Enzyme-PP + Pi T± Enzyme-P + Ppi Pyruvate + ATP I P

Home , Adenylate kinase, Hexokinase, Pyrophosphate

THE MECHANISM OF THE PYRUVATE, PHOSPHATE DIKINASE REACTION* BY HERBERT J. EVANS AND HARLAND G. WOOD DEPARTMENT OF BIOCHEMISTRY, CASE WESTERN RESERVE UNIVERSITY, CLEVELAND, OHIO Communicated October 11, 1968 Abstract and Summary.-Pyruvate, phosphate dikinase has been purified from propionic acid bacteria and evidence is presented in this report to show that the over-all reaction involves the following reactions:

Enzyme + ATP ; enzyme-PP + AMP (1) Enzyme-PP + Pi T± enzyme-P + PPi (2) Enzyme-P + pyruvate T± enzyme + P-enolpyruvate (3) Mg2 + Sum: Pyruvate + ATP + Pi P-enolpyruvate + AMP + PPj. (4) The enzyme combines with ATP to form an enzyme-diphosphate compound and AMP, with the ,f and y phosphates from ATP bound to the enzyme (reaction 1). The a-phosphate of the enzyme-diphosphate then combines with orthophosphate to give pyrophosphate and enzyme-phosphate (reaction 2). The ,8-phosphate is then transferred from the enzyme-P to pyruvate to produce free enzyme and P-enolpyruvate (reaction 3). The sum of the three reactions is reaction 4, which is the over-all reaction catalyzed by pyruvate, phosphate dikinase. The propionic acid bacteria grow well on lactate and pyruvate and therefore must be able to convert pyruvate to P-enolpyruvate, which is required for the net- formation of 4-carbon compounds, carbohydrates, glycerol, and other cell materials. Investigation (unpublished) has revealed that the conversion of pyruvate to P-enolpyruvate does not occur via oxalacetate, nor by a reversal of the pyruvate kinase reaction. We have found1 that the conversion resembles that catalyzed by P-enolpyruvate synthase, which was discovered in E. coli by Cooper. and Kornberg2 and which catalyzes the following reaction: Mg2 +

Pyruvate + ATP I P-enolpyruvate + AMP + Pi. (5) Unlike the P-enolpyruvate synthase-catalyzed reaction it was found, however, that there is a requirement for Pi in the forward reaction catalyzed by the enzyme from propionibacteria, and that PPi is a product. The stoichiometry is as shown in reaction 4 (manuscript in preparation). An enzyme that catalyzes reaction 4 was independently discovered in various tropical grasses by Hatch and Slack.3 Reeves4 reported that the enzyme is present in Entamoeba histolytica and suggested the trivial name "pyruvate, phosphate dikinase." 1448 Downloaded by guest on September 23, 2021 VOL. 61, 1968 BIOCHEMISTRY: EVANS AND WOOD 1449

The mechanism of the P-enolpyruvate synthase reaction of E. coli has been investigated by Cooper and Kornberg5 and Berman et al.,8 who presented evidence for the following partial reactions:

Enzyme + ATP ; enzyme-PP + AMP (6) Enzyme-PP + H20 z± enzyme-P + Pi (7)

