoyl domains (L2 then L1) sequentially connected by mobile linker regions. E2 facilitates markedly enhanced function of the pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). Human E2 structures were prepared with only one lipoyl domain (L1 or L2) or with alanines substituted at the sites of lipoylation (Lys-46 in L1 or Lys-173 in L2). The L2 domain and its lipoyl group were shown to be essential for markedly enhanced PDP function and were required for greatly up-regulated PDK function. The complete absence of the L1 domain reduced the enhancements of both of these activities but not the maximal effector-stimulated PDK activity through acetylation of L2. With nonlipoylated L2 present, lipoylated L1 supported a lesser enhancement in PDK function with significant stimulation upon acetylation of L1. Prevention of L1 lipoylation in K46AE2 removed this competitive L1 role and enhanced L2-facilitated PDK activity beyond that of native E2 when PDK activity was measured in the absence or in the presence of stimulatory effectors. Thus, the E2-L2 domain has a paramount role in facilitating enhanced PDK and PDP function but inclusion of E2-L1 domain, even in a noninteracting (nonlipoylated) form, contributes to the marked elevation of these activities. The dihydrolipoyl acetyltransferase (E2 component) is a 60-mer assembled via its COOH-terminal domain with exterior E1-binding domain and two lipoyl domains (L2 then L1) sequentially connected by mobile linker regions. E2 facilitates markedly enhanced function of the pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). Human E2 structures were prepared with only one lipoyl domain (L1 or L2) or with alanines substituted at the sites of lipoylation (Lys-46 in L1 or Lys-173 in L2). The L2 domain and its lipoyl group were shown to be essential for markedly enhanced PDP function and were required for greatly up-regulated PDK function. The complete absence of the L1 domain reduced the enhancements of both of these activities but not the maximal effector-stimulated PDK activity through acetylation of L2. With nonlipoylated L2 present, lipoylated L1 supported a lesser enhancement in PDK function with significant stimulation upon acetylation of L1. Prevention of L1 lipoylation in K46AE2 removed this competitive L1 role and enhanced L2-facilitated PDK activity beyond that of native E2 when PDK activity was measured in the absence or in the presence of stimulatory effectors. Thus, the E2-L2 domain has a paramount role in facilitating enhanced PDK and PDP function but inclusion of E2-L1 domain, even in a noninteracting (nonlipoylated) form, contributes to the marked elevation of these activities. The mammalian pyruvate dehydrogenase complex (PDC) 1The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase component; E2, dihydrolipoyl acetyltransferase component; L1, NH2-lipoyl domain of E2; L2, interior lipoyl domain of E2; B, E1 binding domain of E2; I, oligomer-forming, transacetylase-catalyzing COOH-terminal inner domain of E2; H1, H2, and H3, connecting hinge (or linker regions) sequentially located between the globular domains of E2; L1E2, E2 oligomer lacking L2 domain; L2E2, E2 oligomer lacking L1 domain; PDK, pyruvate dehydrogenase kinase; PDK1, PDK2, PDK3, and PDK4, PDK isozymes; E3, dihydrolipoyl dehydrogenase; E3BP, E3-binding protein (formerly protein X); PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase component; E2, dihydrolipoyl acetyltransferase component; L1, NH2-lipoyl domain of E2; L2, interior lipoyl domain of E2; B, E1 binding domain of E2; I, oligomer-forming, transacetylase-catalyzing COOH-terminal inner domain of E2; H1, H2, and H3, connecting hinge (or linker regions) sequentially located between the globular domains of E2; L1E2, E2 oligomer lacking L2 domain; L2E2, E2 oligomer lacking L1 domain; PDK, pyruvate dehydrogenase kinase; PDK1, PDK2, PDK3, and PDK4, PDK