Vol. 74, No. 10, pp 42&S-4227, October 1977 Biochemistry Acyl group and electron pair relay system: A network of interacting lipoyl moieties in the pyruvate and a-ketoglutarate dehydrogenase complexes from Escherichia coli (multienzyme complexes/thiol-disulfide interchange/crosslinking of subunits) JIMMY H. COLLINS AND LESTER J. REED Clayton Foundation Biochemical Institute and Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712 Contributed by Lester J. Reed, August 18, 1977

ABSTRACT The dihydrolipoyl transacetylase component complex by [2-'4C]pyruvate (6) and of the pyruvate-dependent of the Escherichia coli pyruvate dehydrogenase complex [pyr- reaction of N-ethyl[2,3-14C]maleimide with the complex (7) uvate:lipoate oxidoreductase (decarboxylating and acceptor- indicate the presence of two functionally active lipoyl acetylating), EC 1.2.4.11 bears two sites on each of its 24poly- moieties peptide chains that undergo reductive acetylation by on each transacetylase chain. 2-4C pyruvate and thiamin pyrophosphate, acetylation by In this communication, we confirm and extend this latter [lMClacetyl-CoA in the presence of DPNH, and reaction with finding, and we present evidence indicating that the trans- N-ethyl[2,3-14Clmaleimide in the presence of pyruvate and succinylase component of the a-ketoglutarate dehydrogenase thiamin pyrophosphate. The data strongly ir tat these sites complex bears one functionally active lipoyl moiety per chain are covalent y bound lipoyl moieties. The results of similar ex- and that the 48 lipoyl moieties in periments with the E. coli a-ketoglutarate dehydrogenase the transacetylase and the 24 complex [2-oxoglutaratelipoate oxidoreductase (decarboxylating lipoyl moieties in the transsuccinylase comprise a network that and acceptor-succinylating), EC 1±2.4.2] indicate that its dihy- functions as an acyl group and electron pair relay system drolipoyl transsuccinylase component bears only one lipoyl through thiol-disulfide and acyl-transfer reactions among all moiety on each of its 24 chains. Chagng of the 48 acetyl ac- of the lipoyl moieties. ceptor sites on the transacetylase or the 24 succinyl acceptor sites on the transsuccinylase by pyruvate or cc-ketoglutarate, re- spectively, and thiamin pyrophosphate was observed in the MATERIALS AND METHODS presence. of only a few functionally active pyruvate dehydro- The specific activities of the pyruvate and a-ketoglutarate genase or a-ketoglutarate dehydro enase chains. Extensive dehydrogenase complexes were 40-49 and 30-35 ,mol of crosslinking of the transacetylase chains was observed when DPNH per min/mg of protein, respectively (8, 9). The ra- the pyruvate dehydrogenase complex was treated with pyruvate dioactive componltds were obtained from Amersham/Searle, and thiamin pyrophosphate or with DPNH in the presence of N,N'-o- or N,N-p-phenylenedimaleimide, respectively. When and N,N'-o- and NN'-p-phenylenedimaleimides (o-PDM and the a-ketoglutarate dehydrogenase complex was treated with p-PDM) were from Aldrich. Thiamin thiazolone pyrophosphate DPNH in the presence of NN-p-phenylenedimaleimide, only was prepared as described by Butler et al. (10). transsuccinylase monomers and crosslinked transsuccinylase The specific radioactivities of the "4C-labeled compounds dimers were detected. It appears that the 48 lipoyl moieties in were determined as follows. The concentrations of stock solu- the transacetylase and the 24 lipoyl moieties in the transsuc- tions of [2-14C]pyruvate and a-[5-14C]ketoglutarate were cinylase comprise an interacting network that functions as an acyl group and electron pair relay system through thioldisulfide measured spectrophotometrically as DPNH produced by oxi- and acyl-transfer reactions among all of the lipoyl moieties. dative decarboxylation in the presence of the pyruvate or a- ketoglutarate dehydrogenase complex (9). Samples of known The pyruvate dehydrogenase complex [pyruvate:lipoate oxi- 14C content were diluted with an accurately measured amount doreductase (decarboxylating and acceptor-acetylating), EC of pure pyruvate or a-ketoglutarate and converted to the 1.2.4.1] and the a-ketoglutarate dehydrogenase complex [2- semicarbazone, and the derivative was recrystallized to constant oxoglutarate:lipoate (decarboxylating and acceptor-succinyl- specific radioactivity. The concentration of N-ethyl[2,3- ating), EC 