Enzyme-P + pyruvate T± enzyme + P-enolpyruvate (8) Mg2+ Sum: Pyruvate + ATP + H20 ' P-enolpyruvate + AMP + Pi. (5) Consideration of the difference in the over-all reaction catalyzed by P-enolpyruvate synthase (reaction 5) and that catalyzed by pyruvate, phosphate dikinase (reaction 4) suggested that the latter reaction might occur as shown in reactions 1-3. The difference in the two mechanisms is the second partial reaction which is an hydrolysis in the P-enolpyruvate synthase mechanism (reaction 7), and a phosphorolysis in the dikinase mechanism (reaction 2). Evidence sup- porting these views is reported herein. Materials and Methods.-Radiochemicals: [U-"4C]-AMP, [1-"4C]-pyruvate (New Eng- land Nuclear), and [32P]-orthophosphate (Tracerlab) were commercial products. [32P]- P-enolpyruvate and [S2P]-PP, were prepared enzymatically by exchange reactions starting with ['2P]-orthophosphate of high specific activity and unlabeled P-enolpyruvate, PPi, and bicarbonate in an incubation containing P-enolpyruvate carboxytransphos- phorylase, which had been purified from propionibacteria.' At the end of the incubation, the products were separated by chromatography on anion-exchange resin as described below. Enzyme: Pyruvate, phosphate dikinase was purified 30-fold from P. shermanii that were grown on a lactate medium. The methods included DEAE-cellulose adsorption and elution, ammonium sulfate fractionation, and TEAE-cellulose adsorption and elution. The enzyme was estimated to be approximately 70% pure by the criteria of ultracen- trifugation and cellulose acetate electrophoresis (manuscript in preparation). Where indicated, the enzyme was made phosphate-free by passing it through a 0.8 X 27.5-cm column of Sephadex G-50 equilibrated with 50 mM Tris-HCl (pH 7.6), 8 mM MgC12, and 0.5 mM dithioerythritol. Separation of products: For separation of products, the terminated reaction mixture was diluted sufficiently to bring the ionic strength below 0.002 M and then passed through a 0.6 X 6.0-cm column of AG-1-X2, 200-400-mesh anion-exchange resin (Bio-Rad) in the chloride form. The column was eluted with a linear gradient from 0 to 0.04 N HCl. AMP, Pi, and glucose-6-P came off together in the early fractions. Later fractions contained (in the order of elution): ADP, P-enolpyruvate, PPi, and ATP. Pi, PPi, P-enolpyruvate, and ATP exchange: The products of the exchange experiments were subjected to the molybdate-extraction method of Walters and Cooper8 that sepa- rates orthophosphate from phosphate esters. The molybdate treatment was performed on samples both with and without prior treatment with inorganic pyrophosphatase (Wor- thington). By treatment with pyrophosphatase, the PP1 was eliminated from the phosphate ester fraction. Radioactivity: Radioactivity was measured by drying samples on aluminum planchets and determining the radioactivity on a Nuclear-Chicago low-background gas-flow counter. Results.-Exchange of [14C]-AMP with A TP: E. coli P-enolpyruvate synthase catalyzes the exchange of [14C ]-AMP into ATP according to reaction 6.5 Downloaded by guest on September 23, 2021 1450 BIOCHEMISTRY: EVANS AND WOOD PROC. N. A. S.

The exchange requires a high concentration of Pi, which Cooper and Kornberg propose to be necessary to maintain the level of enzyme-PP by reversal of reaction 7. The [14C]-AMP exchange into ATP is demonstrated for pyruvate, phosphate dikinase in Table 1. It is seen that neither pyruvate nor Pi is required for this exchange and therefore the exchange occurs as proposed in reaction 1. PP1 inhibits the exchange, possibly due to chelation of magnesium ions. ADP was included in the exchange experiments as a control to determine how much of the exchange into ATP resulted from the adenylate kinase reaction (reaction 9), since adenylate kinase is still a minor contaminant of our purified enzyme. Mg2+ ATP + AMP 2=ADP. (9) If the exchange occurred solely by reaction 9, the specific activity of ADP should be at least equal to that of ATP. It may be seen from Table 1 that the specific activity of ADP is less than one quarter that of the ATP in the complete system. Exchange of 32PJ into PPj: The proposed mechanism for pyruvate, phosphate dikinase predicts that 32P, should exchange with PP1 by means of reaction 2. This exchange also requires the presence of one of the enzyme-phosphate intermediates, i.e. either enzyme-P or enzyme-PP. The exchange was therefore performed in two experiments, the results of which are presented in Table 2. In experiment 1, Table 2, the exchange of 32P, with PP1 was carried out in the TABLE 1. Exchange of [14C]-AMP into ATP. --Specific Activity (cpm/IsM) AMP ADP ATP Complete 2310 250 1130 Complete + Pi 2160 172 1122 Complete + PP1 4520 75 93.3 Complete - enzyme 4970 39.5 50.5 The complete system contained (in Mmoles per ml): Tris-HCl buffer (pH 7.6), 60; MgCl2, 5.6; ATP, 1.5; ADP, 1.5; [14C -AMP, 0.5; and 1.20 units of phosphate-free enzyme (0.40 mg of protein). Where indicated, 2.0 Mmoles per ml of potassium phosphate (pH 7.0) or 1.5 pmoles per ml of sodium pyrophosphate were added. Incubation was for 60 min at 310. TABLE 2. Exchange of [82Pj] into PP1. Cpm/Ml Incubation P-enol- PPi pyruvate ATP Expt. 1 Complete (enzyme, ATP, [32Pi] and PPi) 31,500 - 2400 Complete - ATP 900 0 Complete - enzyme 500 300 Expt. 2 Complete (enzyme, PEP, [32Pi] and PP') 45,400 0 Complete - PEP 600 Complete - enzyme 300 Amounts (in smoles per ml), expt. 1: Tris-HOl buffer (pH 7.4), 46; MgCl2, 3.6; PPi, 2.3; ATP, 2.3; pH 7.4 [32p]-KPi, 1.8 (5.9 X 106 cpm/Lxmole); and 0.225 unit of enzyme (0.075 mg protein), all in a final volume of 2.20 ml. Incubation was for 90 min at 250. Expt. 2: Tris-HCl buffer (pH 7.4), 50; MgCl, 5.0; PPi, 1.0; pH 7.4 [32P]-KPi, 0.2 (2.5 X 106 cpm/,umole); P-enolpyruvate, 2.0; and 0.55 unit of enzyme (0.19 mg of protein); all in a final volume of 1.20 ml. Incubation was for 180 min at 250. Downloaded by guest on September 23, 2021 VOL. 61, 1968 BIOCHEMISTRY: EVANS AND WOOD 1451

