Mechanism of Action of the Pyruvate Dehydrogenase Multienzyme

Home , Dehydrogenase, Flavin mononucleotide, Pyruvate dehydrogenase

Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 4877-4880, October 1978 Biochemistry Mechanism of action of the pyruvate dehydrogenase multienzyme complex from Escherichia coli (protein modification/subunit interactions/enzyme mechanisms/flavoprotein) KIMON J. ANGELIDES AND GORDON G. HAMMES Department of Chemistry, Cornell University, Ithaca, New York 14853 Contrbuted by G. G. Hammes, August 4,1978

ABSTRACT The extent of cooperativity among the poly- catalytic sites of the three enzymes and between the lipoic acids peptide chain components in the overall reaction catalyzed by and the catalytic sites are too long to be consistent with this the pyruvate- dehydrogenase multienzyme complex from Escherichia coli has been studied. Selective inactivation of the mechanism (5, 9-11). Other recent results also have suggested pyruvate dehydrogenase component with thiamin thiazolone a more complex mechanism. (6, 8). pyrophosphate demonstrates that no cooperativity between this The present studies were undertaken to elucidate the degree component and the overall catalytic reaction occurs: the amount of interdependence of the individual components in the overall of overall complex activity is directly proportional to the fraction catalytic reaction in the intact pyruvate dehydrogenase com- of active pyruvate dehydrogenase component. The transacety- Rather than pursuing lase component has two lipoic acid residues on each of its plex. these relationships by self-assembly polypeptide chains that can be modified by Nf3Hjethylmalei- of a dissociated enzyme complex, we chose to modify specifi- mide in the presence of pyruvate and thiamin pyrophosphate. cally the individual components without dissociating the native The kinetics of the loss of overall complex activity due to mod- complex into individual subunit structures. Three sets of ex- ification of the lipoyl residues on the transacetylase component periments are described. Selective inactivation of E1 with by maleimide reagents shows that not all lipoic acids are cou- thiamin thiazolone pyrophosphate (TTPP) indicates that no pled into the overall catalytic reaction and that acyl-group and electron pair transfer involving two or more lipoic acids per cooperativity between E1 and the overall reaction occurs and catalytic cycle must occur. Finally, full complex activity is found that each catalytic cycle normally requires an E1 molecule. By when only half the normal flavin content is present. The results means of chemical modification of the lipoyl groups with indicate that extensive communication among lipoic acids in sulfhydryl reagents, the degree of communication among the acyl-group and electron pair transfer must exist in the normal lipoic acids on the core transacetylase component was exam- catalytic mechanism. These results are consistent with the av- ined. The results indicate that not all lipoic acids are coupled erage distances between catalytic sites measured by energy transfer experiments. into the overall catalytic mechanism, and acylgroup and electron pair transfer involving two or more lipoic acids on the E2 The pyruvate dehydrogenase multienzyme complex from core must occur. Finally, we find that the oxidation of dihy- Escherichia coli that catalyzes the overall reaction drolipoate by flavin can occur with one-half the full comple- ment of flavin adenine dinucleotide, further indicating ex- pyruvate + CoA + NAD+ -- acetyl-CoA tensive communication among lipoic acids. + CO2 + NADH + H+ [1] is composed of three enzymes: pyruvate dehydrogenase (E1) EXPERIMENTAL PROCEDURES which decarboxylates pyruvate and uses thiamin pyrophosphate Materials. The pyruvate dehydrogenase multienzyme (TPP) as a coenzyme; dihydrolipoyl transacetylase (E2) which complex from E. coli, strain B (Miles Laboratories) was pre- contains lipoic acid and transfers the acyl group to CoA; and pared and purified as described (9). The specific activity of the dihydrolipoyl dehydrogenase (E3), a flavoprotein that oxidizes complex, determined by using the NAD+ reduction assay at the dihydrolipoates formed. The E2 forms a structural core of 300, was 30-36 gmol of NADH/min per mg of protein. the assembled complex to which E1 and E3 bind. The E2 core The N-ethylmaleimide (MalNEt) was from Aldrich, and the probably has octahedral symmetry which is consistent with 24 tritiated compound (140 Ci/mol) was from New England polypeptide chains of E2 per molecule (cf. ref. 1). The total Nuclear. All