isozymes; E3, dihydrolipoyl dehydrogenase; E3BP, E3-binding protein (formerly protein X); PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. has a strategic role in controlling the oxidative consumption of glucose (1Randle P.J. Priestman D.A. Patel M.S. Roche T.E. Harris R.A. α-Keto Acid Dehydrogenase Complexes. Birkhäuser Verlag, Basel1996: 151-161Google Scholar). To limit consumption of body carbohydrate reserves, PDC activity is controlled by an intricately regulated cycle carried out by dedicated kinase and phosphatase components. PDC activity is reduced due to phosphorylation of the pyruvate dehydrogenase (E1) tetramers and increased by production of nonphosphorylated tetramers. Phosphorylation proceeds in a kinetically preferred order at three sites on the α subunit of E1 (2Yeaman S.J. Hutcheson E.T. Roche T.E. Pettit F.H. Brown J.R. Reed L.J. Watson D.C. Dixon G.H. Biochemistry. 1978; 17: 2364-2370Crossref PubMed Scopus (203) Google Scholar, 3Sale G.J. Randle P.J. Biochem. J. 1982; 120: 535-540Google Scholar), an α2β2 structure; however, incorporation of a phosphate into each site is capable of causing inactivation (4Korotchkina L.G. Patel M.S. J. Biol. Chem. 1995; 270: 14297-14304Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The dihydrolipoyl acetyltransferase (E2) component forms the structural core of the complex. It consists of four independently folded domains set apart from each other by interdomain linker (or hinge) regions, each having substantial reach (>40 Å) and high mobility (Fig.1, E2). The largest domain, located at the COOH terminus, forms a catalytically active trimer which assembles at the 20 vertices of a pentaganol dodecahedron to form a 60-mer inner core structure with icosahedral symmetry. Then a flexible segment (H3) connects to an E1-binding domain followed by two lipoyl-bearing domains (L2 and then L1 at the NH2 terminus) sequentially connected by two more flexible hinge regions (H2 and H1). In the outer surface of the E260 structure, these mobile, multisegment NH2-terminal structures intercede in dynamic processes associated with catalytic transfers and regulatory interconversions (5Perham R.N. Patel M.S. Roche T.E. Harris R.A. α-Keto Acid Dehydrogenase Complexes. Birkhäuser Verlag, Basel1996: 1-15Google Scholar, 6Roche T.E. Liu J. Ravindran S. Baker J. Wang D. Patel M.S. Roche T.E. Harris R.A. α-Keto Acid Dehydrogenase Complexes. Birkhäuser Verlag, Basel1996: 33-52Google Scholar). Here we further define the latter adaptor protein roles in the regulation of PDC. Bovine kidney pyruvate dehydrogenase kinase (PDK) activity and pyruvate dehydrogenase phosphatase (PDP) activity are greatly enhanced in the presence of E2. Furthermore, E2 mediates acetyl-CoA and NADH stimulation of PDK activity and facilitates Ca2+stimulation of PDP activity (6Roche T.E. Liu J. Ravindran S. Baker J. Wang D. Patel M.S. Roche T.E. Harris R.A. α-Keto Acid Dehydrogenase Complexes. Birkhäuser Verlag, Basel1996: 33-52Google Scholar). These regulatory inputs constitute important and sensitive response mechanisms in the control of cellular energy metabolism. The marked reduction in PDC activity due to elevated NADH:NAD+ and acetyl-CoA:CoA ratios stimulating PDK activity is a strategic response resulting from increased fatty acid oxidation and serves to preserve body carbohydrate stores (1Randle P.J. Priestman D.A. Patel M.S. Roche T.E. Harris R.A. α-Keto Acid Dehydrogenase Complexes. Birkhäuser Verlag, Basel1996: 151-161Google Scholar, 6Roche T.E. Liu J. Ravindran S. Baker J. Wang D. Patel M.S. Roche T.E. Harris R.A. α-Keto Acid Dehydrogenase Complexes. Birkhäuser Verlag, Basel1996: 33-52Google Scholar). To meet transitional energy needs, PDC activity is increased due to elevation of intramitochondrial Ca2+ in association with a wide variety of signal transduction cascades (7Denton R.M. McCormack J.G. Annu. Rev. Physiol. 