presence of ATP so that enzyme-PP would be formed by reaction 1. Little exchange of 32P, into PPi occurs if either ATP or enzyme is omitted. In the complete system ATP also becomes labeled to a small extent. This occurs because PP1 is a symmetrical molecule, and thus, by a reversal of reaction 2, the enzyme-PP becomes labeled. The enzyme-PP in turn labels ATP by a reversal of reaction 1. In experiment 2, Table 2, the 32Pi exchange into PPi was carried out in the presence of P-enolpyruvate. In this case, enzyme-P was formed by a reversal of reaction 3. The results show exchange only in the presence of both P-enolpyruvate and enzyme. The amount of label in P-enolpyruvate was negligible. P-enolpyruvate should not become labeled according to the proposed mechanism because the enzyme-P bond is not broken in reaction 2. Exchange of [14C]-pyruvate into P-enolpyruvate: The last partial reaction (reaction 3) in the proposed mechanism of pyruvate, phosphate dikinase predicts the exchange of ["4C]-pyruvate into P-enolpyruvate in the absence of nu- cleotides. The mixture for the exchange contained the following (in 11moles per ml) in a final volume of 1.00 ml: Tris-HCl buffer (pH 7.6), 50; P-enolpyruvate, 1.0; [1-14C]-pyruvate, 0.5; and 0.40 unit of enzyme (0.13 mg of protein). The mixture was incubated for 60 minutes at 300. At the end of the exchange reaction, radioactivity associated with pyruvate was removed by oxidation with ceric sulfate in a modification of the method of Fromageot and Desnuelle.9 With this method all of the [1-14C]-pyruvate and [14C]-parapyruvate could be removed, leaving a large part of the P-enolpyruvate intact. At the start of the incubation period, there were 44,000 cpm in [1-14C]-pyruvate. During the incubation, 12,500 cpm, or 28 per cent, were transferred to P-enolpyruvate. No 14C was found in P-enolpyruvate if the enzyme was omitted. The fate of the phosphate of P-enolpyruvate in the reverse over-all reaction: The reverse over-all reaction was carried out in the presence of [32P]-P-enolpyruvate by linking the reaction to lactate dehydrogenase. Compounds from the starting material, the terminated reaction, and the terminated reaction after treatment with hexokinase were separated after adding carrier glucose-6-P, Pi, PP1, P-enolpyruvate, ADP, and ATP. It is shown in Table 3 that the enzyme transfers the 32p from [32P ]-P-enolpyruvate to ATP in the products. After hexokinase treatment of the terminated reaction, the label was found in ADP, an indication that the ATP formed in the enzyme reaction was labeled in the d-phosphate. These findings are in accord with reactions 3, 2, and 1. The fate of the phosphates of PPj in the reverse over-all reaction: The reverse over-all reaction was also carried out starting with [32P ]-PPi. This experiment is summarized in Table 4. It is seen that the enzyme distributed the label from [32P ]-PP1 into Pi and ATP in the terminated reaction. Treatment of the terminated reaction with hexokinase transferred the label of ATP to glucose-6-P, an indication that the ATP formed in the enzyme reaction was labeled in the -y-phosphate, which is as predicted by reactions 3, 2, and 1. The formation of an enzyme-P compound: The mechanism proposed for pyruvate, phosphate dikinase, like that for P-enolpyruvate synthase, includes the formation of two phosphorylated intermediates, enzyme-P and enzyme-PP. Downloaded by guest on September 23, 2021 1452 BIOCHEMISTRY: EVANS AND WOOD PROC. N. A. S.

