A-KETO ACID DEHYDROGENASE COMPLEXES, X. Complex

a-KETO ACID DEHYDROGENASE COMPLEXES, X. REGULATION OF THE ACTIVITY OF THE PYRUVATE DEHYDROGENASE COMPLEX FROM BEEF KIDNEY MITOCHONDRIA BY PHOSPHORYLATION AND DEPHOSPHORYLA TION* BY TRACY C. LINNt FLORA H. PETTIT, AND LESTER J. REED

CLAYTON FOUNDATION BIOCHEMICAL INSTITUTE AND DEPARTMENT OF CHEMISTRY, UNIVERSITY OF TEXAS (AUSTIN) Communicated by Roger J. Williams, October 2S, 1968 Abstract and Summary.-This paper reports the discovery that the activity of the multienzyme pyruvate dehydrogenase complex from beef kidney mitochondria is regulated by a phosphorylation-dephosphorylation reaction sequence. The site of this regulation is the pyruvate dehydrogenase component of the complex. Phosphorylation and concomitant inactivation of pyruvate dehydrogenase are catalyzed by an ATP-specific kinase (i.e., a pyruvate dehydrogenase kinase), and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase (i.e., a pyruvate dehydrogenase phosphatase). The kinase and the phosphatase appear to be regulatory subunits of the pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex (PDC) catalyzes a coordinated sequence of reactions that can be represented by over-all reaction (1). CH3COCO2H + CoA-SH + DPN + CH3CO-S-CoA + C02 + DPNH + H+. (1) This paper reports the discovery that the activity of PDC from beef kidney mitochondria is subject to regulation by a phosphorylation-dephosphorylation reaction sequence. The highly purified PDC is inactivated by incubation with low concentrations of ATP, and the inactive preparations are reactivated by incubation with 10 mM Mg++. Inactivation involves phosphorylation of the pyruvate dehydrogenase (PDH) component of the complex, and reactivation accompanies dephosphorylation. The phosphorylation and dephosphorylation reactions are catalyzed, respectively, by a kinase and a phosphatase which appear to be associated closely with or integral parts of the PDH. The significance of these findings with respect to regulation of pyruvate metabolism in kidney mitochondria is discussed below. Materials and Methods.-a_-P32-ATP and y-P'2-ATP were obtained from International Chemical and Nuclear Corp.; ,B, y-P'2-ATP came from Schwartz BioResearch; protamine sulfate was from Eli Lilly and Co.; and Sepharose 4B came from Pharmacia Fine Chemi- cals. The DPN-reduction assay for the intact PDC, which is based on reaction (1), and other assays were carried out as described previously.2 3 Protein-bound radioactivity was determined by precipitation of the radioactive protein on filter-paper disks.4 Purification of PDC and KGDC: Beef kidney mitochondria were isolated in 0.25 M sucrose, washed with 20 mM phosphate buffer (pH 7.0 and 6.3), and frozen and thawed essentially as described in a previous publication.5 The thawed suspension was made 50 mM with respect to NaCl and was clarified by centrifugation. The mitochondrial ex- 234 Downloaded by guest on September 26, 2021 VOL. 62, 1969 BIOCHEMISTRY: LINN, PETTIT, AND REED 235

tract was adjusted to pH 6.2, and 0.002 vol of a 2%0o aqueous solution of protamine sulfate was added dropwise with stirring. The precipitate was discarded, the supernatant fluid was made 10 mM with respect to MgCl2, and 0.007 vol of protamine sulfate solution was added. The yellow precipitate was collected and extracted with 0.1 M phosphate buffer, pH 7.0, which contained 0.075% (w/v) of sodium ribonucleate (about 4 ml per 100 ml of the mitochondrial extract). The extract was diluted with water to a protein concentration of 5-7 mg/ml, and the solution was centrifuged for 2.5 hr at 40,000 rpm in the no. 40 rotor of a Spinco model L ultracentrifuge. The yellow pellet was dissolved in 20 mM phosphate buffer, pH 7.0, and the solution (about 5 mg of protein per ml) was subjected to isoelectric precipitation. The a-ketoglutarate dehydrogenase complex (KGDC) and PDC were collected at pH's of about 5.9 and 5.5, respectively. The over-all recovery of PDC activity from the mitochondrial extracts varied from 70 to 90%; the recovery of KGDC activity was 50-70%. The specific activities of the highly purified preparations were about 10 (micromoles of DPNH formed per minute per mg of protein)3 at 25°, and about 20 at 37°. Resolution of PDC: A solution of PDC (about 90 mg of protein) in 4.5 ml of 0.1 M glycine buffer, pH 9.0, containing 1.0 M NaCl and 2 mM dithiothreitol was applied to a column (2.5 X 38 cm) of Sepharose 4B, which had been equilibrated with the same buffer. The flow rate was about 8 ml/hr, and fractions of about 3.7 ml were collected. Results.-The sedimentation velocity patterns of the purified PDC and KGDC are shown in Figure 1A and B, respectively.

