From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506
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ABSTRACT |
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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.
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INTRODUCTION |
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The mammalian pyruvate dehydrogenase complex
(PDC)1 has a strategic role
in controlling the oxidative consumption of glucose (1). 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 subunit of E1
(2, 3), an
2
2 structure; however,
incorporation of a phosphate into each site is capable of causing
inactivation (4).
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 (5, 6). Here we further define the latter adaptor protein roles in the regulation of PDC.
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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 (6). 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 (1, 6). 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 (7). Increasing Ca2+ from <0.1 µM to >1.5 µM can enhance PDP activity more than 10-fold (8).
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 (9-11). L2 preferentially binds PDK through an interaction that requires the lipoyl cofactor of L2 (9); 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 (10). 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+ (11). 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) (12-15). In work in progress,2 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.3 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 (16). 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 (9-11), 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.
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EXPERIMENTAL PROCEDURES |
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Materials-- 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 (16-18). 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 (9-11, 16, 19).
Polymerase Chain Reaction-- Polymerase chain reaction (PCR) was performed according to Innis et al. (20) 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 IUs Pfu 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.
Construction of the Expression Vectors for L1E2 and L2E2-- 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 (16)) 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 by NcoI 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.
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Production of L1E2 and L2E2-- 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 (16). 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 (16, 19).
Purification of L1E2 and L2E2-- 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 mM potassium 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 atProduction of K46AE2 and K173AE2-- As will be described in detail elsewhere,4 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 glutathione S-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 NH2 terminus. 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 (16). E2, K43AE2, and K173AE2 prepared by this approach were used for studies shown in panel B of Figs. 5-7. Other properties of these and several other constructs developed will be described elsewhere.4Binding of Bovine E1 to L1E2 and L2E2-- 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 (16, 23). SDS-PAGE analysis with silver staining (24) was conducted as described in Fig. 3.
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E2, E3, and PDC Activities and Capacity for Acetylation of L1E2 and L2E2-- 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 (10, 16, 25). Reconstituted PDC activity (16) 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).5 The rate of utilization of lipoyl groups in a cyclic E3 reaction (16, 19) was measured using 150 pmol of L1E2, L2E2, or E2.
Kinase Activation and Regulation--
PDK activity, in the
absence of effectors, was determined as described previously (16, 28)
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 mM
dithiothreitol, 0.05 mM EDTA, 0.2% Pluronic-F-68, and
0.2% Triton X-100. Reactions were started by addition of
[-32P]ATP (~3 × 105 cpm/pmol) to
give a final volume of 50 µl and then terminated and worked up as
described previously (28, 29). For evaluating effects of NADH and
acetyl-CoA on PDK activity (10), 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.
Phosphatase Activation-- PDP activity was determined as described previously (11, 16) 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.
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RESULTS AND DISCUSSION |
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Expression and Purification of E2 Oligomers with One Lipoyl Domain-- 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 in Escherichia coli strain BL-21 reacted selectively with L1- or L2-specific monoclonal antibodies (19), respectively, that the single lipoyl domain-containing E2 structures had about the expected mobilities, and that at least 70% of L2E2 and a lower portion (~50%) of L1E2 remained in the soluble fraction upon centrifugation of cell lysates. By using the purification steps described under "Experimental Procedures," nearly homogeneous L1E2 (Mr = 48.4 ± 1.5) and L2E2 (Mr = 51.2 ± 1.5) were obtained as indicated by their silver-stained patterns following SDS-PAGE separation (Fig. 3). The observed molecular weights are somewhat larger than calculated molecular weight values of 44.4 and 46.2 for L1E2 and L2E2, respectively, as is expected due to hinge regions causing E2 structures to run anomalously slow (30, 31). The recombinant L2E2 was recovered completely