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MICHAEL J. DANSON*, ALAN R. FERSHTtt, AND RICHARD N. PERHAM*. *Department of Biochemistry, University of Cambridge, Cambridge CB2 lQW, ...
Proc. Nati. Acad. Sci. USA

Vol. 75, No. 11, pp. 5386-5390, November 1978 Biochemistry

Rapid intramolecular coupling of active sites in the pyruvate dehydrogenase complex of Escherichia col: Mechanism for rate enhancement in a multimeric structure (multienzyme complex/lipoic acid transacetylation/pulsed-quenched flow)

MICHAEL J. DANSON*, ALAN R. FERSHTtt, AND RICHARD N. PERHAM* *Department of Biochemistry, University of Cambridge, Cambridge CB2 lQW, England; and tMeaical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, England

Communicated by M. F. Perutz, August 21, 1978

In the absence of CoA and presence of pyrABSTRACT uvate, the lipoic acid residues covalently bound to the lipoate acetyltransferase core component acetyl-CoA:dihydrolipoate S-acetyltransferase, EC 2.3.1.12) of the pyruvate dehydrogenase multienzyme complex of -Escherichia coli become reductively acetylated. A study of a series of reassembled complexes varying only in their content of pyruvate decarboxylase [pyruvate:ipoate-oxidoreductase (decarboxylating and acceptor-acetylating) EC 1.2.4.11 showed that the initial direct reductive acetylation of Ii oic acid residues can be followed by extensive intramolecular transacetylation reactions between lipoic acid residues on neighboring polypeptide chains of the lipoate acetyltransferase core [Bates, D. L., Danson, M. J., Hale, G., Hooper, E. A. & Perham, R. N. (1977) Nature (London) 268,313-316]. Pulsedquenched-flow measurements of the rates of the acetylation reactions in the various complexes now demonstrate that the intramolecular transacetylation reactions are not rate-determining in the normal reaction mechanism of the enzyme. There is therefore the potential for rapid multiple coupling of active sites in the lipoate acetyltransferase core. The rate constant for the overall complex reaction, measured by stopped-flow fluorimetry, is found to be approximately twice that for the reductive acetylation reaction measured by pulsed-quenched flow. This result could mean that CoA is an allosteric stimulator of the reductive acetylation part of the overall reaction or that there are two active sites on each chain of the lipoate acetyltransferase component working in parallel. A system of ra pid functional connection of active sites in a multienzyme complex ensures that sequential reactions can be success ully coupled even under conditions of low substrate concentrations for the different steps. The substantial rate enhancement thus achieved offers a plausible explanation for the unusual complexity of the quaternary structure of the enzyme.

The pyruvate dehydrogenase multienzyme complex of Escherichia coli contains three different polypeptide chains, responsible for the three component enzymic activities [for reviews, see refs. 1 and 2]. These are pyruvate decarboxylase [El; pyruvate:lipoate oxidoreductase (decarboxylating and acceptor acetylating), EC 1.2.4.1], lipoate acetyltransferase (E2; acetyl CoA:dihydrolipoate S-acetyltransferase, EC 2.3.1.12), and lipoamide dehydrogenase (ES; NADH:lipoamide oxidoreductase EC 1.6.4.3). Together, they catalyze the oxidative decarboxylation of pyruvate via the following sequence of reactions: Mg2+

Pyruvate + (TPP)-El -1 (hydroxyethyl-TPP)-El + CO2 [1]

(hydroxyethyl-TPP)-El + (lipS2)-E2 (TPP)-E1 + (acetyl-S-lip-SH)-E2 [2] (acetyl-S-lipSH)-E2 + CoA