other biochemicals were from Sigma. Other number of polypeptide chains in the intact complex is still a chemicals were the best reagent grades available, and deionized matter of debate. Reed et al. (2) have proposed 24:24:12 as the distilled water was used for all solutions. TTPP was prepared E1:E2:E3 polypeptide chain ratio in the native structure. Bates as described by Gutowski and Lienhard (12) and was further et al. (3) have concluded that the chain ratio varies between purified on an Amberlite CG-50 ion-exchange column in the 1:1:1 and 2:1:1 for the native complex. Several laboratories have hydrogen-ion form. The product was eluted with deionized shown that two lipoic acid residues are present per polypeptide distilled water. This preparation of TTPP showed a single chain of E2 (4-6) and that these lipoic acid residues can be en- UV-absorbing component with an RF of 0.48 on thin-layer zymatically acetylated (6-8). chromatography [Eastman cellulose plates; ethanol/n-buta- A model for the mechanism of action of this enzyme has been nol/0.15 M sodium citrate, pH 4, 10:1:6 (vol/vol)]. proposed (1) in which a single lipoic acid residue rotates be- Methods. The overall enzyme activity was determined with tween the catalytic sites of all three enzymes. Previous work in the NAD+ reduction assay (13). The activity of the pyruvate this laboratory, utilizing fluorescence resonance energy transfer dehydrogenase component was measured with the ferricyanide measurements, has shown that the average distances between assay (14), and the lipoamide dehydrogenase activity was de-

The publication costs of this article were defrayed in part by page Abbreviations: E1, pyruvate dehydrogenase; E2, dihydrolipoyl trans- charge payment. This article must therefore be hereby marked "ad- acetylase; E3, dihydrolipoyl dehydrogenase; MalNEt, N-ethylmalei- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate mide; TTPP, thiamin thiazolone pyrophosphate; TPP, thiamin py- this fact. rophosphate. 4877 Downloaded by guest on October 2, 2021 4878 Biochemistry: Angelides and Hammes Proc. Natl. Acad. Sci. USA 75 (1978) termined by measuring the reduction of lipoamide (13). Protein move more of the flavin adenine dinucleotide, the modified concentrations were determined by using the Lowry method enzyme complex was sometimes subjected to a second acid and (15) with bovine serum albumin as a standard. salt treatment. An additional 10% of the flavin adenine dinu- The E1 component of the enzyme complex was specifically cleotide was removed by this further treatment. Reincorpora- inactivated by titration with the active-site-directed inhibitor tion of the flavin adenine dinucleotide into E3 at 40 was ac- TTPP. Comparison of the dissociation constants of TTPP and complished by either incubating the enzyme with selected TPP shows that this inhibitor binds at least 20,000 times more amounts of flavin adenine dinucleotide or by adding an excess tightly than does the coenzyme (12). A specific concentration of flavin adenine dinucleotide to the enzyme and terminating of TTPP in 0.5 mM MgCl2, pH 7.0 or 8.0/0.02 M potassium the reincorporation at selected time intervals by passage phosphate at 40 was incubated with the enzyme. After 30 and through a Sephadex G-25 column with 0.02 M potassium 60 min, aliquots were withdrawn and assayed for overall phosphate (pH 7.0) as the eluant buffer. The eluant was col- complex activity and E1 activity at pH 7.0 and 8.0 (in 0.02 M lected, and the enzyme-containing fractions were identified potassium phosphate). Essentially the same activities were by monitoring the fluorescence of flavin adenine dinucleotide found with both aliquots. (360 nm excitation, 520 nm emission). Overall complex activity The enzyme complex was pretreated with unlabeled MalNEt and E1 and E3 activities were then measured. The specific ac- for 4 hr at 40 in the absence of substrates and was subsequently tivity of E1 gives a good measure of the amount of complex labeled with [3H]MalNEt in the presence of TPP and pyruvate destroyed during the procedure. Less than a 5% decrease in E1 to selectively modify the lipoic acids (5). The reaction mixture activity was observed. The amount of flavin adenine dinu- contained 2.08 mg of enzyme per ml, 0.54 mM TPP, 1.9 mM cleotide bound to the reconstituted complex was determined pyruvate, and 2.3 mM MgCl2 in 0.02 M potassium phosphate by two methods. One involved measurement of the flavin flu- (pH 7.0), and the reaction was initiated by addition of MalNEt orescence of the reconstituted complex relative to that of the to a final concentration