A B B~~~~ ICD; E 70S 455 7.65 7.7S 325

FIG. .-Sedimentation velocity patterns obtained with enzyme preparations in 50 mM phosphate buffer, pH 7.0, at 200. Sedimentation coefficients are corrected to water at 200, but not to zero protein concentration. (A) PDC; (B) KGDC; (C) PDH; (D) phosphorylated PDH; and (E) transacetylase. Inactivation of PDC by ATP: During attempts to improve procedures for extraction of PDC fro'.n kidney mitochondria,5 large variations were encountered in the activity (reaction (1)) and stability of PDC in the extracts. Further investigation revealed that PDC (but not KGDC) is inactivated by low levels of ATP. The effect of ATP on the enzymatic activity of purified PDC is shown in Figure 2. Activity was not restored by dilution, by dialysis, or by gel filtration oIn Sephadex G-25. AMP, ADP, CTP, GTP, and UTP showed essentially no inhibition at concentrations up to 1 mM; ITP showed slight inhibition. Phosphorylation of PDC by A TP: The apparent irreversible inactivation of PDC by ATP suggested that a part of the ATP molecule was covalently attached to PDC. To test this possibility, radioactive ATP was used. Typical results are presented in Table 1. Essentially no radioactivity was incorporated into protein from a-P32-ATP. The counts originating from the doubly labeled (g,7)- ATP were approximately half of the counts originating from -y-P32-ATP. Since both samples of ATP had approximately the same specific radioactivity, these Downloaded by guest on September 26, 2021 236 BIOCHEMISTRY: LINN, PETTIT, AND REED PROC. N. A. S.

FIG. 2.-Effect of ATP on PDC activity. Reaction mixtures contained 20 mM >_8C\ potassium- phosphate buffer, pH 7.0; 1 mM MgCl2; 10 mM dithiothreitol; 0.26 mg of purified PDC; and varying amounts of ATP < \\in a\volume\ of 1.0 ml. The mixtures were > \ incubated at 250. At the indicated times, W40 \\ > \ - 20-IAI samples were assayed for DPN-reduc- _J Yk \ >tion activity. r \ N*-N, Control; 0-C, plus 0.001 mM 20 \ t Ad - ATP; *-4, plus 0.005 mM ATP; A-A, plus 0.01 mM ATP; A-A, plus 0.1 mM I 4 ATP; 0-0, plus 1 mM ATP. 0 2 3 4 5 6 M IN UTES data demonstrate that the terminal phosphoryl moiety of ATP is transferred to PDC. No detectable change in the sedimentation coefficient of PDC resulted from phosphorylation. Preliminary experiments have revealed that no radioactive material is released from phosphorylated PDC during incubation with 0.25 M HC1 for 19 hours at 300. Similar treatment with 0.25 M NaOH resulted in a complete loss of protein-bound radioactivity. Treatment with 0.1 M hydroxylamine, pH 7.0, for 10 minutes at room temperature released less than 5 per cent of the protein- bound radioactivity. These results suggest that the phosphoryl moiety is attached to PDC in ester linkage. Dephosphorylation of P32-labeled PDC: Some preparations of phosphorylated PDC were reactivated by incubation with Mg++. The optimum concentration of Mg++ was about 10 mM. Restoration of activity (reaction (1)) was accompanied by a release of protein-bound radioactivity. The data presented in Figure 3 illustrate the time course of the reciprocal changes in enzyme activity and protein-bound phosphoryl groups. The radioactive material released during incubation of P32-labeled PDC with Mg++ appears to be inorganic orthophosphate. When an incubation mixture was subjected to a procedure6 for determination of inorganic orthophosphate as isobutanol-benzene-soluble phosphomolybdic acid, 88 per cent of the radioactivity was found in the organic phase. In a control experiment with 7-P32-ATP, but without enzyme, over 97 per cent of the radioactivity was recovered in the aqueous phase. TABLE 1. Transfer of label from radioactive ATP to PDC. Protein-bound radioactivity, ATP cpm/mg protein a-P32 15 #,aP32 2,680 ..,p32 6,260 The specific radioactivities (cpm/momole) of the labeled ATP were: a-P32-ATP, 1730; 6,,y-P32- ATP, 963; and y-P32-ATP, 1100. The reaction mixtures contained 50 mM phosphate buffer, pH 7.0; 0.25 mM radioactive ATP; and 6.2 mg PDC in a volume of 0.6 ml. The mixtures were incubated for 10 min at 250, and aliquots were assayed for DPN-reduction activity and for protein-bound radioactivity. All three incubation mixtures were inactive in the DPN-reduction assay. Downloaded by guest on September 26, 2021 VOL. 62, 1969 BIOCHEMISTRY: LINN, PETTIT' AND REED 237

