Isolation of a Pyrophosphoryl Form of Pyruvate, Phosphate Dikinase from Propionibacteria* (Phosphoenolpyruvate/ATP/Enzyme) YORAM MILNER and HARLAND G

Proc. Nat. Acad. Sci. USA Vol. 69, No. 9, pp. 2463-2468, September 1972

Isolation of a Pyrophosphoryl Form of Pyruvate, Phosphate Dikinase from Propionibacteria* (phosphoenolpyruvate/ATP/enzyme) YORAM MILNER AND HARLAND G. WOOD Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 Contributed by Harland G. Wood, June 15, 1972

ABSTRACT Pyruvate, phosphate dikinase from Pro- Enzyme-P + pyruvate ;. enzyme + P-enolpyruvate [ic] pionibacterium shermanii catalyzes the formation of P- enolpyruvate, AMP, and inorganic pyrophosphate from P-enolpyruvate synthase of Escherichia coli, which catalyzes pyruvate, ATP, and orthophosphate; the mechanism reaction 2, likewise uses a mechanism that does not involve a involves three partial reactions and three forms of the enzyme: pyrophosphoryl-enzyme, phosphoryl-enzyme, stable, free diphosphoryl-enzyme (3). and free enzyme. The phosphoryl-enzyme was prepared by -- AMP Pi incubation with P-enolpyruvate and isolated by gel- ATP + pyruvate P-enolpyruvate + + [2] chromatography. The phosphoryl-enzyme was converted These observations led us to reinvestigate the validity of the to 32P31P-enzyme and [32P]Pi by incubation with [32P]PPi; 1 mol of pyrophosphoryl-enzyme was formed per mol of Evans-Wood mechanism. Steady-state and exchange kinetics enzyme of molecular weight 150,000. The labeled enzyme have shown clearly that the pyruvate, phosphate dikinase of released its radioactivity upon incubation with Pi or propionibacteria proceeds via a tri(uni,uni) ping-pong se- AMP to produce the expected [33PJPPi or [y-y3P]ATP, re- quence, as expected from the Evans-Wood mechanism (4). We spectively. Hydrolysis of the pyrophosphoryl-enzyme with here the isolation of the form of the dilute acid yielded PPi. The #,'y-methylene analogue of report pyrophosphoryl ATP was reactive in exchange reactions with [14C]AMP. To enzyme. our knowledge, this is the first proven example of a pyrophosphoryl-enzyme. METHODS Pyruvate, phosphate dikinase catalyzes the following overall Materials. Radiochemicals were purchased from New reaction England Nuclear, including Na4 2P207, 1770 Ci/mol; diam- monium [U-_4C]AMP; 422 Ci/mol; and [3-'4C]sodium ATP + pyruvate + Pi i. AMP pyruvate, 5 Ci/mol. [3-14C]P-enolpyruvate was synthesized + P-enolpyruvate + PPi [1] by incubation of the above [3-14C]pyruvate with pyruvate, phosphate dikinase, ATP, Pi, Mg++, NH4+, and inorganic Evans and Wood (1) have presented evidence by deter- pyrophosphatase. [14C]PEP was purified by chromatography mination of the requirements for exchange reactions that the on QAE-Sephadex-A25 (Pharmacia), as described in Fig. 3. mechanism involves three partial reactions: Lithium afl-methylene adenosine-5'-tetraphosphate (AP- CH2PPP); lithium ap-methylene-adenosine 5'-triphosphate Enzyme + ATP ¢± enzyme-P-O-P + AMP [la] (APCH2PP); and #,,y-methylene-adenosine 5'-triphosphoric acid (APPCH2P) were obtained from Milles Laboratories Enzyme-P-O-P + Pi T± enzyme-P + PPi [lbJ Inc. Na2ATP, NaAMP, P-enolpyruvate tricyclohexylamine, and sodium pyruvate were purchased from Sigma Chemical, Enzyme-P + pyruvate >z. enzyme + P-enolpyruvate [lc I Co. All other chemicals were of analytical grade from various They also succeeded in isolating the phosphoryl-enzyme firms. by incubation of the enzyme with [12PJP-enolpyruvate, and Protein was estimated by the modified biuret procedure chromatography on Sephadex G-50. In addition, they showed (5), with bovine serum albumin as a standard. that the distribution of 82p from various labeled substrates into products was in agreement with the above mechanism. Equilibrium Exchange Rates were calculated by the method However, an attempt to isolate the pyrophosphoryl-enzyme used by Yagil and Hoberman (6). by incubation of pyruvate, phosphate dikinase with [-y- Radioactivity Measurements were made by liquid scintilla- 12P]ATP failed for unknown reasons (1). tion on excised portions of paper chromatograms with a The pyruvate, phosphate dikinase from plants, on the Nuclear Chicago Unilux I Counter. other hand, uses (2) only two partial reactions, and a diphos- phorylated form of the enzyme was not implicated: Assay of Pyruvate, Phosphate Dikinase. The mixture at 250 in a total volume of 0.5 ml contained: 30 mM Tris-malate Enzyme + ATP + Pi :± enzyme-P + AMP + PPi [ld] buffer (pH = 6.7), 16 mM MgCl2, 2.4 mM P-enolpyruvate, 6 mM PPi, 4 mM AMP, and proper dilutions of the enzyme. * This is paper III in the series, "Pyruvate, phosphate dikinase," The reaction was terminated by addition of 0.3 ml of dinitro- Numbers I and II are refs. 1 and 7. phenylhydrazine reagent (0.1% in 2 N HCl), followed by 2463 Downloaded by guest on September 29, 2021 2464 Biochemistry: Milner and Wood Proc. Nat. Acad. Sci. USA 69 (1972)