intact. However, as occurred in the recombinant production of whole E2 (Ref. 16 and Fig. 3, lane 5), two of the bands in the L1E2 preparation were determined by NH2-terminal sequencing to be truncated products (within L1 (E2") in Fig. 3 and just before the I domain in H3 (E2I) in Fig. 3). Since these partial structures retain the inner domain, they can participate in the L1E2 assemblage and contribute to acetyltransferase activity. NH2-terminal sequencing of L1E2 gave a mixed sequence that fit about 50% each of MGSLPPHQK and GSLPPHQK, indicating about half the start Met residue was retained. L2E2 only gave a GSSYPPHM sequence, indicating all the start Met was removed. From the use of densitometric area scanning, bands densities were estimated in Coomassie-stained SDS-PAGE patterns of L1E2. Full-sized L1E2 was about 80 ± 5% of the band density and therefore about 76 ± 5% on a molar basis. L2E2 was about 94 ± 5% of the protein.Properties of L1E2 and L2E2--
Besides having high
acetyltransferase-specific activities (L1E2, 15.76 ± 0.61 A232 nm; L2E2, 16.93 ± 0.93
A232 nm), the lipoyl domain of each
engineered oligomer served as effective substrates in the cyclic E3
assay. At equivalent full-sized subunit levels, substrate-limited E3
rates were 52-60% of that observed for E2 oligomer with L2E2 serving
as a somewhat better substrate than L1E2 on a per mg basis (Table
I). Upon applying a correction for
contaminants and lipoyl domain-deficient subunits in the L1E2 preparation, the observed E3 rate supported by L1E2 is somewhat greater
(9% higher) than that for L2E2 in this substrate-limited reaction. E3
catalytic utilizations (estimated Km and Vm values) for the free individual lipoyl domains
(L1 and L2) were very
similar.6 Thus, this measure
supports at least high lipoylation of full-sized L1E2 as L2E2. The
higher E3 reaction rate for the bilipoyl domain containing E2 substrate
versus the rates using the monolipoyl domain oligomers
indicates that the unbound E3 is responding to the levels of the
coupled lipoyl domains almost as if they were in independent
structures. This is consistent with the high mobility of the linker
regions aiding independent encounters of the co-tethered lipoyl domains
with E3.
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L1E2 and L2E2 Enhancement of PDK Activity-- The capacity of L1E2 and L2E2 oligomers to support enhanced PDK activity was compared with mature E2 using reaction condition 1. The concentration of L1E2 was based on the mass proportion of L1E2 subunits estimated following corrections for other polypeptides based on area densitometric analysis of a Coomassie-stained SDS-polyacrylamide gel. As shown in Fig. 5, panel A, 2.5 and 5 µM subunit concentration for mature E2 enhanced PDK activity by 2.2- and 4-fold, respectively. The same subunit levels of L2E2 enhanced kinase activity by 1.2- and 2-fold, respectively, and 7.5 µM L2E2 gave a 2.4-fold enhancement. In marked contrast, L1E2 gave only a very small enhancement of PDK activity. Thus, the presence of the PDK-binding L2 domain in the truncated oligomer enhances PDK activity but not nearly as effectively as mature E2. A major contribution to the observed enhancements most likely results from increased encounters due to concentrating PDK and E1 at the oligomer surface on the flexibly held L2 and B domains, respectively. The much smaller effect of L1E2 further supports the primary role of L2 domain in PDK binding. The very small activation (maximally 15%) by L1E2 probably reflects binding of a low proportion of PDK which is consistent with the results of Liu et al. (9) using fusion protein held lipoyl domains. The possibility that binding of E1 to the B domain alters the conformation or presentation of E1 in a way that makes E1 a better substrate for unbound PDK cannot be eliminated by these data.
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Enhancement of PDK Activity by Full-sized E2 with One Lipoyl Group-- Because the above results suggest a need for the L1 domain for full PDK activity and yet the lipoyl prosthetic group is required both for the strong binding of PDK to L2 and weak binding to L1 (9), we evaluated the effects of substitution in full-sized E2 constructs of alanines at the lipoylation site in either lipoyl domain. For direct comparison of these results to those showing regulatory effects (below), the relative PDK activations are shown for studies in the high salt buffer (condition 2). As shown in Fig. 5, panel B, 3.5-10.5 µM E2 increased PDK activity from nearly 3-fold to over 6-fold. Higher PDK activities (2.5-fold) but lower fold activations were obtained when assays were conducted under the nonphysiological low salt assays (condition 1) with this E2 preparation (data not shown). For the studies in Fig. 5, panel B, E2 constructs, produced with a His tag which was then removed, contained much lower levels of modified E2 subunit with a partial L1 domain4 than the L1-containing constructs shown in Fig. 3, lanes 3 and 5. However, the E2 oligomer with fewer partial E2 subunits was not significantly more effective than the previous E2 preparation (Fig. 3, lane 5) in activating PDK or PDP (below).
As shown in Fig. 5, panel B, A173KE2 (L2 not lipoylated) was not nearly as effective in supporting PDK function as unmodified E2. This further supports the lipoyl group of L2 being critical for fully activated PDK function. Nevertheless, A173KE2 was much more effective in activating PDK than L2-truncated L1E2 (condition 1, Fig. 5, panel A, or condition 2, Fig. 6, panel A, in the absence of effectors). Thus, the presence of a nonbinding L2 domain does enhance the functionality of L1 although it remains low.