of 0.31 mM. At selected time intervals native enzyme. Alternatively, the enzyme was precipitated with the reactionmixture was quenched with dithiothreitol (100-fold 50% trichloroacetic acid and centrifuged at 18,000 X g for 15 excess over MalNEt) and assayed for overall complex activity. min. The precipitate was resuspended in 5% trichloroacetic acid In addition, the activities of E1 and E3 were assayed to establish and recentrifuged. The supernatant was collected in the dark, that no inactivation of either component occurred upon in- and the flavin adenine dinucleotide in the supernatant was corporation of maleimide. The reaction mixtures were pre- hydrolyzed to flavin mononucleotide either by 1 M HCl at 500 cipitated by injection of cold 10% trichloroacetic acid into the for 1 hr (17) or by enzymatic hydrolysis at pH 7.0 with phos- vials. The protein was collected on Whatman glass microfiber phodiesterase (Naja naja venom; 15 mg/ml in 0.02 M potas- filters (GF/C or GF/A) and washed with 25 ml of cold 10% sium phosphate, pH 7.0). The flavin mononucleotide was trichloroacetic acid, 10 ml of water, and then 10 ml of petro- measured by comparing its fluorescence with that of standard leum ether. The dry filters were placed in vials of Aquasol and flavin mononucleotide solutions. Both methods of determining allowed to swell overnight; the radioactivity was determined flavin content gave identical results. Flavin fluorescence was in a Beckman LS-250 liquid scintillation counter. Analysis by measured with a Hitachi Perkin-Elmer MPF-3 spectrofluori- sodium dodecyl sulfate/polyacrylamide gel electrophoresis meter with excitation at either 360 nm or 445 nm and the indicated that all of the radioactivity was on E2 (5). emission scanned from 480 to 580 nm. The removal of flavin adenine dinucleotide from the enzyme complex was achieved by treatment of the multienzyme RESULTS complex with 43% saturated (NH4)2SO4 at pH 3.2 for 10 min Selective Inactivation of E1. The inhibitor TTPP was found on ice (16). The precipitated enzyme was resuspended in 0.02 to inactivate the overall complex activity and the activity of E1 M potassium phosphate (pH 7.0) and dialyzed at 40 to remove stoichiometrically. The activity of EF was unaffected by TTPP. the ammonium sulfate. This treatment removed 75-80% of the The results obtained at pH 7.0 and 8.0 are summarized in Fig. total flavin adenine dinucleotide content. In an attempt to re- 1A. Fig. 1B shows the variation of the overall enzyme activity

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0 20 40 60 80 100 0 1.0 2.0 3.0 4.0 Percent Activity of El TTPP (pM) FIG. 1. Selective inactivation of E1 by TTPP. A mixture of varying amounts of TTPP and pyruvate dehydrogenase complex (0.7 mg/ml) was incubated in 0.02 M potassium phosphate, pH 7.0 or 8.0/1 mM MgCl2 at 4°. The overall complex and E1 activities were assayed at both pH 7.0 (0) and 8.0 (-) in 0.02 M potassium phosphate. (A) Percentage overall activity vs. percentage E1 activity. (B) Titration of the pyruvate dehydrogenase multienzyme complex with TTPP. Percentage overall activity vs. concentration of TTPP is shown. All activities are referred to the unmodified complex under identical conditions. Downloaded by guest on October 2, 2021 Biochemistry: Angelides and Hammes Proc. Natl. Acad. Sci. USA 75 (1978) 4879

Displacement of the TTPP did not occur within the time 0-9 course (<2 min) of the assay because no evidence of a lag period 32 -0o-o, was observed upon addition of TPP. However, when 4.4 mM TPP was incubated for 10-20 min with the TTPP-inactivated 28 complex, about 10% of both overall and E1 activities were restored, indicating that some TTPP can be displaced by TPP. x 24 A sample to which no TTPP was added served as a control, and 0. - 0 all activities are expressed relative to the activity of this con- E 01 trol. 0 20 0 TPP-Pyruvate-Dependent Incorporation of MaINEt. In- 0 Oa activation of the complex was achieved by reductive acetylation 6 of the lipoic acid moieties on the transacetylase followed by 0~~~~~ maleimide incorporation onto the free sulfhydryl group of the was per 12 _ o lipoic acid. A total of 10 nmol of MalNEt incorporated mlg of protein, which corresponds to about 46 mol of label per ._ mol of complex if a molecular weight of 4.6 X 106 is assumed. 