1.2.4.2] from Escherichia coil each consists of three 14C]maleimide was determined spectrophotometrically (11). enzymes that, acting in sequence, catalyze the reactions shown A sample of known 14C content was diluted with pure N-eth- in Fig. 1. Each complex is organized about a core consisting of ylmaleimide and converted to S-(N-ethylsuccinimido)-L-cys- dihydrolipoyl transacetylase or dihydrolipoyl transsuccinylase teine (12), and the derivative was recrystallized to constant to which the other two enzymes (pyruvate dehydrogenase or specific radioactivity. The results of this method were in good a-ketoglutarate dehydrogenase and dihydrolipoyl dehydro- agreement with the specific radioactivity determined by genase) are joined by noncovalent bonds (1). The transacetylase analysis of the S-[(1,2-dicarboxy)ethyl] cysteine derivative (7) and transsuccinylase each consists of 24 apparently identical on a Beckman 120C amino acid analyzer equipped with an polypeptide chains in an arrangement having octahedral in-line scintillation counter. [1-"4C]Acetyl-CoA was diluted 1:10 symmetry (2). The cofactor lipoic acid is covalently attached with nonradioactive acetyl-CoA, and the concentration was by an amide bond to a lysine residue in the two transacylases. determined spectrophotometrically with phosphotransacetylase The lipoyl moiety is thought to rotate among the catalytic and arsenate (13). Measurement of protein-bound radioactivity centers of the three enzymes in each complex (3). Studies of the and performic acid-labile acyl groups on filter paper disks was mobility of the spin-labeled lipoyl moieties in the pyruvate carried out as described by Pettit et al. (9). Radioactivity was dehydrogenase complex are consistent with this view (4, 5). determined in a Beckman LS-230 scintillation counter with Studies of the acetylation of the pyruvate dehydrogenase ACS cocktail (Amersham/Searle). The costs of publication of this article were defrayed in part by the Abbreviations: o-PDM, N,N'-o-phenylenedimaleimide; p-PDM, payment-of page charges. This article must therefore be hereby marked N,N'-p-phenylenedimaleimide; NaDodSO4, sodium dodecyl sulfate; "adcement" in accordance with 18 U.S.C. §1734 solely to indicate Mr. molecular weight. this fact. 4223 Downloaded by guest on September 28, 2021 4224 Biochemistry: Collins and Reed Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Stoichiometry of substrate-dependent incorporation of DPN* acyl groups and N-ethylmaleimide

5 [14C]acyl groups [14C]NEM OH [H DPNH + Ht incorporated, incorporated, [RCH-TPP] [Lps2] dehydrogenase Additions mol/mol complex mol/mol complex Co2 [FAD] dehydrogenasePyruvate Dihydrolipoyl SS Pyruvate dehydrogenase complex 1 and 2 tronsocetylase [Lip(SH) ] II o~~~-Ke togluaote trnussuccinylasean RC-SCoA ['4C]Pyruvate 47.8 4 1.4 (11) RCC02H dehydrogenase 0 ['4C]Pyruvate + NEM 49.6 4 1.4 (8) [RC-SLipSH] Pyruvate + [14C]NEM 51.1 + 1.9 (4) [TPP] [14C]NEM 5.7 + 0.9 (3) 0 O CoASH a-Ketoglutarate dehydrogenase complex RCCO2H + CoASH + DPN+ -4- RC-'SCoA t CO2 + DPNH + HR 23.3 I 0.6 (3) FIG. 1. Reaction sequence in pyruvate and a-ketoglutarate oxi- [14C]KG dation. TPP, thiamin pyrophosphate; LipS2 and Lip(SH)2, lipoyl [14C]KG + NEM 24.4 + 0.5 (3) moiety and its reduced form. KG + [14C]NEM 26.3 + 1.1 (3) [14C]NEM 3.8 I 0.7 (2) protein-bound acetyl groups were stabilized in the presence of Pyruvate dehydrogenase complex (PDC) or a-ketoglutarate de- N-ethylmaleimide. In parallel experiments, treatment of the hydrogenase complex (KGDC) at 1.0 to 2.0 mg/ml was treated at 40 native complex with N-ethyl[2,3-14C]maleimide in the presence under N2 with 0.25 mM [2-14C]pyruvate or a-[5-14C]ketoglutarate of nonradioactive pyruvate and thiamin pyrophosphate resulted (KG), respectively, in the presence and absence of 0.5 mM nonra- dioactive N-ethylmaleimide (NEM), and with 0.5 mM N-ethyl[2,3- in incorporation of about 51 mol of the radioactive maleimide 14C~maleimide in the presence and absence of0.25 mM nonradioactive per mol of complex. In the absence of pyruvate about 6 mol of pyruvate or a-ketoglutarate in 50 mM potassium phosphate buffer the maleimide were incorporated. These data demonstrate the (pH 7.0) containing 0.2 mM thiamin pyrophosphate, 1 mM MgCl2, incorporation of about 45 mol of the radioactive maleimide per and 1 mM EDTA. Incorporation of [14C]acetyl or [l4C]succinyl