60.

0 10 20 30 40 50 60 70 80 M INUTES FIG. 3.-Time course of the phosphorylation and dephosphorylation of PDC. The reaction mixture contained 20 mM phosphate buffer, pH 7.0; 0.5 mM MgCl,; 0.04 mM 7 P32-ATP (34,534 cpm/m~smole); 'and 5.0 mg of purified PDC in a volume of 2.0 ml. The mixture was incubated at 150. At the indicated times, 2O-,sl samples were assayed for DPN-reduction activity, and SO-sl samples were assayed for protein- bound radioactivity. At the time interval indicated by the vertical arrow, sufficient MgCl, was added to give a final concentration of 10 mM. To obtain a common ordinate, enzyme assays and protein-bound radioactivity have been expressed as per cent of maximum activity. *-*, DPN-reduction assay, O-O. protein-bound radioactivity. Some preparations of phosphorylated PDC underwent little, if any, reactiva- tionB~~~in the presence of Mg++. Further investigation revealed that such preparations were reactivated, with concomitant dephosphorylation, when incubated with Mg++ and certain protein fractions obtained in the later steps in the purification of PDC. These fractions did not contain PDC or KGDC, and the activity was heat-labile. Typical data are presented in Table 2. These results indicate that phosphoryiation and dephosphorylation of PDC are catalyzed by two different enzymes-an ATP-specific kinase and a Mg++-dependent phosphatase. Both of these enzymes appear to be integral parts (i.e., regulatory subunits) of the PDC molecule, but the phosphatase appears to be less strongly bound than the kinase, with the result that variable amounts of the phosphatase are released during the purification of PDC. TABLE 2. Phosphatase requirement for reactivation of phosphorylated PDC. Protein-bound radioactivity, Relative enzymatic A System mpmoles/mg protein activity, % PD)C+ ATP 5.1 0 (P'2)PDC + Mg++ 5.1 0 (P"a)PDC + Mg++ + phosphatase 0.2 100 (A) The reaction mixture contained 20 mM phosphate buffer, pH 7.0; 1 mM MgCls, 10 mM dithiothreitol; 0.02 mM 'y-P3" ATP (61,000 cpm/mjymole); and 1.04 mg PDC in a volume of 2.0 ml. The mixture was incubated at 250 for 10 min. and aliquots were taken for assay of DPN-reduction activity and protein-bound radioactivity. (B) To two 0.5-mi aliquots was added sufficient MgCI, to give a final concentration of 10 mM. To one of these aliquots was added 0.5 ml of a phosphatase preparation (1.1 mg protein) in 20 mM phosphate buffer, and to the second aliquot was added 0.5 ml of 20 mM phosphate buffer. Incuba- tion was continued at 250 for 15 min, and aliquots were assayed. Downloaded by guest on September 26, 2021 238 BIOCHEMISTRY: LINN, PETTIT, AND REED PROC. N. A. S.