(lip(SH)2)-E2 + acetyl-SCoA [31

(Lip(SH)2)-E2 + (FAD)-E3 + NAD+ (lip S2)-E2 + (FAD)-E3 + NADH + H+ [4] Overall reaction: Pyruvate + CoA + NAD+ acetyl-SCoA + NADH + H+ + CO2 in which lipS2 is an oxidized lipoic acid residue, lip(SH)2 is a dihydrolipoic acid residue, and TPP is thiamin pyrophosphate. The substrate is carried by lipoic acid residues covalently attached to lysine residues on the E2 polypeptide chains (3). Measurements of the mobility of spin-labeled lipoyl groups are consistent with this "swinging arm" mechanism (4, 5). We had examined the fate of pyruvate decarboxylated within the pyruvate dehydrogenase complex in the absence of CoA (reactions 1 and 2) and found that a single El dimer can bring about the reductive acetylation of multiple copies of the E2 chains in the core of the complex (6, 7). We therefore proposed that the lipoyllysine swinging arms can participate in two types of subunit interactions: each El dimer can interact directly with the lipoic acid residues of a small number of E2 chains, followed by the transfer of acetyl groups between the lipoic acid residues of neighboring E2 chains in the complex. We referred to these internal reactions as "transacetylations" (6). This work has been confirmed by Collins and Reed (8). In the present paper we show that the intramolecular transacetylation reactions are sufficiently fast to take part in the overall enzymic reaction, and we incorporate them into a reaction scheme that can account for the complex subunit structure of the enzyme. MATERIALS AND METHODS Reagents. [2-14C]Pyruvate (sodium salt) (CFA.79) was obtained from The Radiochemical Centre (Amersham, Bucks, United Kingdom) and assayed spectrophotometrically as NADH produced in the presence of lactate dehydrogenase. DL-Dihydrolipoamide was prepared by the reduction of lipoamide with sodium borohydride (9). Enzyme and Enzyme Assays. Pyruvate dehydrogenase multienzyme complex was purified from a pyruvate dehydrogenase constitutive mutant of E. coli as described by Reed and Mukherjee (10). The mutant organism was kindly provided by H. L. Kornberg. From the native enzyme, four partly reassembled pyruvate dehydrogenase complexes (R1-R4), varying only in their El content, were prepared as described by Bates et al. (6). The ratios of the polypeptide chains in these complexes were determined by the radioamidination method of Abbreviations: El, pyruvate decarboxylase; E2, lipoate acetyltransferase; E3, lipoamide dehydrogenase; lipS2, oxidized lipoic acid residue; lip(SH)2, dihydrolipoic acid residue; TPP, thiamin pyrophosphate. * Present address: Department of Chemistry, Imperial College of Science and Technology, London, England.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Table 1. Polypeptide chain stoichiometries and overall complex activities of native and partly reassembled pyruvate dehydrogenase complexes Polypeptide chain stoichiometry El Enzyme* E2 E3 Enzymic activityt 0.21 h 0.04 Ri 1.0 0.89 + 0.03 11.9 + 0.4 0.39 ± 0.02 R2 1.0 0.95 + 0.02 20.8 + 0.8 0.71 0.04 1.0 0.97 I 0.08 R3 34.7 + 1.8 1.29 + 0.07 R4 1.0 0.80 + 0.10 90.9 + 2.5 1.24 : 0.08 1.0 0.95 ± 0.03 Native 80.8 + 1.7 Polypeptide chain stoichiometries, measured by radioamidination (11), are based on E2 as unity. Results are ±SEM. For the enzymic activities, each SEM is a combination of the SEMs of the stopped-flow in fluorimetric assays, protein concentrations, and the polypeptide chain stoichiometries. * R1-R4 are the partly reassembled complexes. t At 250C, shown as ,umol of NADH per ,umol of E2 chain per sec measured by stopped-flow fluorimetry in 4.8 mM pyruvate.