groups mol of complex in a pyruvate-dependent reaction. The py- was determined after 1 min incubation by the filter paper disk pro- ruvate-dependent incorporation of N-ethyl[2,3-14C]maleimide cedure described in Materials and Methods. Incorporation of CoA (0.1 mM) and [14C]NEM was determined on aliquots of the incubation mixtures that was inhibited at least 95% by addition of had been quenched with 5 mM 2-mercaptoethanol after the overall arsenite (1 mM) 20 min prior to addition of the maleimide. This activity of the complex had decreased to less than 5% of the original observation provides further evidence for the selective modi- activity [about 15 min (4, 16)]. Activity losses due to incubation with fication of the lipoyl moieties by N-ethylmaleimide, because pyruvate or a-ketoglutarate or NEM individually were less than 10%. arsenite is known to react with the dithiol form of lipoic acid Results are means I SD; the number of determinations is given in (17). When the pyruvate dehydrogenase complex was treated parentheses. with both [2-'4C]pyruvate and N-ethyl[2,3-14C]maleimide in the presence of thiamin pyrophosphate, the amount of radio- Sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel activity incorporated into the complex corresponded closely electrophoresis was carried out in 4% or 5% (wt/vol) gels as to the sum of the radioactivities incorporated in parallel ex- described by Weber and Osborn (14). Enzyme preparations that periments with radioactive pyruvate and nonradioactive had been modified with o-PDM or p-PDM were made 5 mM maleimide and with nonradioactive pyruvate and the ra- in 2-mercaptoethanol and 1% in NaDodSO4 and were heated dioactive maleimide (18). at 1000 for 5 min. Incubation mixtures that contained ra- When samples of the acetylated or N-ethylmaleimide- dioactive reagents were filtered on 1.5 X 20 cm columns of modified complex were analyzed by NaDodSO4/polyacryl- Sephadex 0-25 equilibrated with 50 mM phosphate buffer (pH amide gel electrophoresis, followed by scintillation counting 6.0) at 40 prior to gel electrophoresis. Samples of known protein of the radioactivity in the stained protein bands, 90 to 96% of and radioactivity content were made 1% in 2-mercaptoethanol the radioactive label was found in the transacetylase component and 1% in NaDodSO4 and were applied, without-heating, to 5% of the complex. The recovery of the radioactivity applied to the polyacrylamide gels prepared with pH 6.0 buffer and with gels was 90-96%. Because the transacetylase consists of 24 ap- N,N'-diallyl tartardiamide (Eastman) instead of methylene-bis parently identical polypeptide chains (8), the simplest inter- acrylamide (15). The Coomassie blue-stained bands were ex- pretation of our data is that the sites of incorporation of acetyl cised from the gel and dissolved by incubation for 30 min at 230 groups and N-ethylmaleimide are the lipoyl moieties and that with 0.5 ml of 2% periodic acid for radioactivity measure- each transacetylase chain bears two lipoyl moieties. As indicated ment. in Fig. 1, reductive acetylation of a lipoyl moiety by pyruvate RESULTS (reactions 1 and 2) produces an S-acetyldihydrolipoyl moiety Stoichiometry of Substrate-Dependent Incorporation of which, in turn, reacts with N-ethylmaleimide. Acyl Groups and N-Ethylmaleimide. The stoichiometry of The results of similar experiments with the a-ketoglutarate incorporation of acetyl groups and N-ethylmaleimide into the dehydrogenase complex are summarized in Table 1. About 24 pyruvate dehydrogenase complex is summarized in Table 1. mol of [4-14C]succinyl groups were incorporated per mol of About 48 mol of [I-_4C]acetyl groups were incorporated per complex [Mr, 2.8 X 106 (9)] within 1 min at 40 and pH 7.0 in mol of complex [molecular weight (Mr) 4.6 X 106(8)] within the presence of a-[5-'4C]ketoglutarate and thiamin pyro- 1 min in the presence of [2-'4C]pyruvate and thiamin pyro- phosphate and in the absence or presence of nonradioactive phosphate under N2 at 40 and pH 7.0. About 90% of the pro- N-ethylmaleimide. Treatment of the complex with N-ethyl- tein-bound acetyl groups were released within 1 min in the [2,3-14C]maleimide in the presence and absence of nonra- presence of either 0.1 mM CoA, 0.1 mM CoA and 1 mM DPN+, dioactive a-ketoglutarate and thiamin pyrophosphate resulted or 0.1 mM CoA and 1 mM arsenite, and about 95% were