1990; 52: 451-466Crossref PubMed Scopus (266) Google Scholar). Increasing Ca2+ from 1.5 μmcan enhance PDP activity more than 10-fold (8Denton R.M. Randle P.J. Martin B.R. Biochem. J. 1972; 128: 161-163Crossref PubMed Scopus (325) Google Scholar). In dissecting the intercession of E2 in these regulatory mechanisms, we have used recombinant lipoyl domain constructs to establish that the L2 domain of E2 has a crucial role in these processes (9Liu S. Baker J.C. Roche T.E. J. Biol. Chem. 1995; 270: 793-800Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11Chen G. Wang W. Liu S. Chang C. Roche T.E. J. Biol. Chem. 1996; 271: 28064-28070Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). L2 preferentially binds PDK through an interaction that requires the lipoyl cofactor of L2 (9Liu S. Baker J.C. Roche T.E. J. Biol. Chem. 1995; 270: 793-800Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar); we have suggested that this is critical to E2 activation of PDK activity. Effector stimulation of bovine kidney PDK by NADH and acetyl-CoA ensues from using these products in reducing and acetylating, respectively, the L2 lipoyl prosthetic group (10Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Reduction and acetylation are sequentially catalyzed by the reverse of the dihydrolipoyl dehydrogenase (E3) and E2 components. Additionally, the L2 domain exclusively binds PDP through a Ca2+-dependent interaction, and we have suggested that this is critical to stimulation of PDP in response to increased Ca2+ (11Chen G. Wang W. Liu S. Chang C. Roche T.E. J. Biol. Chem. 1996; 271: 28064-28070Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This marked enhancement in PDP activity only occurs when E2 retains its lipoyl prosthetic groups. Although interaction of PDK and PDP with L2 and effector stimulation of PDK by NADH and acetyl-CoA have been demonstrated with the isolated lipoyl domain, the marked activations of PDK and PDP function elicited by the E2–60-mer are not achieved with the isolated domain. It is now known that there are at least 4 PDK isozymes (PDK1, PDK2, PDK3, and PDK4) (12Popov K.M. Kedishvili N.Y. Zhao Y. Shimomura Y. Crabb D.W. Harris R.A. J. Biol. Chem. 1993; 268: 26602-26606Abstract Full Text PDF PubMed Google Scholar, 13Popov K.M. Kedishvili N.Y. Zhao Y. Guidi R. Harris R.A. J. Biol. Chem. 1994; 269: 29720-29724Abstract Full Text PDF PubMed Google Scholar, 14Popov K.M. Zhao Y. Shimomura Y. Kuntz M.J. Harris R.A. J. Biol. Chem. 1992; 267: 13127-13130Abstract Full Text PDF PubMed Google Scholar, 15Rowles J. Scherer S.W. Xi T. Majer M. Nickle D.C. Rommens J.M. Popov K. Harris R.A. Riebow N.L. Xia J. Tsui L.-C. Bogardus C. Prochazka M. J. Biol. Chem. 1996; 271: 22376-22382Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In work in progress, 2J. C. Baker, J. Dong, and T. E. Roche, unpublished observations. 2J. C. Baker, J. Dong, and T. E. Roche, unpublished observations. we have developed unique preparations of the human isozymes and are characterizing the capacity of these recombinantly produced PDK isozymes to undergo activation by E2. Considerable isozyme variability has been observed; we are evaluating whether some unexpected outcomes reflect normal isozyme properties or are an artifact due to the physical state of the recombinantly prepared kinase isozymes. 3PDK3 is markedly activated (>10-fold) by E2, and this is a stable response; PDK1 and PDK2 are activated up to 4-fold by E2 when first prepared, but these responses fade with time. 3PDK3 is markedly activated (>10-fold) by E2, and this is a stable response; PDK1 and PDK2 are activated up to 4-fold by E2 when first prepared, but these responses fade with time. Thus, the present studies are conducted with bovine kidney kinase. Recombinantly produced human E2 (free of tightly bound E3BP) provides high activation of bovine PDK and PDP activity (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar). Here, we