Resolution of PDC: Phosphorylation of PDC did not affect its dihydrolipoyl transacetylase or dihydrolipoyl dehydrogenase (flavoprotein) activities.2' 5 This observation pointed to the pyruvate dehydrogenase component of PDC as the site of the phosphorylation and dephosphorylation reactions. To confirm this possibility, procedures were developed for separation of PDC into its component enzymes. The resolution of PDC preparations exhibiting kinase but not phosphatase activity by gel filtration on Sepharose 4B at pH 9 is illustrated in Figure 4. All three components-PDH, transacetylase, and flavoprotein-were re- quired to reconstitute the CoA- and DPN-linked oxidation of pyruvate (reaction (1)). The sedimentation-velocity patterns of the highly purified PDH and the

FIG. 4.-Elution profile of com- I#, ponent enzymes of PDC on Sepha- 2.0_ rose 4B. The resolution was carried out as described in Materials and Methods. The first major peak E 1.6I (fractions 20-30) contained the OD and the second 1.2 jtransacetylase, major peak (fractions 37-46) was a 1.2 Of t mixture of the pyruvate dehydro- z ~ 1 1 genase and the flavoprotein. The c 0.8I latter two enzymes were separated or 8 \ 1 4by fractionation with ammonium sulfate. PDH was collected be- 0v4 - tween 0.25 and 0.35 saturation, and the flavoprotein was collected be- tween 0.50 and 0.90 saturation. 0 15 30 45 60 The last, minor peak appeared to be FRACTION NUM BER ribonucleic acid, which was added during the purification procedure. transacetylase are shown in Figure 1C and E, respectively. The molecular weight of PDH, determined by the high-speed equilibrium centrifugation procedure of Yphantis,7 is about 160,000. When phosphorylated (P32) PDC was resolved on Sepharose 4B, essentially all of the protein-bound radioactivity was found in the PDH fraction. Similar results were obtained when phosphorylated PDC was resolved at pH 9.5 on columns of calcium phosphate gel suspended on cellulose, as described previously for the resolution of PDC from E. coli2' 8 and from pig heart.' The sedimentation-velocity pattern of the phosphorylated PDH is shown in Figure 1D. No detectable change in the sedimentation coefficient of PDH resulted from phosphorylation. The phosphorylated PDH contained about two equivalents of protein-bound phosphoryl groups per 160,000 gm of protein. Phosphorylation and dephosphorylation of PDH: The PDH fraction, obtained as described above, underwent phosphorylation in the absence of the transacetylase and the flavoprotein fractions. However, the rate of phosphorylation was increased significantly in the presence of the transacetylase fraction (Table 3A). Control experiments showed that radioactivity was not incorporated into the transacetylase fraction. These results are interpreted as indicating that (a) the regulatory subunit which catalyzes the phosphorylation reaction (i.e., the kinase) is an integral part of the PDH molecule; and (b) binding of PDH to the Downloaded by guest on September 26, 2021 VOL. 62, 1969 BIOCHEMISTRY: LINN, PETTIT, AND REED 239