re- in incorporation of about 26 and 4 mol, respectively, of the leased by treatment with performic acid. The stoichiometry radioactive maleimide per mol of complex. These data dem- of acetyl group incorporation was essentially the same in the onstrate the incorporation of about 22 mol of the radioactive absence and presence of N-ethylmaleimide. However, the maleimide in an a-ketoglutarate-dependent reaction. About Downloaded by guest on September 28, 2021 Biochemistry: Collins and Reed Proc. Natl. Acad. Sd. USA 74 (1977) 4225 incorporated. In a subsequent experiment, 5 mM N-ethyl- maleimide was included in the reaction mixture to inhibit re- lease of the protein-bound acetyl groups. Incubation was con- tinued until the overall activity of the complex had decreased to less than 5% of the original activity, and the mixture was passed through a column of Sephadex G-25. Radioactivity and protein determinations indicated that the complex contained about 44 [14C]acetyl groups per molecule. Analysis by Na- DodSO4/gel electrophoresis showed that about 95% of the ra- dioactive label had been incorporated into the transacetylase component. These data provide support for the reversibility of reactions 5,4, and 3 (Fig. 1) and for the presence of two func- tionally active lipoyl moieties per transacetylase chain. Evidence for a Network of Interacting Lipoyl Moieties in the Transacetylase and in the Transsuccinylase. Although the native pyruvate dehydrogenase complex contains 24 pyr- uvate dehydrogenase chains (i.e., 12 dimers) (8), charging of the 48 acetyl acceptor sites on the transacetylase by pyruvate and thiamin pyrophosphate was observed with reconstituted TIME (min) subcomplexes containing, on average, three or only one pyr- FIG. 2. Reductive acetylation of transacetylase by [2-_4C]pyr- uvate dehydrogenase dimer per transacetylase molecule (Fig. uvate in the presence of a few functional pyruvate dehydrogenase 2A). The rate of acetyl group incorporation was slower than that chains. (A) Mixtures containing 0.22 AM dihydrolipoyl transacetylase (LTA) and 0.22MM (0) or 0.66MM (0) pyruvate dehydrogenase were observed with 12 dimers (Fig. 2B) because the rate-limiting step incubated in 50 mM phosphate buffer (pH 7.0), 0.2 mM thiamin py- is the generation of the intermediate, hydroxyethylthiamin rophosphate, 1 mM MgCl2, and 1 mM EDTA for 30 min at 4°. Acet- pyrophosphate, by the pyruvate dehydrogenase (Fig. 1). ylation was then initiated by addition of 0.25 mM [2-14C]pyruvate. Further support for a network of interacting lipoyl moieties At the indicated times, 20-gl aliquots were assayed for protein-bound in the transacetylase was obtained from charging experiments acetyl groups. (B) A mixture containing 0.5 mg (0.11 nmol) ofpyruvate using native pyruvate that had been dehydrogenase complex (PDC), 50 mM phosphate buffer (pH 7.0), dehydrogenase complex 1 mM MgCl2, 1 mM EDTA, and 1.06 Mg (2.32 nmol) of thiamin thia- titrated with thiamin thiazolone pyrophosphate to inactivate zolone pyrophosphate in a total volume of 0.5 ml was incubated for about 21 of its 24 pyruvate dehydrogenase chains. This transi- 30 min at 4°. The overall activity of the inhibited complex, measured tion state analog is a powerful competitive inhibitor of thiamin as the initial rate in the DPN-reduction assay (9), was 11% of the ac- pyrophosphate. The dissociation constant (KD) of the analog tivity of a control lacking the inhibitor. Acetylation was initiated by is about 0.5 nM at pH 6.6, whereas the KD of thiamin pyro- the addition of 0.2 mM thiamin pyrophosphate and 0.25 mM [2- phosphate is about 10 (20). When the inhibited complex was "4C]pyruvate. Protein-bound acetyl groups in the inhibited (A) and AM untreated (A) complexes were measured at the indicated times. After treated with [2-14C]pyruvate and thiamin pyrophosphate, in- 30 min of incubation, the overall activity of the inhibited complex was corporation of about 44 acetyl groups per molecule of complex still 11% of the activity of the control. was observed (Fig. 2B). About 91% of the protein-bound acetyl groups were released rapidly in the presence of excess CoA. 94% of the protein-bound succinyl groups were both CoA-labile Incubation of the inhibited complex with untreated pyruvate (in the absence of N-ethylmaleimide) and performic acid-labile. dehydrogenase (molar ratio, 1:24) at 40 for 5 hr had little effect, Analysis of the radioactive complexes by NaDodSO4/gel if any, on the overall activity of the complex. This control electrophoresis demonstrated that 90-99% of the radioactive showed that, under these conditions, no exchange occurred label had been incorporated into the transsuccinylase compo- between the free, fully active pyruvate dehydrogenase and the nent of the complex. Because the transsuccinylase consists of inhibited pyruvate dehydrogenase bound to the transacetylase. 