8 °900 The kinetics of inactivation are complex: a typical plot of the 0 0. loss of overall complex activity vs. time is shown in Fig. 2. Under Un 4 - -0--,~~o---O identical conditions, inactivation of the complex was more rapid with aromatic maleimides [pyrene- and N-(4-dimethyl- amino-3,5-dinitrophenylmaleimide] than with MalNEt. Both 0 E1 and E3 activities were unaffected by reaction with MalNEt 0 40 80 120 160 200 240 280 but the overall complex activity was decreased. A plot of the Time (min) overall activity vs. the fraction of unmodified lipoic acids is FIG. 2. Time course of the inactivation of the pyruvate dehy- presented in Fig. 3. The sigmoidal character of this plot indi- drogenase complex by MalNEt. The specific activity (in gmol of cates that the loss of overall complex activity was not propor- NADH/min per mg of protein) vs. time is shown for a solution con- tional to the loss of free lipoic acid. Furthermore, only after taining 0.31 mM [3H]MalNEt, 2.08 mg of prelabeled enzyme per ml, approximately 10% of the lipoic acids were modified by the 1.9 mM pyruvate, 0.54 mM TPP, 2.3 mM MgCl2, and 0.02 M potas- maleimides did a loss occur in the overall catalytic activity of sium phosphate (pH 7.0) at 4°. the complex. The remainder of the time course of the inactivation required at least two exponential decay terms for a with the concentration of TTPP at both pH 7.0 and 8.0. On the mathematical description of the kinetic curve. The overall basis of a molecular weight of 4.6 X 106 for the enzyme com- activity'did not drop to zero even after 4 hr, although it did so plex, complete loss of overall activity corresponds to 24 mol of either at sufficiently long times or after addition of fresh TPP TTPP per mol of complex. and pyruvate. A Hill-type plot of the logarithm of the relative

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301 0 0~ 0~ 301 0 0' 201 / 20 101 OZ0 10 0 o 0 0.2 0.4 0.6 0.8 1.0 . I I I I I I I Fraction of Unmodified Lipoic Acid 0 0.2 0.4 0.6 0.8 1.0 FIG. 3. Percentage overall complex activity vs. fraction of un- Fraction Reconstituted FAD modified lipoic acid. Labeling and stoichiometry measurements were FIG. 4. Percentage of pyruvate dehydrogenase complex activity as described in Experimental Procedures, and the reaction conditions vs. fraction of flavin adenine dinucleotide reconstituted. The line were similar to those in Fig. 2. drawn is a theoretical curve calculated as described in the text. Downloaded by guest on October 2, 2021 4880 Biochemistry: Angelides and Hammes Proc. Natl. Acad. Sci. USA 75 (1978) activity vs. the logarithm of the fraction of unmodified lipoic ments have indicated that lipoic acids within a molecule are acids had a maximum slope of 2.2, indicating that >2.2 lipoic sufficiently close to permit eximer formation (5); more recent acids are required for each catalytic cycle. energy transfer measurements have shown directly that one Dependence of Overall Complex Activity on Flavin Ade- lipoic acid is within 24 A of another lipoic acid (unpublished nine Dinucleotide. When the overall complex activity was data). Thus, transacetylation involving at least two lipoic acids measured with varying amounts of flavin adenine dinucleotide appears to be part of the normal catalytic reaction. present on the enzyme, full activity was attained under con- The finding that only half of the normal flavin content is ditions such that not all of the flavin adenine dinucleotide of necessary for full activity is most readily explained by postu- the native structure was restored. The results obtained are lating extensive reoxidation among lipoic acids with a single shown in Fig. 4 as a plot of the percentage of overall activity flavin able to service at least eight lipoic acids [assuming the vs. the fraction of reconstituted flavin. The turnover rate of the subunit stoichiometry of Reed et al. (2)]. This also suggests that fully reconstituted enzyme was identical, within experimental lipoic acid oxidation is probably not rate determining in the error, to that of an untreated "native" complex, provided that overall reaction. Previous work has suggested that a full com- the reconstitution was done within 30 min after loss of the flavin plement of flavin molecules is not necessary for activity (16), adenine dinucleotide. The curve shown in Fig. 4 was calculated but reconstitution did not yield a fully active enzyme as in this by assuming a binomial distribution of the flavin adenine di- work. nucleotide on the enzyme for a given fraction of total sites oc- In summary, we have demonstrated that cooperativity cupied and assuming enzyme with 6-12 flavins is fully active (presumably transacetylation) among a minimum of two lipoic but that with 0, 1, 2, 3, 4, or 5 flavins have relative activities of acids is essential for normal catalytic activity. Furthermore, a 0, 0.05, 0.1, 0.3, 0.6, and 