TABLE 3. Phosphorylation and dephosphorylation of PDH. Protein-bound radioactivity, System mumoles/mg PDH A 10 min 30 min 60 min PDH + ATP 2.1 4.5 6.2 PDH + LTA + ATP 10.5 14.3 14.3 B (P'2)PDH + Mg++ 6.3 6.3 (P32)PDH + phosphatase + Mg++ 5.6 4.1 (P32)PDH + LTA + Mg++ 9.0 8.4 (P32)PDH + LTA + phosphatase + Mg++ 1.1 0.3 (A) The two reaction mixtures contained 20 mM phosphate buffer, pH 7.0; 1 mM MgCl2; 2 mM dithiothreitol; 0.015 mM 'y-P32-ATP (59,253 cpm/mismole); 0.54 mg/ml of PDH; and, where indicated, 0.79 mg/ml of dihydrolipoyl transacetylase (LTA). The two mixtures were incubated at 250. At 10- and 30-min intervals, 50-IA aliquots were assayed for protein-bound radioactivity. (B) At 60 min, four 0.5-ml aliquots of the first incubation mixture (PDH + ATP) were taken, and to each aliquot were added the indicated components in a total volume of 1.0 ml. The amounts were Mg++, 10 mM; LTA, 0.3 mg; and phosphatase, 2.0 mg. Incubation was continued at 25°, and aliquots were taken for assay at the indicated times. transacetylase5' 8 9 stabilizes the active conformation of PDH, and thereby facilitates the phosphorylation reaction. Consistent with the tentative con- clusion that PDH contains both a catalytic subunit and a regulatory subunit (i.e., the kinase) is the observation that preparations of PDC underwent a significant loss of kinase activity, but not DPN-reduction activity (reaction (1)), during storage at 5°. Kinase activity was largely restored by incubating the aged preparations with a thiol, e.g., 2 mM dithiothreitol. Preparations of phosphorylated PDH underwent dephosphorylation at a slow rate in the presence of Mg++ and the phosphatase. The rate of dephosphorylation was markedly accelerated when the transacetylase fraction was included in the incubation mixture (Table 3B), and complete reactivation of the PDH was observed. These results provide additional evidence that the PDH component of PDC is the site of the phosphorylation and dephosphorylation reactions and indicate that binding of the PDH (and presumably the regulatory enzymes) to the transacetylase is essential for rapid phosphorylation and dephosphorylation. Discussion.-The data reported in this communication indicate that the activity of the pyruvate dehydrogenase complex from beef kidney is regulated by a phosphorylation-dephosphorylation reaction sequence. The site of this regulation is the pyruvate dehydrogenase component of PDC. It is noteworthy that PDH catalyzes the first and, apparently, irreversible step in pyruvate oxidation. It appears that phosphorylation of PDH is catalyzed by an ATP-specific kinase (i.e., a pyruvate dehydrogenase k nase) and that dephosphorylation is catalyzed by a Mg++-dependent phosphatase (i.e., a pyruvate dehydrogenase phosphatase). The PDH phosphatase has been separated from the other component enzymes of PDC. The PDH kinase appears to be an integral part of the PDH molecule. The kinase, the catalytic subunit of PDH, and the phosphatase may well con- stitute an organized unit in the intact PDC. Certainly, highly favorable posi- tioning of these three components must be assumed in order to account for the occurrence of the phosphorylation-dephosphorylation reaction sequence. In this connection, the high degree of structural organization in beef kidney PDC Downloaded by guest on September 26, 2021 240 BIOCHEMISTRY: LINN, PETTIT, AND REED PROC. N. A. S.

should be emphasized. The transacetylase component of the complex appears to consist of 20 morphological subunits that are situated at the vertices of a pentagonal dodecahedron.' The molecules of PDH (about 20), their associated regulatory enzymes, and the flavoprotein molecules appear to be distributed in a regular manner on the surface of the transacetylase. Both the PDH kinase and the PDH phosphatase are MIg++-dependent. The data obtained thus far indicate that the phosphatase requires about ten times as high a Mg++ concentration as the kinase for optimal activity. This difference suggests that the activity of the phosphatase is regulated by the intramitochondrial concentration of free Mg++ which, in turn, may be dependent on the ATP/- ADP ratio. It is known that ATP has a much greater affinity for Mg++ than has ADP.10 According to this rationale, the phosphatase will tend to be active when the ATP/ADP ratio is low and, presumably, the concentration of free Mg++ is high. Whether the PDH phosphatase and the PDH kinase are subject to other control mechanisms remains to be determined. Regulation of the activity of beef kidney PDC by a phosphorylation-dephosphorylation reaction sequence is reminiscent of the interconversions of phosphorylase b and phosphorylase all and the interconversions of the glucose-6-P dependent and glucose-6-P independent forms of glycogen synthetase.12 These interconversions are catalyzed by protein-specific kinases and phosphatases. The pattern of regulation of PDC activity resembles that observed with glycogen synthetase in that phosphorylation of both PDC and glycogen synthetase results in formation of a less active (or inactive) form of the enzyme. In other experiments,13 to be reported later, it was observed that partially purified preparations of PDC from pork liver mitochondria were inactivated by incubation with y-P32-ATP, and the inactive preparations were reactivated by incubation with 10 mM\1 Mg++. Inactivation was accompanied by incorpora- tion of radioactivity into protein, and reactivation was accompanied by release of protein-bound radioactivity. Thus, it appears that the activity of PDC is regulated in a similar manner in both kidney and liver. This control mechanism is of considerable interest with respect to regulation of the direction of pyruvate metabolism in kidney and liver tissue, i.e., whether pyruvate undergoes oxidation (to acetyl CoA) or carboxylation (to oxalacetate).14 15 It should be noted that these two organs are primarily responsible for gluconeogenesisl6 and that both PDC and pyruvate carboxylase, a key enzyme of the gluconeogenic path- way, are located in the mitochondria. There appears to be an inverse relation- ship in the regulation of PDC (i.e., its pyruvate dehydrogenase component) and pyruvate carboxylase. The former enzyme will tend to be active when the (intramitochondrial) ATP/ADP ratio is low, whereas the latter enzyme will tend to be active when the ATP/ADP ratio is high.'4 This rationale also provides a reasonable explanation of the observations", 17-20 that oxidation of fatty acids, acylcarnitines, and intermediates of the tricarboxylic acid cycle by liver and kidney mitochondria inhibits pyruvate oxidation (and stimulates pyruvate carboxylation), and that this inhibition is prevented by uncoupling agents such as pentachlorophenol'3 and dinitrophenol.19 Substrate oxidation generates ATP which, in turn, inhibits PDO (and stimulates conversion of pyruvate to Downloaded by guest on September 26, 2021 VOL. 62, 1969 BIOCHEMISTRY: LINN, PETTIT, AND REED 241