24 apparently identical polypeptide chains (9), we interpret our This control also renders unlikely the possibility that the single results to indicate that each transsuccinylase chain bears one pyruvate dehydrogenase dimer attached to the transacetylase lipoyl moiety. We reported previously that about 12 mol of molecule (Fig. 2A) could dissociate from and reassociate with succinyl groups were incorporated per mol of complex in 2-5 binding sites on the transacetylase and thereby charge all the min at pH 7.4 and 250 (9). This low value may be due, at least acetyl acceptor sites. It should be noted that the KD of pyruvate in part, to the instability of the protein-bound succinyl groups dehydrogenase is about 2 nM at pH 7.0 (P. F. Davis and L. J. under these conditions, particularly in the absence of N-eth- Reed, unpublished data). ylmaleimide. The a-ketoglutarate dehydrogenase complex was treated DPNH-Dependent Acetylation by [1-14CJAcetyl-CoA. with thiamin thiazolone pyrophosphate to inactivate about 9 Maximum incorporation of acetyl groups required the presence of its 12 a-ketoglutarate dehydrogenase chains. When the in- of DPN+ as well as DPNH and the addition of acetyl-CoA prior hibited complex was incubated with a-[5-14C]ketoglutarate and to DPNH. Presumably, DPN+ prevents inhibition of the fla- thiamin pyrophosphate, rapid incorporation of about 20 suc- voprotein by DPNH-i.e., conversion to the four-electron re- cinyl groups per molecule of complex was observed (Fig. 3). duced form which is catalytically inactive (19). In a typical Substrate- and DPNH-Dependent Crosslinking of experiment, the reaction mixture contained pyruvate dehy- Transacylase Chains by o-PDM and p-PDM. The pyruvate drogenase complex at 2 mg/ml, 1 mM MgCI2, 0.2mM thiamin dehydrogenase complex was incubated at room temperature pyrophosphate, 0.3 mM DPN-, 0.1 mM DPNH, and 0.25 mM under anaerobic conditions with pyruvate and thiamin pyro- [1-14C]acetyl-CoA in 50 mM phosphate buffer (pH 7.0) at 40. phosphate in the presence of 0.2 mM o-PDM. Aliquots were DPNH was added last. Within 15 sec, 47 acetyl groups were removed at different time intervals and made 5 mM with re- incorporated per molecule of complex, followed by a gradual spect to 2-mercaptoethanol to stop the reaction. The samples release of the protein-bound acetyl groups to a value of about were assayed for overall activity and were analyzed by Na- 35 at 5 min. In the absence of DPNH, no acetyl groups were DodSO4/gel electrophoresis. About 98% of the overall activity Downloaded by guest on September 28, 2021 4226 Biochemistry: Collins and Reed Proc. Natl. Acad. .ci. UISA 74 (1977)

0 - , C,-Ix _ Y, ; _ :-. 0'E 16 .w i E Mc, ,_4w t t CM _ _a _ Co _ I c E AM mm _w

:3 III ! ~- 5 10 15 30 TIME (min) .~~~~~~~~~~~~~ FIG. 3. Reductive succinylation of transsuccinylase by a-[5- '4C]ketoglutarate in the presence of a few functional a-ketoglutarate A B C D E F G H dehydrogenase chains. A mixture containing 1.0 mg (0.36 nmol) of gel electrophoretic patterns 50 mM phosphate buffer FIG. 4. NaDodSO4polyacrylamide a-ketoglutarate dehydrogenase complex; of dimaleimide-modified complexes. The incubation mixtures con- 1 mM MgCl2, 1 mM EDTA, and 2.61 ug (5.71 nmol) of (pH 7.0), tained protein at 1 mg/ml in 50 mM sodium phosphate buffer, pH was thiamin thiazolone pyrophosphate in a total volume of 1.0 ml were incubated at 230 under N2, and pyrophosphate. 