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Mechanism of Action of the Pyruvate Dehydrogenase Multienzyme

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Mechanism of Action of the Pyruvate Dehydrogenase Multienzyme 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 elec- tron 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).
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