oxalacetate). Uncoupling agents, by suppressing ATP formation, prevent inhibition of PDC. Abbreviations used: PDC, pyruvate dehydrogenase complex; PDH, pyruvate dehydrogenase; DPN, diphosphopyridine nucleotide; KGDC, a-ketoglutarate dehydrogenase complex; LTA, dihydrolipoyl transacetylase. * Supported in part by a grant (GM-06590) from the U.S. Public Health Service. t Postdoctoral fellow of the U.S. Public Health Service. 1 Reed, L. J., and D. J. Cox, Ann. Rev. Biochem., 35, 57 (1966). 2Reed, L. J., and C. R. Willms, in Methods in Enzymology, ed. W. A. Wood (New York: Academic Press, 1966), vol. 9, p. 247. 3 Schwartz, E. R., L. 0. Old, and L. J. Reed, Biochem. Biophys. Res. Commun., 31, 495 (1968). 4Kingdon, H. S., B. M. Shapiro, and E. R. Stadtman, these PROCEEDINGS, 58, 1703 (1967). I Ishikawa, E., R. M. Oliver, and L. J. Reed, these PROCEEDINGS, 56, 534 (1966). 6 Penniall, R., Anal. Biochem., 14, 87 (1966). 7Yphantis, D. A., Biochemistry, 3, 297 (1964). 8 Koike, M., L. J. Reed, and W. R. Carroll, J. Biol. Chem., 238, 30 (1963). 9 Hayakawa, T., and M. Koike, J. Biol. Chem., 242, 1356 (1967). 0 Burton, K., Biochem. J., 71, 388 (1959). '1 Krebs, E. G., and E. H. Fischer, in Advances in Enzymology, ed. F. F. Nord (New York Interscience, 1962), vol. 24, p. 263. 12Friedman, D. L., and J. Larner, Biochemistry, 2, 669 (1963). 13 Linn, T. C., F. H. Pettit, and L. J. Reed, unpublished data. 14Keech, D. B., and M. F. Utter, J. Biol. Chem., 238, 2609 (1963). 15 Walter, P., V. Paetkau, and H. A. Lardy, J. Biol. Chem., 241, 2523 (1966). 16 Krebs, H. A., Proc. Roy. Soc. (London) Ser. B., 159, 545 (1964). 17 Haslam, R. J., in Regulation of Metabolic Processes in Mitochondria, ed. J. M. Tager, S. Papa, E. Quagliariello, and E. C. Slater (Amsterdam: Elsevier Publishing Co., 1966), p. 108. 18 Nicholls, D. G., D. Shepherd, and P. B. Garland, Biochem. J., 103, 677 (1967). 19 Konig, T., and Gy. Szabados, Acta Biochim. Biophys. Acad. Sci. Hung., 2, 253 (1967). 20 von Jagow, G., B. Westermann, and 0. Wieland, European J. Biochem., 3, 512 (1968). Downloaded by guest on September 26, 2021