incubation for 15 min at room temperature. The amount of pyruvate formed was estimated I dinitrophenylhydrazone at 415 nm in a Gilford 300 spectrophotometer after addition E of 0.2 ml of water (6415 = 1 1 X 106). Ie* 70 Purification of Pyruvate, Phosphate Dikinase. The enzyme ,, E 50 1.0- E was purified essentially as described by Evans and Wood _"I 0.8- O (7) from Propionibacterium shermanii grown on a glycerol- 0'CL 'E. 'E 3 0 0.6- a. yeast extract medium. It had a specific activity of 1.0 E = 0.48 W (Amol/ min per mg), and the purity was about 50% as judged by IE10 0.2 E acrylamide gel electrophoresis in the presence of sodium do- 8 l2 16 20 24 28 decyl sulfate. The molecular weight was about 150,000 by FRACTION N2 gel filtration. Accordingly, 1 nmol of enzyme was equivalent FIG. 1. Isolation of pyrophosphoryl-pyruvate, phosphate to 0.3 mg of protein (0.3 units of enzyme). dikinase. Phosphoryl-enzyme (0.88 nmol, 0.26 mg of protein) in 2.0 ml containing 200 nmol of [32P]PPi (3.3 X 101 cpm/nmol), Preparation and Isolation of the Phosphoryl-Enzyme. This 2 jsmol of MgCI2, 10 umol of (NH4)2SO4, and 15 ,mol of Tris-malate form of the enzyme was prepared by reversal of reaction lc. (pH 6.9) was incubated for 8 min at 250, then passed through a The enzyme (155 nmol, about 46 mg of protein) in 2.0 ml 1.1 X 55 cm Sephadex B-50 (fine) column equilibrated with final volume, containing 160 nmol of [3-14C]P-enolpyruvate 15 mM Tris-malate (pH = 6.9), 1 mM MgCl2, 0.3 mM EDTA, (2.14 X 103 cpm/nmol), 2 ,umol of MgCl2, 5 Jmol of (NH4)2- enzyme was same 0.1 mM 2-mercaptoethanol. The eluted with the SO4, and 15 ,umol of Tris-malate (pH = 6.9) was incubated buffer, at the rate of 8 ml/hr per cm2. 1.85-ml fractions were for 15 min at 250. The mixture was then passed through a collected. 32p incorporated into the enzyme was calculated from Sephadex G-50 column (1.1 X 55 cm) equilibrated in 15 mM the specific activity of the ["2P]PPi (3.3 X 106 cpm/nmol) and Tris-malate (pH = 6.9), 2 mM MgC12, 0.5 mM EDTA, the units of enzyme (1 unit = 0.3 nmol of enzyme). For example, the peak fraction (no. 11) had 32.5 X 104 cpm/ml and 0.071 0.1 mM 2-mercaptoethanol. No radioactivity was found in units/ml. Thus, 0.2 nmol of Pi were incorporated (32.5 X 104/ the enzymatic peak, showing that P-enolpyruvate was not at- 1.65 X 106) into 0.24 nmol (0.071/0.3) of enzyme. The value of tached to the protein. Separate tests showed that [4C ]- 1.65 X 106 is used in these calculations because the specific pyruvate was released in an amount about equivalent to activity of Pi would be half that of the PPi. the nmol of enzyme present. The phosphoryl enzyme is relatively stable, and was kept frozen as a stock solution for further experiments. RESULTS Formation of P-Enolpyruvate from Phosphoryl-Enzyme TABLE 1. Distribution of radioactivity