7.0/1 mM EDTA. Mixtures A-F incubated for 24 hr at 40 to displace bound thiamin mixtures G-I were incubated at 4°. The reaction with the dimaleimide was about 23% of the The overall activity of the inhibited complex was stopped at the indicated times by addition of 50-gl aliquots to 5 activity of a control lacking the inhibitor. Succinylation was initiated mM 2-mercaptoethanol. Lanes: (A) Pyruvate dehydrogenase complex and 0.25 mM a- by the addition of 0.2 mM thiamin pyrophosphate after 1 hr in presence or absence of 0.2 mM o-PDM. The three bands groups in the inhibited [5-14C]ketoglutarate. Protein-bound succinyl are, from top to bottom, pyruvate dehydrogenase monomer (Mr, () and untreated (0) complexes were measured at the indicated 96,000), dihydrolipoyl transacetylase monomer (Mr, 70,000), and times. After 30 min of incubation, the overall activity ofthe inhibited dihydrolipoyl dehydrogenase monomer (Mr, 56,000) (8). (B) Pyruvate complex was still about 23% of the activity of the control. dehydrogenase complex after 12 min in presence of0.2 mM o-PDM, 1 mM pyruvate, and 0.2 mM thiamin pyrophosphate. (C) Pyruvate dehydrogenase complex after 1 hr in presence of0.2 mM p-PDM. (D) was inhibited within 10 min in the presence of o-PDM and Pyruvate dehydrogenase complex after 10 min in presence of 0.2 mM pyruvate, but little loss of activity occurred in the absence of p-PDM and 0.1 mM DPNH. (E) Reconstituted subcomplex oftrans- pyruvate. The gel electrophoretic patterns showed that the loss acetylase and pyruvate dehydrogenase (chain ratio, 24:8) after 1 hr after of activity was accompanied by disappearance of the mono- in presence of 0.2 mM o-PDM. (F) Reconstituted subcomplex 20 min in presence of 0.2 mM o-PDM, 1 mM pyruvate, and 0.2 mM meric transacetylase band and the appearance of oligomeric thiamin pyrophosphate. (G) a-Ketoglutarate dehydrogenase complex transacetylase species, including material that did not enter the after 1 hr in presence of 1 mM DPNH. The three bands from top to gel (Fig. 4). Little change, if any, was observed in the pyruvate bottom are a-ketoglutarate dehydrogenase monomer (Mr, 95,000), dehydrogenase and dihydrolipoyl dehydrogenase bands. dihydrolipoyl dehydrogenase monomer (Mr, 56,000), and dihydroli- Similar results were obtained with a reconstituted subcomplex poyl transsuccinylase monomer (Mr, 42,000) (9). (H) a-Ketoglutarate consisting of transacetylase and pyruvate dehydrogenase (chain dehydrogenase complex after 1 hr in presence of 0.2 mM p-PDM. The upper band is crosslinked a-ketoglutarate dehydrogenase dimer. (I) was the incubation ratio, 24:8). When pyruvate omitted from a-Ketoglutarate dehydrogenase complex after 1 hr in presence of 1 mixtures, no oligomeric species of the transacetylase were de- mM DPNH and 0.2 mM p-PDM. The crosslinked transsuccinylase tected on the gel. Production of oligomeric species of crosslinked dimer is indicated by the arrow. The four sets of gels (A and B, C and transacetylase chains was also observed when the complex was D, E and F, and G-I) were prepared at different times. treated with DPNH in the presence of p-PDM. p-PDM ap- constituted subcomplex consisting of transsuccinylase and peared to be more effective than o-PDM in the DPNH-de- flavoprotein (chain ratio, 24:2) (data not shown). pendent crosslinking of transacetylase chains, whereas o-PDM appeared to be more effective in the pyruvate-dependent DISCUSSION crosslinking. Because the two reactive maleimide groups are The results of experiments with the E. coli pyruvate dehydro- separated from each other in o-PDM by a distance of 5.2 A and genase complex indicate that the transacetylase component in p-PDM by 10.4 A (R. Pettit, personal communication; ref. bears two sites per polypeptide chain that undergo reductive 21), o-PDM may tend to react intramolecularly with the two acetylation by pyruvate and thiamin pyrophosphate, acetyla- thiol groups in a dihydrolipoyl moiety. tion by acetyl-CoA in the presence of DPNH, and reaction with We interpret these observations to indicate that the pyruvate- N-ethylmaleimide in the presence of pyruvate and thiamin or DPNH-dependent crosslinking of transacetylase chains re- pyrophosphate. These sites are presumably lipoyl moieties (Fig. sulted from reaction of their S-acetyldihydrolipoyl or dihy- 1). These data confirm and extend the findings of Danson and drolipoyl moieties with the dimaleimide. The observation that Perham (7) and Speckhard et al. (6). The results of similar ex- crosslinked transacetylase species were produced that did not periments with the a-ketoglutarate dehydrogenase complex enter the polyacrylamide gel during electrophoresis indicates indicate that the transsuccinylase component bears only one that crosslinking of many transacetylase chains occurred. These lipoyl moiety per chain. Accurate determination