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L1E2 and L2E2 Facilitation of Effector Stimulation of PDK-- By using physiological level of potassium salts (condition 2) required for significant effector stimulations of PDK activity, we evaluated the capacity of truncated E2 oligomers to support stimulations by NADH or acetyl-CoA, alone or in combination with a low level (2 µM) of oligomeric E2 structures. As shown in Fig. 6, panel A, very small enhancements of PDK activity were supported by L1E2 despite effective reductive acetylation of its lipoyl groups by the combination of E3-catalyzed reduction and E2-catalyzed acetylation of the lipoyl groups of L1 (Table I). However, in the presence of L2E2, NADH and acetyl-CoA gave a pronounced stimulation of PDK activity, and the combination of both effectors so markedly stimulated PDK activity that it rose to essentially the same level as supported by mature E2 (Fig. 6, panel A). In the case of L2E2, NADH gave a fractional stimulation higher than that achieved with E2, but the absolute stimulated activity was still below that with E2.
Previous studies (10) have shown that acetyl-CoA stimulation requires transacetylase activity (catalyzed by trimers in the E2I assemblage) and that acetylation of a low proportion of sites in E2 can mediate kinase stimulation (33). Consistent with this trend of low levels of acetylated E2 being effective in enhancing kinase activity, marked stimulation with NADH and acetyl-CoA was observed with low, 2 µM L2E2 (Fig. 6, panel A). The combination of results contribute further support for the conclusion that acetylation leads to development of an altered, higher affinity interaction of PDK with one or more reductively acetylated L2 domains in the L2E2-60-mer. Acetyl-CoA stimulation in the absence of NADH is a function of the portion of reduced lipoyl groups in the E2 preparations. That portion is normally low, but reductants that reduce lipoyl groups raise this stimulation to the level achieved in the presence of NADH (34, 35).Effector Stimulation of PDK Mediated by E2, K173AE2, and
K46AE2--
With the full-sized E2 sources included at 7 µM, both modified forms facilitated NADH and acetyl-CoA
stimulation of PDK, Fig. 6, panel B. As found in the absence
of effectors (Fig. 5, panel B), the enhanced PDK activity
mediated by K46AE2 remained greater than that supported by native E2
for all conditions. However, the fractional enhancement by effectors
was higher when native E2 mediated the product stimulation of PDK
activity (e.g. the combination of products gave 2.9-fold
stimulation with E2 versus 2.5-fold stimulation with
K46AE2). The column step used in the preparation of these E2s included
10 mM -mercaptoethanol which apparently left several
lipoyl groups in the reduced form thereby allowing some E2-catalyzed
acetylation of lipoyl moieties with acetyl-CoA to thereby provide
substantial stimulation of PDK in the absence of NADH.
Ca2+-dependent Activation of PDP by the Different E2 Constructs-- E2 greatly increases the rate of dephosphorylation of E1b by a process involving Ca2+-dependent binding of PDP to the L2 domain of E2. Fig. 7, panel A, compares the effects of E2 and the E2 structures engineered to contain one lipoyl domain. L1E2 failed to activate PDP, whereas 1.7-5.0 µM L2E2 enhanced PDP activity 2-6-fold, further supporting a highly specific interaction with the L2 domain. However, equivalent levels of full-sized E2 supported even higher enhancements of PDP activity (4-7.5-fold).7
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Basis of Greater Effectiveness of K46AE2 Than L2E2 in Supporting PDK and PDP Function-- The activation of PDK and PDP requires the lipoyl prosthetic groups on the lipoyl domains of E2 (11, 36), and PDK and PDP do not bind to delipoylated L1 (9, 11). Removal of L1 reduced PDP and PDK function, whereas prevention of lipoylation of L1 allowed fully activated PDP function and supported enhanced PDK function exceeding native E2. The latter gain in PDK activation and maximal stimulation can be explained by a lack of competitive binding of PDK to L1. However, to explain the diminished PDK and PDP activations upon removal of the L1 domain requires either that the bilipoyl domain E2 is more effective in enhancing PDK and PDP activity due to an active role of L1 or that these activities are diminished by the marked change in the environment at the surface of E2 following removal of the L1 domain in L2E2. Although many explanations are possible, for simplicity we would emphasize the latter. Each lipoyl domain has a high proportion of charged residues (>25%) with an excess of acidic over basic amino acids.8 Thus, 60 L1 concentrated at the surface of the E2 oligomer can change electrostatic interactions and additionally introduce molecular crowding effects that could alter component interactions. Considering components must move for one PDK or PDP molecule to efficiently modify many bound E1, it seems likely that the absence of L1 in L2E2 alters the environment at the surface of the complex to such an extent that it would alter these dynamic protein-protein interactions. PDK activation may be complex since PDK isomers are oligomers9 and can probably bind at least temporarily to more than one lipoyl domain (9). Whatever role L1 has in aiding PDK function, it is by-passed upon stimulation of PDK by acetylation, supporting a preferential interaction of the PDK used in these studies with acetylated-L2 (cf. below).