from and Pyruvate. According to reaction lc, phosphoryl-enzyme pyruvate should [I2P]pyrophosphoryl-enzyme after paper chromatography plus yield P-enolpyruvate. Phosphoryl- of hydrolyzed and unhydrolyzed ["2P]pyrophosphoryl-enzyme enzyme (20 nmol, about 6.0 mg of protein) was incubated for 5 min at 250 in 1 ml of 15 mM Tris-malate (pH 6.9), Pi PPi P of origin 1 mM MgCl2, 0.25 mM EDTA, 0.05 mM 2-mercaptoethanol containing 2.0 ,mol of [3-14C]pyruvate (3.85 X 103 cpm/ nmol). 5 uA was spotted on paper, and the pyruvate and P- nmol nmol nmol enolpyruvate were separated; their radioactivities were de- Enzyme cpm enzyme cpm enzyme cpm enzyme termined. 308 cpm was found in the P-enolpyruvate spot, 25 ,dI, or 61,500 cpm for the 1.0 ml of reaction mixture. Thus, there Treated was conversion of 16 nmol of pyruvate to P-enolpyruvate, with equivalent to 80% of the 20 nmol of phosphoryl-enzyme. HCl 798 0.11 4520 0.62 485 0.07 10 ,uA, Formation of the Pyrophosphoryl-Enzyme. This form of Not the enzyme was prepared by reversal of reaction lb by in- treated 286 0.08 1000 0.27 1560 0.42 cubation of phosphoryl-enzyme with [32P]PPi and isolation of the labeled enzyme by chromatography on Sephadex G-50. Enzyme (5.9 pmol) in 25 Al of fraction 11 of Fig. 1 was incubated The radioactivity and enzymatic activity were determined; with 25 ul of 0.02 N HCl for 6.0 hr at 21°, and 40 1Al was spotted the elution profiles are shown in Fig. 1. The mol of P incor- on a cellulose 6065 while thin-layer plate (Eastman, sheets) porated per mol of enzyme was essentially constant, and continuously being dried with an air stream. 10 ,ul of untreated was close to 1.0. 0.70 nmol of enzyme were recovered in the fraction 11 (2.4 pmol of enzyme) was also spotted. The specific when the the [32P]Pi and activity of the 32P was 1.56 X 106 at this time. Pi and PPi enzymatic peak; peak containing markers were applied on both sides of the samples. The chromato- [32P]PPi was chromatographed on paper and the radioac- gram was developed for 4 hr by ascending chromatography in tivity was determined, 0.72 nmol of 2Pi were found. Thus, the isopropanol-20%0, trichloroacetic acid-27%, NH40H 75:25:0.25. formation of pyrophosphoryl-enzyme and the release of Pi Pi and PPi were revealed by the Hanes-Isherwood spray (8), from PPi were in accord with reaction lb. and the equivalent areas from the sample of the reactions were Is Not a cut out and the radioactivity was determined. The origin of the Evidence that Pyrophosphoryl-Enzyme Diphos- chromatogram was likewise cut out to determine radioactivity phoryl-Enzyme. The pyrophosphoryl-enzyme readily hydro- remaining attached to the enzyme. lyzes in dilute acid; the product is predominantly PPi (Table Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Pyrophosphoryl-Enzyme from Propionibacteria 2465