of the lipoyl observations are consistent with the presence of two lipoyl content of the two transacylases has proved to be difficult. moieties per transacetylase chain, because the presence of only Variable results have been obtained from microbiological assays one lipoyl moiety per chain would preclude production of any of acid hydrolysates of the two complexes and from measure- crosslinked transacetylase species larger than a dimer. ments of the content of radioactive lipoic acid in the complexes In contrast to the results obtained with the pyruvate dehy- isolated from E. coli cells that were grown in the presence of drogenase complex, only monomeric and dimeric transsuc- [35S2]lipoic acid (8). Under the conditions used in the latter cinylase species were observed when the a-ketoglutarate de- study, the exogenous, radioactive lipoic acid apparently did not hydrogenase complex was incubated at 40 with DPNH in the completely inhibit the biosynthesis or incorporation of endog- presence of p-PDM (Fig. 4). No crosslinked transsuccinylase enous lipoic acid. dimer was detected in the absence of DPNH. Transsuccinylase An important finding in the present investigation is that all monomers and crosslinked dimers, but no larger oligomeric 48 lipoyl moieties on the transacetylase and all 24 lipoyl moieties species, were also observed in similar experiments with a re- on the transsuccinylase are capable of interacting with each Downloaded by guest on September 28, 2021 Biochemistry: Collins and Reed Proc. Nat!. Acad. Sci. USA 74 (1977) 4227 LipS2 Ac-SLipSH UpS2 Lip(SH)2 phate binding sites on the pyruvate dehydrogenase complex is more than 30 A and probably close to 45 A. This distance is Ac-SLipS-SLipSH HSipS-SLipSH considerably larger than the 28 A predicted by our model of Ac-SLipSH LipS2 Lip(SH)2 LipS2 a single rotating lipoyl moiety interacting with successive active sites on the complex. However, the results of Moe et al. (24) are FIG. 5. Scheme illustrating thiol-disulfide interchange and compatible with the distances expected for the alternative acyl-transfer reactions among lipoyl moieties. formulation of the model (23) involving transfer of acyl groups other. It appears that this communication network operates and electron pairs among the lipoyl moieties. through thiol-disulfide interchange and acyl-transfer reactions The presence of a network of interacting lipoyl moieties in among the lipoyl moieties. These conclusions are based on the the transacetylase and in the transsuccinylase suggests that a following observations. pyruvate ehydrogenase (or a-ketoglutarate dehydrogenase) (i) Treatment of a reconstituted subcomplex of the transa- molecule and a flavoprotein molecule need not be in juxtapo- cetylase and pyruvate dehydrogenase containing on average sition to a particular lipoyl moiety for the overall reaction to only one pyruvate dehydrogenase dimer per transacetylase occur. This may have conferred some selective advantage on molecule (i.e., a chain ratio of 2:24) with [2-14C]pyruvate and the organization of the dihydrolipoyl transacylase chains into thiamin pyrophosphate resulted in incorporation of about 48 large, cube-like structures. radioactive acetyl groups per molecule of transacetylase. We thank Karen Hobson and William Lee for excellent technical (ii) Titration of the pyruvate dehydrogenase complex with assistance, Arloa Bergquist for. assistance in determining the specific sufficient thiamin thiazolone pyrophosphate to inhibit about radioactivity of N-ethyl[2,3-14C]maleimide, Dr. Flora Pettit for advice 21 of its 24 pyruvate dehydrogenase chains, followed by ad- and assistance, and Dr. Marvin Hackert and Robert Oliver for helpful dition of [2-14C]pyruvate and thiamin pyrophosphate, resulted discussions. We are grateful to Dr. Perry Frey for sending us a copy 44 molecule of ref. 6 before its publication. This work was supported in part by in rapid incorporation of about acetyl groups per Grant GM06590 from the U.S. Public Health Service. of complex. 1. Reed, L. J. & Oliver, R. M. (1968) Brookhaven Symp. Biol. 21, (iii) Treatment of the a-ketoglutarate dehydrogenase 397-411. complex with sufficient thiamin thiazolone pyrophosphate to 2. DeRosier, D. J., Oliver, R. M. & Reed, L. J. (1971) Proc. Nati. inhibit approximately 9 of its 12 a-ketoglutarate dehydrogenase Acad. Sci. USA 68,1135-1137. chains, followed by addition of a-[5-14C]ketoglutarate and 3. Koike, M., Reed, L. J. & Carroll, W. R. (1963) J. Biol. Chem. 238, thiamin pyrophosphate, resulted in rapid incorporation of about 30-39. 