0.8, respectively. Thus, apparently only significant number of lipoic acids is not essential for overall half of the flavin content of the native enzyme is necessary for catalytic activity although they can be reductively labeled. full activity of the complex. Oxidation-reduction among lipoic acids also can occur, pro- viding further evidence for an extensive communication net- DISCUSSION work between lipoic acids on the E2 core. Finally a single E1 The fact that the loss in E1 activity is directly proportional to normally services a single catalytic cycle. Such a mechanism the loss in the overall enzyme activity indicates that a single E1 is consistent with the average distances between catalytic sites molecule is involved in each catalytic cycle. This is consistent measured by energy transfer experiments. with previous measurements using TTPP (12) and reconstitu- This work was supported by grants from the National Institutes of tion experiments (8). The suggestion has been made that a single Health (GM 13292) and the National Science Foundation (PCM 77- E1 can service multiple lipoic acids (6, 8). Collins and Reed (6) 11392). have found that the complex can be fully acetylated even 1. Reed, L. J. (1974) Acct. Chem. Res. 7, 40-46. though E1 was 90% inhibited by TTPP. However, the acety- 2. Reed, L. J., Pettit, F. M., Eley, M. M., Hamilton, C., Collins, J. lation was quite slow (reaction time, #30 min) and might be H. & Oliver, R. M. (1975) Proc. Natl. Acad. Sci. USA 72, due to dissociation or migration of TTPP on the complex. Such 3068-3072. a slow rate cannot be of significance in the normal activity of 3. Bates, D. L., Harrison, R. A. & Perham, R. N. (1975) FEBS Lett. the enzyme. Bates et al. (8) have found that a complex of E2 and 60,427-430. 4. Danson, M. J. & Perham, R. N. (1976) Biochem. J. 159, 677- E3 partially reconstituted with E1 can be fully acetylated, al- 682. though this might be due to migration of E1 on the complex. 5. Shepherd, G. & Hammes, G. G. (1977) Biochemistry 16, The roleof transacetylation in the normal activity of the com- 5234-5241. plex is not clear from this experiment. 6. Collins, J. H. & Reed, L. J. (1977) Proc. Nati. Acad. Sci. USA 74, The time course of lipoic acid labeling and the correlation 4223-4227. between activity and extent of labeling indicates that at least 7. Speckhard, D. C., Ikeda, B. M., Wong, S. S. & Frey, P. A. (1977) three classes of lipoic acids exist: approximately 10% of the lipoic Biochem. Biophys. Res. Commun. 77,708-713. acids are not involved in the overall activity; the labeling of the 8. Bates, D. C., Danson, M. J., Hale, G., Hooper, E. A. & Perham, other lipoic acids indicates at least two additional classes of lipoic R. N. (1977) Nature (London) 268,313-316. 9. Shepherd, G. & Hammes, G. G. (1976) Biochemistry 15,311- acids-i.e., lipoic acids in different environments. The sensi- 317. tivity of this reaction to environment is indicated by the 10. Shepherd, G., Papadakis, N. & Hammes, G. G. (1976) Bio- markedly different rates of reaction with aromatic maleimides. chemistry 15,2888-2893. The existence of multiple classes of lipoic acids is supported by 11. Moe, 0. A., Jr., Lerner, D. A. & Hammes, G. G. (1974) Bio- energy transfer experiments in which different fractions (i.e., chemistry 12,2552-2557. fast-reacting or slow-reacting) of the lipoic acids are labeled 12. Gutowski, J. A. & Leinhard, G. E. (1976) J. Biol. Chem. 251, with an energy donor; the different fractions of donors are 2863-2866. found to be at different distances from the catalytic sites of E1 13. Reed, L. J. & Willms, C. R. (1966) Methods Enzymol. 9,246- and E3 (unpublished data). A recent measurement of the 265. equivalents of NADH produced per catalytic cycle suggests that 14. Schwartz, E. R., Old, L. 0. & Reed, L. J. (1968) Biochem. Bio- phys. Res. Commun. 31,495-500. half of the lipoic acids are not involved in the overall catalytic 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. reaction (18), which is consistent with the finding that lipoic (1951) J. Biol. Chem. 193,265-275. acids are not necessary for the catalytic reaction. Finally, the 16. Koike, M. & Reed, L. J. (1960) J. Biol. Chem. 235, 1931-1938. relationship between the extent of lipoic acid labeling and the 17. Koziol, J. (1971) Methods Enzymol. 18B, 253-281. overall activity clearly demonstrates that at least two lipoic acids 18. Frey, P. A., Ikeda, B. H., Gavino, G. R., Speckhard, D. C. & are involved in each catalytic cycle. Fluorescence measure- Wong, S. S. (1978) J. Biol. Chem., in press. 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