9 ATP 40 E E c I0 30 >,

3 E0 200 z 0 To 10 o Pi C-) .E

30 40 50 60 70 80 FRACTION NP

TUBE Ng FIG. 3. Synthesis of ATP from [32Pjpyrophosphoryl-enzyme and AMP. 0.3 nmol of enzyme of combined fractions 11 and 12 of FIG. 2. Rechromatography of the [32P]pyrophosphoryl- Fig. 1 were incubated with 5 umol of MgCl2 and 9 umol of AMP enzyme after incubation with cold pyrophosphate. Enzyme-P32P for 10 min at 250, then placed on a QAE-Sephadex A-25 column was prepared as in Fig. 1 from 1.78 nmol of enzyme-P by incuba- (1 X 17 cm). A linear gradient of 550 ml of 0.4 M triethylam- monium acetate = tion with [B2PJPPi, and subsequent gel chromatography on (pH 7.3) into 600 ml of water was used at a Sephadex G-50. 1.5 ml of the eluate, containing 0.5 nmol of rate of 48 ml/hr. 10-ml fractions were collected. enzyme and 0.45 nmol of bound 32p, was incubated with 20,umol of imidazole buffer (pH = 7.0), 10 ,mol PPi, 10 /Amol MgCl2 in 2.0 ml for 30 min at 270. The mixture was chromato- raphy on Sephadex G-50 (Fig. 2). The ratio of mol of P graphed as in Fig. 1, and 0.41 nmol of enzyme was recovered. bound per mol of enzyme was reduced to about 0.7 by this treatment. It is evident that the pyrophosphate was not in a Michaelis-AMenten type with the enzyme. 1). In both the hydrolyzed and complex The loss unhydrolyzed samples in- of 32p could be due to spontaneous hydrolysis of the covalent organic Pi and PPi were observed, apparently because of labile or there may have a hydrolysis by the bond, been trace of Pi in the pyro- acidic solvent used in the chromatography; phosphate that caused the loss of [32P]Pi by reaction (lb). however, there was much more radioactivity in the PPi and much less at the origin with the hydrolyzed sample. Thus, Formation of ATP from the [32P]Pyrophosphoryl-Enzyme the majority of the radioactivity (78%) was associated with and AMIP and Proof That the 32p Is in a Single Position. By inorganic pyrophosphate in the hydrolyzed sample, a result reversal of reaction la, the pyrophosphoryl-enzyme and that shows conclusively that the enzyme is in the pyrophos- AMP should yield ATP; Fig. 3 shows the results of such an phoryl form. The amount of 32p incorporated was 0.83 nmol/ experiment. The 32p was released from the enzyme quantita- nmol of enzyme in Fraction 11 of Fig. 1, of which 0.80 nmol tively by incubation with AMP, and was found to be in ATP was accounted for in the hydrolyzed preparation. The lability by chromatography on QAE-Sephadex. From 0.3 nmol of of histidyl phosphate to acid is well known (9), and this N-P enzyme, 0.32 nmol of ATP was isolated, showing there was bond might be relatively more labile than the P-O-P bond. transfer of two phosphoryl moieties per mol of enzyme to However, it is not known what type of bond is involved in the AMP as in reaction la. The resulting ATP fractions were pyrophosphoryl-enzyme. pooled and lyophylized. The ATP was dissolved in 1.0 ml By a "chasing experiment" with excess cold PPi, we dem- of 0.01 M Tris-malate (pH = 6.9) containing 1 mM MgCl2, onstrated that the pyrophosphoryl group is in a stable 5 lumol of glucose, and 5 units of yeast hexokinase. After nonexchangeable linkage with the enzyme; about 70% of 10 min of incubation, a 5-ul sample was placed on Whatman the 32 p remained bound to the enzyme, when it was incubated no. 3 paper and subjected to descending chromatography with 2 X 104 molar excess of cold PPi, followed by chromatog- in methanol-water 8:2; 95% of the counts migrated to a TABLE 2. Rates of exchange of 82p into PPi catalyzed by different forms of pyruvate, phosphate dikinase Equilibrium rate of Amount of exchange enzyme Pi PPi (,gmol/min Form of enzyme (milliunits) P-enolpyruvate (cpm/2 IA) (cpm/2,u1) per unit) 1 Enzyme 1.20 None 60,000 314 0.001 2 Enzyme 1.20 Present 52,800 7020 0.085 3 Enzyme-P 0.45 None 39, 900 2670 0.058 4 Enzyme-P 0.45 Present 58,200 3080 0.075 ; Enzyme-PP 0.70 None 59, 900 4410 0.081 6 Enzyme-PP 0.70 Present 55,300 4620 0.091 7 None 59,100 117 0.000