20 succinyl groups per molecule of complex. 4. Grande, H. J., Bresters, T. W., DeAbreu, R. A., DeKok, A. & (iv) Treatment of the pyruvate dehydrogenase complex with Veeger, C. (1975) Eur. J. Biochem. 59, 355-363. [1-14C]acetyl-CoA in the presence of DPNH resulted in rapid 5. Ambrose, M. C. & Perham, R. N. (1976) Biochem. J. 155, com- 429-432. incorporation of about 47 acetyl groups per molecule of 6. Speckhard, D. C., Ikeda, B. H., Wong, S. S. & Frey, P. A. (1977) plex. Because there are only about 12 flavoprotein chains per Biochem. Biophys. Res. Commun., in press. molecule of complex (8, 22), it appears that each flavoprotein 7. Danson, M. J. & Perham, R. N. (1976) Biochem. J. 159, 677- chain can interact with (i.e., reduce) four lipoyl moieties or, 682. more likely, that thiol-disulfide interchange occurs between 8. Eley, M. H., Namihira, G., Hamilton, L., Munk, P. & Reed, L. lipoyl moieties. J. (1972) Arch. Biochem. Biophys. 152, 655-669. (v) Treatment of the pyruvate dehydrogenase complex with 9. Pettit, F. H., Hamilton, L., Munk, P., Namihira, G., Eley, M. H., the dimaleimides o-PDM or p-PDM in the presence of pyruvate Willms, C. R. & Reed, L. J. (1973) J. Biol. Chem. 248, 5282- and thiamin pyrophosphate or of DPNH, respectively, resulted 5290. chains. Treatment 10. Butler, J. R., Pettit, F. H. & Reed, L. J. (1977) Biochem. Biophys. in extensive crosslinking of the transacetylase Res. Commun. 74, 1667-1674. of the a-ketoglutarate dehydrogenase complex with p-PDM 11. Gregory, J. D. (1955) J. Am. Chem. Soc. 77,3922-3923. in the presence of DPNH produced crosslinked transsuccinylase 12. Smyth, D. G., Nagamatsu, A. & Fruton, J. S. (1960) J. Am. Chem. dimers but no larger oligomeric species. Soc. 82,4600-4604. We propose that intrachain and interchain transfers of acetyl 13. Stadtman, E. R. (1957) in Methods in Enzymology, eds. Co- groups and electron pairs can occur between S-acetyldihy- lowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. drolipoyl moieties and oxidized lipoyl moieties and between 3, pp. 931-941. dihydrolipoyl moieties and oxidized lipoyl moieties in the 14. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406- transacetylase as illustrated in Fig. 5. We propose an analogous 4412. transfer of succinyl groups and electron pairs among the 24 li- 15. Anker, H. S. (1970) FEBS Lett. 7,293. in 16. Brown, J. P. & Perham, R. N. (1976) Biochem. J. 155, 419- poyl moieties in the transsuccinylase. However, the latter case 427. only interchain transfers can presumably occur, because each 17. Gunsalus, L. C. (1954) in The Mechanism ofEnzyme Action, eds. transsuccinylase chain bears only one lipoyl moiety. It is con- McElroy, W. D. & Glass, B. (Johns Hopkins Press, Baltimore, ceivable that preferred pathways within the network of lipoyl MD), pp. 545-580. moieties may be utilized when the whole complex is turning 18. Collins, J. H. (1977) Doctoral Dissertation, The University of over in the presence of CoA and DPN+-e.g., interaction of Texas at Austin. lipoyl moieties within a morphological subunit of the transa- 19. Williams, C. H., Jr. (1975) in The Enzymes, ed. Boyer, P. D. cetylase (or transsuccinylase),consistingofthreechains organized (Academic Press, New York), Vol. 13, pp. 89-173. about a 3-fold axis of rotation (1, 2). 20. Gutowski, J. A. & Lienhard, G. E. (1976) J. Biol. Chem. 251, of communication between moieties 2863-2866. The possibility lipoyl 21. Chang, F. N. & Flaks, J. G. (1972) J. Mol. Biol. 68, 177-180. in the pyruvate and a-ketoglutarate dehydrogenase complexes 22. Speckhard, D. C. & Frey, P. A. (1975) Biochem. Biophys. Res. was recognized in earlier papers from this laboratory (3, 23). Commun. 62, 614-620. The postulated communication network of lipoyl moieties 23. Reed, L. J. (1962) in Vitamins and Hormones, eds. Harris, R. S. provides a possible molecular basis for the finding by Moe et & Wool, I. G. (Academic Press, New York), Vol. 20, pp. 1-38. al. (24), from fluorescence energy transfer experiments, that 24. Moe, 0. A., Jr., Lerner, D. A. & Hammes, G. G. (1974) Bio- the apparent distance between FAD and thiamin pyrophos- chemistry 13,2552-2557. Downloaded by guest on September 28, 2021