TABLE 3. Distribution of radioactivity from [32P]-P-enolpyruvate in the products of the reverse reaction. Total Cpm/Ml Incubation P-enol- Pi PPi pyruvate ADP ATP Starting material 480 0 42,200 0 0 Products 846 81 6,990 0 30,900 Products after hexokinase 1,184 96 5,710 24,750 0 The mixture contained (in Mmoles per ml): Tris-HCl buffer (pH 7.6), 50; MgCl2, 2; [32P ]-P enolpyruvate, 0.096; AMP, 1.0; PP,, 1.0; and DPNH, 0.18; and, in units per ml, lactate dehydrogenase, 0.8; and the dikinase, 0.195 (0.065 mg). Incubation was for 20 min at 250 and was terminated by being boiled for 1 min. The hexokinase reaction contained 1 ml of boiled solution, 5 emoles glucose, 4 /Amoles MgC12, and 5.6 units hexokinase in 1.05 ml. Incubation was for 15 min at 300.

TABLE 4. Distribution of radioactivity from [32P]-PPi n the products of the reverse reaction ______Total Cpm/Ml Incubation Glucose- P-enol- 6-P Pi PPi pyruvate ADP ATP Starting material 0 0 34,500 0 0 0 Products 0 18,574 0 0 0 14,280 Products after hexokinase 31,680 0 0 1,140 0 The starting mix contained (in jsmoles per ml): Tris-HCI buffer (pH 7.6), 50; MgCl2, 2; P-enolpyruvate, 1.0; [32P]-PPi, 0.11; AMP, 1.0; DPNH, 0.18; and in units, lactate dehydrogenase, 0.8; and the dikinase, 0.500 (0.165 mg). Incubation was for 15 min at 250. Treatment was as described in Table 3.

Cooper and Kornberg'0 have reported the isolation of a phosphorylated protein by incubation of P-enolpyruvate synthase with [3,7y-32P]-ATP. The formation of a similar intermediate of pyruvate, phosphate dikinase is illustrated in Figure 1. After incubation of the phosphate-free enzyme with [32P]-P-enolpyruvate, separation on Sephadex showed that radioactivity was directly associated with the enzyme. When the enzyme (first peak) was treated with 0.4 N HC1 at 1000 for one minute, the label dissociated from the enzyme and cochromatographed with added orthophosphate carrier on an anion-exchange resin. The same treatment of the second peak gave label only in P-enolpyruvate. Thus the 32p phosphate of P-enolpyruvate was shown to have been converted to enzyme-32P. When the labeled enzyme was incubated with P-enolpyruvate, AMP, and PP1, the 32p was removed from the enzyme and converted to products. Discussion.-The pyruvate, phosphate dikinase reaction, like the P-enolpyruvate synthase reaction, provides means of synthesis that uses two high-energy bonds of ATP to produce P-enolpyruvate from pyruvate. The mechanism proposed in the present work, as well as that of P-enolpyruvate synthase, stipulates that much of the energy of both "high-energy" bonds of ATP is conserved in the enzyme-PP and the enzyme-P intermediates in order that synthesis of P-enolpyruvate, with a free energy of approximately 13 kcal, may occur. The type of bond, or bonds, between the enzyme and phosphates of the intermediates is therefore of much interest. Possibilities include acyl-phosphate linkage (acetyl phosphate is approximately 10.5 kcal), histidyl phosphate, a cyclic anhydride between phosphate and two acidic groups of the enzyme, or some presently unpre- dicted linkage. Downloaded by guest on September 23, 2021 VOL. 61, 1968 BIOCHEMISTRY: EVANS AND WOOD 1453