The mixture (25 ,ul) at 250 contained: Pi, 30 nmol (22,400 cpm/nmol); PPi, 20 nmol; MgCI2, 70 nmol; Tris-malate (pH = 6.7) 1000 nmol, with or without 50 nmol of P-enolpyruvate, and properly diluted enzyme. At 2, 5, 10, 20, 30, 60, and 100 min, 2-1A samples were withdrawn and applied to Whatman no. 3 chromatography paper and dried immediately under hot air after application of Pi and PPi as carriers. The rates of exchange were linear for 60 min, and the values are for 30 min of reaction. Downloaded by guest on September 29, 2021 2466 Biochemistry: Milner and Wood Proc. Nat. Acad. Sci. USA 69 (1972) TABLE 3. Rates of exchange of [14C]pyruvate into P-enolpyruvate catalyzed by different forms of the enzyme

Equilibrium rate of Amount of exchange Form of enzyme Pyruvate P-enolpyruvate (umol/min enzyme (milliunits) Pi (cpm/21pl) (cpm/2 Ad) per unit) 1 Enzyme 0.25 None 7510 1100 4.2 2 Enzyme 0.25 Present 6990 1230 4.3 3 Enzyme-P 0.44 None 6580 1750 4.3 4 Enzyme-P 0.44 Present 6020 1890 4.4 5 Enzyme-PP 0.5 None 7510 740 2.2 6 Enzyme-PP 0.5 Present 5340 3000 4.9 7 - None 8100 102 0

The mixture (25 1d) at 250 contained: [3-14C]pyruvate, 50 nmol (1.0 Ci/mol); P-enolpyruvate, 50 nmol; MgCl2, 50 nmol imidazole buffer (pH = 7.0), 1000 nmol; (NH4)2S04, 60 Amol; Pi (where included), 100 nmol; and enzyme at the proper dilution. Samples were applied to chromatographic paper before carrier pyruvate and P-enolpyruvate. Chromatography was conducted with methanol-water 8:2. Pyruvate and P-enolpyruvate were visualized by UV light, cut out, and their radioactivity determined. The rates were linear during 10 min; the values given are for 5 min of reaction time.

spot indistinguishable from an authentic glucose-6-P marker. of Pi and 2.48 nmol of PPi were recovered, and there was The ADP fraction from the hexokinase reaction did not con- a quantitative transfer of 32p from the [32P]pyrophosphoryl- tain appreciable radioactivity. Thus, the label was clearly enzyme to form [82P]PPi. established to be in the y position of the ATP, in accord 'with the Evans-Wood mechanism. Catalysis of Pi i± PPi Exchange (Reaction lb). Catalysis of the exchange reaction by different forms of the enzyme Formation of [32P]PPi from [82P]Pyrophosphoryl-Enzyme and the requirement for P-enolpyruvate for the exchange and Pi. According to reaction lb, the pyrophosphoryl-enzyme of 32P into PPi is shown in Table 2. Free enzyme did not and Pi should yield PPi. Pyrophosphoryl-enzyme (2.54 pmol, catalyze the exchange of 32Pi into PP unless P-enolpyruvate 2200 cpm) was incubated with 5 Mmol of Pi, 5 /Amol of MgCl2, was added; this finding is in accord with reactions lc and lb, and 5 ,umol of Tris-malate (pH 6.9) in a 20-II final volume because phosphoryl-enzyme is required for this exchange. for 10 min at 260. The sample was treated and chromato- With phosphoryl- or pyrophosphoryl-enzyme there was no graphed as described in the legend of Table 1. 0.104 pmol requirement for P-enolpyruvate for the exchange, since it could occur with either form directly via reaction (lb). Pyruvate ;± P-Enolpyruvate Exchange (Reaction ic). Table 3 shows the requirement of Pi for catalysis of the exchange of [3-'4C]pyruvate into P-enolpyruvate by the three forms of the enzyme. Pi should have little, if any, influence on the exchange by reaction lc with either free enzyme or phosphoryl-enzyme, but should increase the exchange catalyzed by pyrophosphoryl-enzyme -since Pi is required to convert the pyrophosphoryl-enzyme via reaction lb to the phosphoryl-enzyme required in the pyruvate exchange reaction. The exchange was increased by addition of phosphate (Exp. minutes 5 compared to Exp. 6). The exchange that occurred in the FIG. 4. The exchange of [14C]AMP with ATP and with the absence of added Pi may have resulted from a slight con- methylene analogues of ATP. The mixture (25 ul final volume) tamination of the P-enolpyruvate with Pi, causing phos- contained: Tris-malate (pH 7.0), 2 pmol; MgCl2, 40 nmol; phorolysis of enzyme-PP to enzyme-P. (NH4)2S04, 100 nmol; ATP, 25 nmol (or 20 nmol of the ATP analogues); [14C]AMP, 25 nmol (2600 cpm/nmol); and properly The AMP *± ATP Exchange and Exchange with Analogues diluted enzyme. Incubation was at 250, 2-Aul samples were chro- of ATP (Reaction la). A comparison of the rates of exchange matographed in 95% ethanol, 1.0 M ammonium acetate pH = of [14C]AMP into ATP and into the methylene analogues of 7.5 7:3 for 14 hr at room temperature after application of carrier ATP is shown in Fig. 4. Both free enzyme and the pyrophos- AMP and ATP. AMP and ATP were located in UV light, marked, phoryl-enzyme catalyzed a rapid and almost identical rate of cut out, and their 14C content was determined. 12.1 milliunits of exchange, as expected for reaction la. The rate was slower free enzyme or pyrophosphoryl-enzyme (Fraction 11 of Fig. 1) with the phosphoryl-enzyme but was increased by addition and 9 milliunits of phosphoryl-enzyme were used for the AMP F± of pyruvate, which converts the phosphoryl-enzyme to free ATP exchange, and 24.2 milliunits of free enzyme was used for in the exchange with the analogues. ca,,8-methylene adenosine tetra- enzyme. The catalysis' by the phosphoryl-enzyme the occurrence of some phosphate had little or no activity and was comparable to the a,#- absence of pyruvate probably reflects ATP analogue. The symbols used are: E, free enzyme; E-P, free enzyme arising by hydrolysis during storage of the phos- phosphoryl-enzyme and E-PP, pyrophosphoryl-enzyme. phoryl-enzyme and during the reaction. Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Pyrophosphoryl-Enzyme from Propionibacteria 2467