Final Considerations--
Our results emphatically support an
essential mediatory role of the L2 domain and its lipoyl group in
facilitating PDK and PDP operation at accelerated rates and for
conveying the primary control of these regulatory enzymes. The primary
second messenger activation of PDC engages L2 and its lipoyl group in
binding PDP in response to increased free Ca2+ which is
known to be effective in the low micromolar range (7). Stimulation of
PDK activity via increased reduction and acetylation of the lipoyl
group of L2 fulfills a key feedback role in fuel selection by shutting
down of PDC due to -oxidation increasing NADH:NAD+and
acetyl-CoA:CoA ratios. Previously, Ravindran et al. (10) emphasized that reductive acetylation of a low proportion of the lipoyl
groups in an E2-60-mer is sufficient to markedly stimulate PDK
activity. This was based on both the forms of bovine PDK that fractionate with E2 and E1 in PDC resolution. The marked increase in
PDK activity upon acetylation of L2E2 supports a critical change in the
interaction of the PDK associated with the resolved bovine E1 component
with at least one acetylated L2 since PDK activity dramatically rises
to the stimulated level attained with unmodified E2. It remains to be
established whether the four PDK isozymes all respond to E2 and various
regulatory signals in the same manner. Although we have not established
which isozyme we are studying in the E1 preparation, we are certain
that this PDK has a dependence on higher E2 levels for E2 enhancement
of its PDK activity than does the PDK that remains associated with
bovine E2 (16). This apparent weaker affinity for E2 is reminiscent of
the findings by Randle and co-workers (1, 37, 38) that a PDK with lower affinity for PDC (presumably E2) increases in amount under conditions of starvation or diabetes (noninsulin-producing animals). Recently, Wu
et al. (39) have shown that the mRNA and protein levels
for the PDK4 isozyme increase in rat heart under those conditions. We
would note that the magnitude of the stimulations by acetyl-CoA and
NADH found here for the bovine kidney PDK associated with E1 are
somewhat larger than the stimulation found for the bovine kidney PDK
that remains associated with E2 (e.g. Refs. 10 and 25)
further supporting differences in the PDK isozyme composition of these
preparations.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK18320 and by the Agriculture Experiment Station, contribution 97-270-J.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Pharmacology, Health Science Center,
University of Virginia, Charlottesville, VA 22908.
§ To whom correspondence should be addressed: Dept. of Biochemistry, 104 Willard Hall, Kansas State University, Manhattan, KS 66506.
1 The 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.
2 J. C. Baker, J. Dong, and T. E. Roche, unpublished observations.
3 PDK3 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.
4 A. Yakhnin, X. Gong, X. Yan, M. P. Sadler, and T. E. Roche, manuscript in preparation.
5 Porcine E3 is used for comparison to our previous data with recombinant human E2 (16). Lindsay and co-workers (26, 27) 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.
6 J. Baker and T. E. Roche, manuscript in preparation.
7 As indicated under "Experimental Procedures" E1b is prepared using E2·E3BP·PDK subcomplex as a source of kinase, and this is then removed by centrifugation. Trace levels of E2 in this E1b preparation (<1% of the protein based on SDS-PAGE pattern from a heavily loaded sample) reduced the activation observed with various E2 sources. The presence of contaminating bovine E2 was also indicated by EGTA reducing PDP activity by 40% in the absence of an added E2 source.
8 In the L1 structure, >25% of its residues are acidic (16 Asp + Glu) or basic (10 Arg + Lys, excluding Lys-46); similarly L2 has 17 acidic and 9 basic residues. For these values, we include residues 1-96 in L1 domain and residues 128-229 in the L2 domain. Residues 82-96 in L1 and residues 209-229 in L2 do not align with domain residues in bacterial lipoyl domains of known structure (5). However, these regions cannot be removed without significant loss of functional properties of L1 and L2 (A. Yakhnin, X. Yong, and T. E. Roche, unpublished data).
9 Recombinant PDK preparations tend to form aggregates. A dimer appears to be the lowest aggregation state.
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