FIG. 1.-Separation of enzyme-bound- 32p on Sephadex G-50. The incubation Z 100 contained (in Mmoles per ml), in a volume MG/ML CPM/03ML of 0.35 ml: Tris-HCL buffer (pH 7.6), 43; MgCl2, 8.6; [3"P]-P-enolpyruvate, 0.023; and 2.150 unit of enzyme (0.715 mg of protein). After 150 min, the mixture was passed through a 0.8 X 27.5-cm column of Sephadex G-50 and the column was washed with 50 mM Tris-HCl (pH 7.6), 8 mM MgCl2, 0.5 mM dithioerythritol. Fractions of 0.36 ml were monitored for 5 10 protein (0-E-0) and radioactivity (@--). ML. Although the diphosphorylated enzyme is depicted as enzyme-PP, this repre- sentation is not meant to imply that it is necessarily an enzyme-pyrophosphate intermediate with one covalent bond connecting the enzyme and pyrophosphate. This is one possible form, but it is also possible that the two phosphates are individually bonded to the enzyme at different sites, and they may or may not be bonded to each other. Although Reeves4 suggests that pyruvate, phosphate dikinase functions pri- marily in the conversion of P-enolpyruvate to pyruvate in Entamoeba histolytica, which appears to lack pyruvate kinase, the major role of the dikinase in propionibacteria appears to be for the opposite conversion. Crude extracts of propionibacteria contain pyruvate kinase in addition to the dikinase. The dikinase is induced tenfold by growing propionibacteria on lactate rather than on glycerol, presumably because the enzyme is essential for a net formation of P-enolpyruvate from lactate. The equilibrium of the dikinase reaction may be close to unity, or may favor pyruvate formation, but P-enolpyruvate formation would be fa- vored by hydrolysis of the pyrophosphate formed in the reaction, as well as by removal of the P-enolpyruvate for biosynthesis of cellular components and re- plenishment of 4-carbon compounds. Abbreviations: ATP, adenosine 5'-triphosphate; AMP, adenosine 5'-phosphate; ADP, adenosine 5'-diphosphate; Pi, inorganic orthophosphate; PPi, inorganic pyrophosphate; DEAE-cellulose, O-(diethylaminoethyl)cellulose; TEAE-cellulose, O-(triethylaminoethyl) cellulose. * This work was supported by a grant from the U.S. Atomic Energy Commission (AT(30-1)- 1320) and a research training grant of the National Institutes of Health (2T01-GN-00035). 'Evans, H. J., and H. G. Wood, Federation Proc., 27, 588 (1968). 2 Cooper, R. A., and H. L. Kornberg, Biochim. Biophys. Ada, 104, 618 (1965). 3 Hatch, M. D., and C. R. Slack, Biochen. J., 106, 141 (1968). 4 Reeves, R. E., J. Biol. Chem., 243, 3202 (1968). 5 Cooper, R. A., and H. L. Kornberg, Biochim. Biophys. Ada, 141, 211 (1967). 6 Berman, K., N. Itada, and M. Cohn, Biochim. Biophys. Ada, 141, 214 (1967). 7 Lochmfiller, H., H. G. Wood, and J. J. Davis, J. Biol. Chem., 241, 5678 (1966). 8 Walters, E., and C. Cooper, Anal. Biochem., 10, 370 (1965). 9 Fromageot, C., and P. Desnuelle, Biochem. Z., 279, 174 (1935). 10 Cooper, R. A., and H. L. Kornberg, Biochem. J., 105, 49C (1967). Downloaded by guest on September 23, 2021