Bates et al. (11). The enzymic activities of the whole complex and the lipoamide dehydrogenase were assayed spectrophotometrically (7). Rates of Incorporation of [2-4C]Pyruvate into Partly Reassembled Pyruvate Dehydrogenase Complexes. The rates of incorporation of acetyl groups from [2-14C]pyruvate into native and partly reassembled complexes were measured at 25°C by using the pulsed-quenched flow apparatus of Fersht and Jakes (12). One syringe contained enzyme (120 ,ug/ml) in 100 mM sodium phosphate buffer, pH 7.0/1.4 mM TPP/20 mM MgCl2/2 mM NAD+. The other syringe contained 0.036 mM [2-14C]pyruvate in glass-distilled water. This low concentration of substrate (final concentration, Km/27) was used to slow the enzyme reaction sufficiently for accurate kinetic measurements to be made. The quenching syringe contained 10% trichloroacetic acid. The effluent from the second mixing chamber was collected in acid-washed tubes, filtered onto nitrocellulose discs, washed with 15 ml of ice-cold 10% trichloroacetic acid, and dried. The radioactivity was measured in a nonrefrigerated Beckman LS-233 liquid scintillation counter using a toluenebased scintillant [12.5 g of 2,5-diphenyloxazole and 0.75 g of 1,4-bis(2-[5-phenyloxazyl])benzene in 2.5 liters of toluene (13)]. Blank samples were set up manually by mixing enzyme with trichloroacetic acid before the addition of [2-14C]pyruvate. The precipitated protein was then treated in exactly the same way as the test samples. Stopped-Flow-Fluorescence Measurements of Overall Complex Activities. The overall complex activities of native and partly reassembled pyruvate dehydrogenase complexes were measured at 25°C by stopped-flow fluorimetric assays of the NADH produced (13, 14). One syringe of the spectrofluorimeter contained enzyme (120 ,Ag/ml) in 50 mM sodium phosphate buffer, pH 7.0/0.7 mM TPP/10 mM MgCl2/1 mM NAD+/1.30 mM CoA/52 mM cysteine hydrochloride. The other syringe contained 9.6 or 0.036 mM sodium pyruvate in the same buffer but without the CoA and cysteine hydrochloride. The increase in fluorescence (excitation at 340 nm with a monochromator and UG 11 filter; emission at 460 nm collected with a 3-73 filter) caused by formation of NADH on mixing was monitored on a storage oscilloscope. The enzyme concentration in each assay was identical with that used in the pulsed-quenched-flow studies. RESULTS Polypeptide Chain Ratios in the Pyruvate Dehydrogenase Complexes. The stoichiometries of the polypeptide chains in the native and partly reassembled pyruvate dehydrogenase complexes are given in Table 1. The overall complex activities

measured in the spectrophotometric assay varied directly with the El content, as reported by Bates et al. (6). There was no significant difference in the lipoamide dehydrogenase enzymnic activities of the various complexes. Rates of Incorporation of [2-14CJPyruvate into the Pyruvate Dehydrogenase Complexes. The results of the pulsedquenched-flow experiments are illustrated in Fig. 1. On mixing 100

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61 16 3Is Time (sl FIG. 1. Incorporation of [2-'4C]pyruvate into a series of partly assembled pyruvate dehydrogenase complexes measured with the pulsed-quenched-flow apparatus. Enzyme was preincubated with TPP, MgCl2, and NAD+ in one syringe and then mixed with [214C]pyruvate from the other syringe to give the following final concentrations: 0.7 mM TPP, 10 mM MgCl2, 1.0 mM NAD+, and 0.018 mM [2-14C]pyruvate. The buffer was 50 mM sodium phosphate at pH 7.0. For each complex, the incorporation of radiolabel is expressed as the percentage of the E2 core not acetylated, taking the total incorporation observed as 100%. Reassembled pyruvate dehydrogenase complexes (R): *, Ri; &, R2; A, R3; and 0, R4. For native enzyme (O), only one of the two estimations is shown, for clarity.