have prepared and evaluated the capacity of reconstructed E2 assemblages modified by selective deletion of one lipoyl domain or by mutation at the site of lipoylation for their capacity to support enhanced PDK and PDP activity and mediate effector stimulations (Fig. 1). The results further support critical roles of the L2 domain in activated PDK and PDP function. Conversion of the lysines that undergo lipoylation to alanines provides additional support for an essential role of lipoyl cofactor of L2 in both PDK and PDP function. The Lys to Ala mutation is less drastic than delipoylation (9Liu S. Baker J.C. Roche T.E. J. Biol. Chem. 1995; 270: 793-800Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11Chen G. Wang W. Liu S. Chang C. Roche T.E. J. Biol. Chem. 1996; 271: 28064-28070Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), a transition from a hydrophobic lipoyl lysine to a positively charged lysyl side chain. The results also indicate some role for the L1 domain but not for the L1 lipoyl group for E2 assemblages maximally enhancing PDK and PDP activity. However, NADH and acetyl-CoA stimulated PDK activity to the maximal extent with the E2 assemblage lacking the L1 domain suggesting the selective interaction of acetylated L2 with bovine PDK markedly alters its function. Bovine kidney PDC, E1 component, E2·E3BP·PDK subcomplex, the recombinant bilipoyl domain region of human E2, and one form of full sized human E2–60-mer were prepared as described previously (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar, 17Roche T.E. Cate R.L. Arch. Biochem. Biophys. 1977; 183: 664-677Crossref PubMed Scopus (67) Google Scholar, 18Linn T.C. Pelley J.W. Pettit F.H. Hucho F. Randall D.D. Reed L.J. Arch. Biochem. Biophys. 1972; 148: 327-342Crossref PubMed Scopus (193) Google Scholar). Porcine heart E3 was from Boehringer Mannheim or Sigma. Pfu DNA polymerase was from Stratagene Inc. DNA oligonucleotides used for plasmid construction were made by Eppendorf. Primers used for PCR reaction or sequencing were from Oligos Etc. or Biotechnology Core Facility at Kansas State University. T4 DNA ligase and BamHI were from Promega Corp.; other restriction enzymes were from New England Biolabs. Other materials used are the same as those described previously (9Liu S. Baker J.C. Roche T.E. J. Biol. Chem. 1995; 270: 793-800Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 11Chen G. Wang W. Liu S. Chang C. Roche T.E. J. Biol. Chem. 1996; 271: 28064-28070Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar, 19Liu S. Baker J.C. Andrews P.C. Roche T.E. Arch. Biochem. Biophys. 1995; 316: 926-940Crossref PubMed Scopus (29) Google Scholar). Polymerase chain reaction (PCR) was performed according to Innis et al. (20Innis M.A. Gelfand D.H. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protein: A Guide to Methods and Applications. Academic Press, San Diego1990: 3-12Google Scholar) with a GeneAmp PCR System 2400 thermocycler from Perkin-Elmer. Primers (200 pmol) having about 50–60% G:C content (Tms >45 °C) were reacted with 1.5 ng of purified template DNA, 200 μm dNTPs, 2.5 IUsPfu DNA polymerase in Pfu buffer (Stratagene). The reaction mixtures, overlaid with 50 μl of mineral oil, were denatured initially and in each cycle for 1 min at 95 °C, reacted for 20 to 30 cycles with 0.5 min annealing at 55 °C, and 0.5 min extension at 72 °C with the final extension reaction proceeding for 2 min. As diagramed on the right side of Fig.2, a cDNA fragment coding for L1 was amplified by PCR with pShE2 plasmid (codes for mature human E2 with the E2 leader sequence removed and a start Met inserted (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar)) as a template using 5′-CATCCATGGGTAGTCTTCCCCCGCATC-3′ (sense) and 5′-GATCGGCCGAGGAATCCAGTGTAT-3′ (antisense) as primers. This introduced flanking NcoI and EagI sites (compatible with EaeI site) at