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).