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of enzyme with substrate, the incorporation of [14C]acetyl groups from [2-14C]pyruvate into each complex followed a single pseudo-first-order exponential. Even when the El/E2 chain ratio was as low as 0.2:1-i.e., 10% of the maximal ratio attainable (6)-at least 97% of the total incorporation of radioactivity could be described by a single rate constant. Moreover, as shown in Fig. 2, the first-order rate constant for the acetylation was directly proportional to the El content of the complex. The fractions of the E2 core acetylated at the end of the reaction for the native complex and the four reassembled complexes were as follows: native, 1.00; RI, 0.64; R2, 0.93; R3, 1.00; R4, 1.00. These values are fully consistent with the extents of acetylation found in our previous experiments (6). The maximal incorporation of radioactivity corresponds to 2.1 ± 0.3 (mean + SEM) acetyl groups per E2 chain, in excellent agreement with our earlier estimations of the acetylatable lipoic acid content of the complex (15). Stopped-Flow-Fluorescence Measurements of the Overall Complex Activities. To correlate the rates of acetylation obtained in the pulsed-quenched-flow studies with the rate of turnover of pyruvate by the same enzymes in the whole complex reaction, the overall enzymic activities of the native and reassembled complexes were measured. The use of stoppedflov fluorimetry enabled these experiments to be carried out with identical concentrations of enzymes and in the same buffer as the pulsed-quenched-flow experiments, except that the measurements of whole complex activities necessarily required the presence of CoA. They were performed at 4.8 mM pyruvate (10 X Km) to enable them to be compared with similar spectrophotometric assays of. the activities. For a direct comparison with the rate of reductive acetylation, native complex was also assayed by stopped-flow fluorimetry at the same pyruvate concentration (0.018 mM) as that used in the pulsedquenched-flow experiments. The overall complex activities are given in Table 1. These activities are in good agreement with those measured in the spectrophotometric assay at 340 nm, which necessarily uses

much lower concentrations of enzyme. For example, spectrophotometric assays of the native complex gave a mean (+SEM) turnover number of 79 +5 ,umol of NADH per ,tmol of E2 chain per sec at 25°C, a value very close to that found by stopped-flow fluorimetry (Table 1). Therefore, as one would expect from previous observations (6) and from the experiments described above, the enzymic activities measured by stopped-flow fluorimetry were directly proportional to the El content of the complex. Spectrophotometric assays at 340 nm of the native enzyme and partly reassembled complex (R3) demonstrated normal Michaelis-Menten kinetics with respect to pyruvate, giving Km values for the substrate of 0.43 ±0.05 and 0.49 +0.03 mM, respectively. From the stopped-flow fluorimetric assays of native complex, the Km was calculated to be 0.48 +0.02 mM, in good agreement with that determined spectrophotometrically. Comparison of the Rates of Acetylation with the Overall Complex Activities. In Fig. 3, the first-order rate constants for the acetylation of the variously assembled complexes in the absence of CoA, determined by the pulsed-quenched-flow experiments at 0.018 mM pyruvate (Km/27), are plotted against the respective enzymic activities given in Table 1 for the overall reaction in the presence of CoA and 4.8 mM pyruvate (10 X Km). The enzymic activity is directly proportional to the rate constant for acetylation. Comparing the data at the same concentration of pyruvate (0.018 mM), by using the MichaelisMenten equation and the measured Km value of 0.48 mM, gives a value of 2.74 +0.14 to 1 for the ratio of specific rate constant (,umol of NADH per ,tmol of E2 chain per sec) of the overall enzymic reaction to the first-order rate constant for the part (acetylation) reaction. This value was confirmed directly for the native complex by measuring the rate of the overall reaction by stopped flow fluorimetry at 0.018 mM pyruvate, from which a ratio of 2.64 +0.10 to 1 was calculated.

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El: E2_ chain ratio rates of acetylation of the partly asthe FIG. 2. Comparison of sembled pyruvate dehydrogenase complexes with their E1/E2 polypeptide chain ratios. The rates of acetylation, determined at 0.018 mM [2-14C]pyruvate, are calculated from the data presented in Fig. 1. The E1/E2 chain ratios are those given in Table 1.

50 75 25 Overall complex activity (pmol NADH/ pmol E2

100

chain/s) FIG. 3. Comparison of the rates of acetylation of the partly assembled pyruvate dehydrogenase complexes with their overall complex activities. The rates of acetylation, determined at 0.018 mM [2-14C]pyruvate, are calculated from the data presented in Fig. 1. The overall complex activities are those given in Table 1 and were determined at 4.8 mM pyruvate.