the 5′- and 3′-ends (sense direction), respectively. The DNA amplified from PCR was digested byNcoI and EagI, purified, and ligated to 1-kilobase pair DNA fragment purified after digestion of pShE2 plasmid with EaeI and BamHI. The resulting DNA fragment coding for L1E2 (Fig. 1) was ligated to pSE420 vector previously digested by NcoI and BamHI to produce pShL1E2. DNA sequencing was performed for the region produced by PCR and flanking ligation sites. As shown on the left side of Fig. 2, a segment including the 5′-region coding for L1 portion of E2 was removed from pShE2 vector by digestion with BspMI in combination with BamHI digestion to produce a 1.3-kilobase pair cDNA fragment. This fragment was ligated to a hybrid of two oligonucleotides that include 16 base pairs encoding the NH2 terminus of L2 plus two other amino acids (Gly and Ser) prior to start codon. This spliced fragment was subcloned into the pSE420 vector previously digested byNcoI and BamHI. The resultant expression vector, pShL2E2 (Fig. 2, left side), encoded L2E2 (Fig. 1). The DNA derived from the synthetic oligonucleotides along with their adjoining ligation sites were sequenced in pShL1E2 and pShL2E2. pShL1E2 and pShL2E2 plasmids were introduced into BL21(DE3) strains by electroporation using the Transfector 300 BTX. Expression of pShL1E2 and pShL2E2 was carried out as described previously for pShE2 to produce E2 (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar). Expression of each of the modified E2 subunits, bearing only one E2 lipoyl domain, was analyzed by dot blotting and Western blotting techniques using lipoyl domain-specific monoclonal antibodies with 150.2 for detecting L1E2, 157.2 plus 315.2 for detecting L2E2 protein, and horseradish peroxidase-conjugated goat anti-mouse IgG (H + L) as the second antibody under conditions previously described (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar, 19Liu S. Baker J.C. Andrews P.C. Roche T.E. Arch. Biochem. Biophys. 1995; 316: 926-940Crossref PubMed Scopus (29) Google Scholar). All steps were performed at 4 °C. Cells were resuspended in 50 mm potassium phosphate buffer, pH 7.2, containing 0.5 mm EDTA, 1 μg/ml aprotinin, and 1 μg/ml leupeptin (buffer A) and then disrupted by sonication. Cell debris was removed by centrifugation (15,000 ×g for 20 min). PEG-8000 was added dropwise to 8% (v/v), and the precipitated protein was recovered by centrifugation (20,000 × g for 20 min). The pellet was in a cloudy state after being resuspended in buffer A. Upon addition of (NH4)2SO4 to 9% saturation, substantial clearing occurred; after ∼20 min, the material still suspended was precipitated and discarded. Addition of (NH4)2SO4 was continued until the concentration of (NH4)2SO4 reached 25% saturation. The precipitated protein was pelleted at 20,000 × g for 20 min and redissolved in 50 mmpotassium phosphate buffer, pH 7.2, containing 0.2 mm EDTA, 1 μg/ml aprotinin, and 1 μg/ml leupeptin. Aliquots of purified samples were stored frozen at −80 °C. The protein concentration was measured by the BCA method. The E2 activity at each fraction step was followed by measuring the PTA activity (21Ono K. Radke G.A. Roche T.E. Rahmatullah M. J. Biol. Chem. 1993; 268: 26135-26143Abstract Full Text PDF PubMed Google Scholar), and the protein pattern was analyzed by SDS-PAGE (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206615) Google Scholar). NH2-terminal sequencing of SDS-PAGE-separated protein bands was conducted as described previously (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar, 19Liu S. Baker J.C. Andrews P.C. Roche T.E. Arch. Biochem. Biophys. 