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Before the significance of this ratio can be fully assessed, the influence of cysteine hydrochloride on the overall complex activities must be determined. Cysteine was added routinely with the CoA in the fluorimetric assays to keep the coenzyme fully reduced, but it was omitted from the pulsed-quenchedflow experiments for which CoA was required to be absent. Spectrophotometric assays (at 340 nm) of the overall enzymic activities of partly assembled and native complexes were therefore carried out in the absence and presence of 26 mM cysteine. Cysteine hydrochloride was neutralized with NaOH and this solution and the assay mixtures were kept under N2 to minimize oxidation of the thiol compounds. All other conditions were identical with those used in the stopped-flow fluorimetric assays. It was found that the inclusion of 26 mM cysteine in the assay mixture increased the overall complex activities 1.31-fold (SEM, +0.07). In both the presence and absence of cysteine, the CoA concentration was saturating with regard to the overall complex activity. The final estimate that can be made from the comparison of the stopped-flow data is that the ratio of the overall pyruvate dehydrogenase activity to the rate of acetylation of the lipoic acid residues is 2.74/1.31, which equals 2.09 ±0.15. DISCUSSION Investigations of the fate of substrate within the pyruvate dehydrogenase complex of E. coli have demonstrated that, in the absence of CoA, acetyl groups can be transferred from the lipoic acid residues on one E2 polypeptide chain to those on neighboring E2 chains of the same core (6, 7). By means of these novel transacetylation reactions, a single El dimer is capable of catalyzing the acetylation of the lipoic acid residues of probably 12 E2 chains. Before we can speculate about the physiological significance of such transacetylation, it is necessary to determine the rate at which it occurs with respect to the other catalytic processes of the enzymic mechanism. The rate of incorporation of acetyl groups into the lipoic acid residues from [2-'4C]pyruvate has now been measured in a pulsed-quenched-flow apparatus. As in our previous studies, it seemed most fruitful to use partly reassembled complexes in these experiments, particularly ones with low El contents in which there is the potential for extensive transacetylation among the E2 component chains. In each complex it was found that the acetylation process could be described by a single exponential curve, even when the El/E2 polypeptide ratio was as low as 0.2:1.0 (Fig. 1). We conclude that the rate at which acetyl groups are transferred between the lipoic acids residues on different E2 chains is equal to, or greater than, the rate at which any lipoic acid receives an acetyl group by direct reaction with an El component. If the initial reductive acetylation of lipoic acid residues by El had been faster than the subsequent internal transacetylation reactions between lipoic acid residues, the incorporation profiles would have been composed of more than one exponential. The faster component would describe the direct reductive acetylation of lipoic acid residues on E2 by El and its amplitude should be proportional to the El content of the complex. This plainly is not so. Given that the rate of acetylation of the E2 core in a partly reassembled complex is not dependent on the rate of the transacetylation processes, one would expect it to depend the rate of flow of substrate through the E2 core as a whole. The catalytic activities of the various complexes were found to be directly proportional to the rate constants for the acetylation of the E2 cores, confirming this prediction (Fig. 3). Thus, the rates of acetylation and the catalytic activities vary directly with the polypeptide chain ratio, El/E2. Comparison of the specific rate constants (,Mmol of NADH on

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per,umol of E2 chain per sec) for the overall enzymic reaction of the complexes with the pseudo-first-order rate constants for the reductive acetylation of the lipoic acid residues gave a ratio of 2.1:1. It is possible that this ratio departs from the obvious value of unity because of experimental error, although this is unlikely in view of the good agreement between the measurements of overall catalytic activity by stopped-flow fluorimetry and by steady-state spectrophotometric assay. The only difference in experimental conditions was the presence of CoA in measurements of the overall catalytic activity of the complexes and its enforced absence from measurements of the rates of acetylation of the E2 core. Thus, it is conceivable that CoA can stimulate the acetylation reaction, presumably by some allosteric effect because it plays no direct part in the mechanism until the lipoic acid residues have become acetylated. A third possibility to account for the measured ratio of 2.1:1 for the rate constants is that two identical part reactions, running in parallel, contribute to the overall complex reaction. It should be be borne in mind that two lipoic acid residues on each E2 polypeptide chain become reductively acetylated in the part reaction (6-8, 15, 16). A clear conclusion to be drawn from our work is that the transacetylation reactions between lipoic acid residues in the E2 core take place faster than the initial reductive acetylation of a lipoic acid residue by reaction with enzyme El. Their part in the enzyme mechanism therefore merits consideration. Internal transacetylation reactions of the same kind have also been detected in the 2-oxoglutarate dehydrogenase complex of E. ccli (8) and they are therefore not limited to the pyruvate dehydrogenase complex. It is generally accepted that the intracellular concentrations of pyruvate and CoA are low in E. coli, which would lead to problems of diffusion in the formation of acetyl-SCoA from these two substrates catalyzed by three successive enzymes. The association of multiple copies of the three enzymes into the 2-oxoacid dehydrogenase complexes, with the potential that we have now demonstrated for rapid transfer of acyl groups from one E2 chain to another within the core, provides a way around such problems. Thus, pyruvate decarboxylated by one El dimer can be connected with CoA bound to any one of probably 12 E2 chains (6, 7), relieving the requirement for pyruvate and CoA to bind to an El chain and an E2 chain that are directly associated with one another. This would bring a substantial rate enhancement to the formation of acetyl-SCoA when the substrates are in low concentration. Previous rationalizations of the existence of multienzyme complexes have turned on the advantages of kinetic enhancements to be gained by reducing diffusion distances between active sites, on substrate channeling, and other ideas (1, 2). The enormous size of the pyruvate and 2-oxoglutarate dehydrogenase complexes has remained a puzzle. The rapid coupling of multiple active sites in the same enzyme particle that we have now documented provides a plausible reason. This work was supported by a Science Research Council Advanced Fellowship to M.J.D. and a research grant (B/RG/6689.6) to R.N.P. from the Science Research Council and by the Wellcome Trust.

1. Reed, L. J. (1974) Acc. Chem. Res. 7,40-46. 2. Perham, R. N. (1975) Philos. Trans. R. Soc. London Ser. B. 272, 123-136. 3. Nawa, H., Brady, W. T., Koike, M. & Reed, L. J. (1960) J. Am. Chem. Soc. 82,896-903. 4. Ambrose, M. C. & Perham, R. N. (1976) Biochem. J. 155, 429-432. 5. Grande, H. J., Van Telgen, H. J. & Veeger, C. (1976) Eur. J. Biochem. 71, 509-518. 6. Bates, D. L., Danson, M. J., Hale, G., Hooper, E. A. & Perham, R. N. (1977) Nature (London) 268,313-316.

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7. Danson, M. J., Hooper, E. A. & Perham, R. N. (1978) Biochem. J. 175, 193-198. 8. Collins, J. H. & Reed, L. J. (1977) Proc. Nati. Acad. Sci. USA 74, 4223-4227. 9. Reed, L. J., Koike, M., Levitch, M. E. & Leach, F. R. (1958) J. Biol. Chem. 232,143-158. 10. Reed, L. J. & Mukherjee, B. B. (1969) Methods Enzymol. 13, 55-61. 11. Bates, D. L., Harrison, R. A. & Perham, R. N. (1975) FEBS Lett. 60,427-430.

Proc. Nati. Acad. Sci. USA 75 (1978) 12. Fersht, A. R. & Jakes, R. (1975) Biochemistry 14, 3350-3356. 13. Fersht, A. R., Ashford, J. S., Bruton, C. J., Jakes, R., Koch, G. L. E. & Hartley, B. S. (1975) Biochemistry 14,1-4. 14. Fersht, A. R., Mulvey, R. S. & Koch, G. L. E. (1975) Biochemistry 14, 13-18. 15. Danson, M. J. & Perham, R. N. (1976) Biochem. J. 159, 677682. 16. Speckhard, D. C., Ikeda. B. H., Wong, S. S. & Frey, P. A. (1977) Biochem. Biophys. Res. Commun. 77,708-713.