1995; 316: 926-940Crossref PubMed Scopus (29) Google Scholar) with thioglycolate increased in the sample loading buffer to 15 mm. The ratio of the different protein bands was analyzed by scanning the band patterns for Coomassie Blue R-250 and silver-stained gels following SDS-PAGE separation on samples from the preparations of L1E2 or L2E2. An Ittis Dos Program was used to do the image density analysis and analyze the intensity ratios. As will be described in detail elsewhere, 4A. Yakhnin, X. Gong, X. Yan, M. P. Sadler, and T. E. Roche, manuscript in preparation. a variety of lipoyl domain mutants has been prepared and tested alone and incorporated into E2 oligomers. Expression vectors for mature E2 and for glutathione S-transferase fused L1 or L2 and have been designed with silent restriction sites to permit transfer of cDNA fragments encoding L1 or L2 mutants from vectors expressing glutathioneS-transferase-lipoyl domains to vectors expressing these mutant domains in whole E2 structures. DNA fragments, encoding K46A-modified L1 or K173A-modified L2, were introduced by this approach into E2 structures. The modified cDNA inserts expressing whole E2 have also been modified to include a removable His tag at the NH2terminus. The E2 assemblages are purified to >98% purity by a two-step procedure which involves fractionation with polyethylene glycol and gel filtration chromatography which is immediately preceded by removal of the His tag.4 The introduction of the His tag greatly increased the recovery of E2 by improving solubility of E2 assemblages and reduced the presence of truncated E2 subunits, probably by reducing the tendency of a codon for Met in the L1 domain operating as an internal start site (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar). E2, K43AE2, and K173AE2 prepared by this approach were used for studies shown in panel B of Figs. Figure 5, Figure 6, Figure 7. Other properties of these and several other constructs developed will be described elsewhere.4 To evaluate E1 binding to the truncated or full-sized human E2 constructs, ∼20 μg of L1E2 or L2E2 subunit with or without E1 (20 μg) was incubated at 4 °C for 120 min. After incubation, the above mixtures and the control E1 sample were each loaded onto the top of a three-step sucrose gradient, and gradient separation was carried out as described (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar,23Powers-Greenwood S.L. Rahmatullah M. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 3655-3657Abstract Full Text PDF PubMed Google Scholar). SDS-PAGE analysis with silver staining (24Oakley B.R. Kirsch D.R. Morris N.R. Anal. Biochem. 1980; 105: 361-363Crossref PubMed Scopus (2442) Google Scholar) was conducted as described in Fig. 3. E2 activity was determined spectrophotometrically by measuring acetyl-dihydrolipoamide production at 232 nm. The acetylation capacities for E2, L1E2, and L2E2 (50 pmol) were determined by E3-catalyzed reduction of lipoyl groups followed by E2-catalyzed acetylation using [1-14C]acetyl-CoA (11.35 cpm/pmol) under conditions of CoA conversion to succinyl-CoA as described previously (10Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar, 25Rahmatullah M. Roche T.E. J. Biol. Chem. 1985; 260: 10146-10152Abstract Full Text PDF PubMed Google Scholar). Reconstituted PDC activity (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar) was measured using 75 pmol of the human constructs (E2, L1E2, or L2E2) or bovine E2-E3BP combined with excess E1 (2 μg/μg E2) and high E3 (1 μg/μg E2). 5Porcine E3 is used for comparison to our previous data with recombinant human E2 (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar). Lindsay and co-workers (26Sanderson S.J. Khan S.S. McCartney R.G. Miller C. Lindsay J.G. Biochem. J. 1996; 319: 109-116Crossref PubMed Scopus (31) Google Scholar, 27McCartney R.G. Sanderson S.J. Lindsay J.G. Biochemistry. 1997; 36: 6812-6819Crossref Scopus (26) Google Scholar) have indicated that, in the absence of the E3BP component, very high levels of bovine E3 (e.g. 100-fold excess) are more effective than similarly high levels of porcine E3 in supporting reconstituted bovine PDC activity. They suggest weak binding of bovine E3 to E2 may occur. The rate of utilization of lipoyl groups in a cyclic E3 reaction (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar, 19Liu S. Baker J.C. Andrews P.C. Roche T.E. Arch. Biochem. Biophys. 1995; 316: 926-940Crossref PubMed Scopus (29) Google Scholar) was measured using 150 pmol of L1E2, L2E2, or E2. PDK activity, in the absence of effectors, was determined as described previously (16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar, 28Li L. Radke G.A. Ono K. Roche T.E. Arch. Biochem. Biophys. 1992; 296: 497-504Crossref PubMed Scopus (9) Google Scholar) after the E2 constructs were incubated at 4 °C for about 120 min (domain truncated E2) or 20 min (mutated E2) to maximize binding of E1 and increase the solubility, particularly of L1E2 and L2E2. 25–30 μg of E1 and the molar levels of E2 construct indicated were added to PDK reaction mixtures. To evaluate maximal PDK activities, assays were conducted in the presence of 20 mm potassium phosphate (condition 1); to evaluate regulatory effects or otherwise assay PDK activity at a near-physiological level of K+, assays were conducted in the presence of 50 mm MOPS-K, pH 7.5, 20 mm potassium phosphate, pH 7.5, 60 mm KCl (condition 2). Under both reaction conditions, assays additionally contained 1 mm MgCl2, 1 mmdithiothreitol, 0.05 mm EDTA, 0.2% Pluronic-F-68, and 0.2% Triton X-100. Reactions were started by addition of [γ-32P]ATP (∼3 × 105 cpm/pmol) to give a final volume of 50 μl and then terminated and worked up as described previously (28Li L. Radke G.A. Ono K. Roche T.E. Arch. Biochem. Biophys. 1992; 296: 497-504Crossref PubMed Scopus (9) Google Scholar, 29Rahmatullah M. Roche T.E. J. Biol. Chem. 1987; 262: 10265-10271Abstract Full Text PDF PubMed Google Scholar). For evaluating effects of NADH and acetyl-CoA on PDK activity (10Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), assays (condition 2) also included 1 μg of E3 and, when indicated, 0.6 mm NADH plus 0.2 mm NAD+ and 50 μm acetyl-CoA. PDP activity was determined as described previously (11Chen G. Wang W. Liu S. Chang C. Roche T.E. J. Biol. Chem. 1996; 271: 28064-28070Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 16Yang D. Song J. Wagenknecht T. Roche T.E. J. Biol. Chem. 1996; 272: 6361-6369Abstract Full Text Full Text PDF Scopus (32) Google Scholar) except that 0.4 mg/ml bovine serum albumin was included along with 2 mg/ml Pluronic-F-68 in reaction mixtures, and 14–17 μg of E1b was included in each assay. In studies involving L1E2 and L2E2, E2 sources were preincubated with E1b at 4 °C for about 120 min before the activity assays, while only 20 min was used with mutated E2 structures. By using the domain codes in Fig. 1 and the approaches outlined under "Experimental Procedures" and Fig. 2, the region coding for the H1-L2 was removed along with reconnecting the coding region for the L1 domain to yield a vector, pShL1E2 (Fig. 2,right side) expressing L1E2 (Fig. 1). L1E2 has one extra amino acid, a glycine at the NH2 terminus following the start Met codon, and was designed with residues 233–240 of H2 hinge region removed so that L1 was connected after Ser-98 to a hinge region, H2′, which starts with an Ala-Ala sequence (residues 241 and 242 of E2). This produced a transition similar to that between L1 and the beginning of H1; H2′ is still a hinge region over 20 residues in length. Similarly, the coding region for L1-H1 was removed from pShE2 expression vector for E2 to produce a vector, pShL2E2 (Fig. 2,left side), coding for L2E2 (Fig. 1). The last two residues of H1 region (Gly-126 and Ser-127) were retained following the start Met in L2E2. The accuracy of the cDNA inserts was confirmed by restriction enzyme digestion and DNA sequencing. Western blotting confirmed that L1E2 or L2E2 expressed inEsc
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