It is of special significance that there was exchange of with that of Pi >. ATP exchange and found that AMP was [14C]AMP with the ,,y-methylene analogue of ATP, and required in order to obtain comparable rates, even when the there was essentially no exchange with the a,o-methylene enzyme was maintained in the phosphoryl form with ATP analogue. These results indicate there was formation of en- or P-enolpyruvate. They concluded that the free pyrophos- zyme-P-C-P, and that the P-C-P bond of the a,fl-analogue phoryl enzyme as such is not an intermediate, and suggested is not cleaved by the enzyme. The exchange with APPCH2P that the reaction occurs by the following sequence, in which was slow, but in 2 hr about 25% had exchanged with [14C]- the dots represent Michaelis-Menton type complexes and AMP. The APPCH2P was found to be free of ATP by assay E is enzyme, Pyr is pyruvate, and PEP is P-enolpyruvate: with hexokinase and glucose 6-P dehydrogenase. In unpub- H20 lished experiments, we also found that the methylene ana- E + ATP 2± E*ATP 2. E-PP 2± logues inhibit the overall reaction. The comparative rates of exchange of AMP with ATP AMP in jmol/min per unit of enzyme were 0.106 for free enzyme or pyrophosphoryl-enzyme, 0.048 for phosphoryl-enzyme, P For and 0.100 for phosphoryl-enzyme plus pyruvate. free E-P E-P + Pi + AMP (2d) enzyme plus ,,'y-APPCH2P, the rate was 0.004. It is evident from Tables 2 and 3, and Fig. 4 that there is AMP a wide difference in the rates of exchange in the different partial reactions. Pyruvate exchanges rapidly with P-enol- E-P + Pyr z E-P E * PEP E + PEP (2c) pyruvate, AMP exchanges more slowly with ATP, and P exchanges still more slowly with PPi. The exchange of Pi Pyr with PPi was about 10% of the rate of the overall back- reaction, or 30% of the overall forward-reaction, since the If Pi is substituted for the water in reaction 2d, reaction le is ratio of the rate of the back to forward reaction is about 3 obtained, in which the products are the same as those pro- to 1. The slow rates of exchanges may be due to product in- posed by Andrews and Hatch (2) for the plant pyruvate, hibition by the various substrates under conditions neces- phosphate dikinase (see reaction Id). sary to perform'such operations. Pi E + ATP T EATP E-PP DISCUSSION Although isolations of phosphoryl-enzyme have been re- AMP

ported in numerous instances (10), to our knowledge this is PP the first example of the isolation of a free, active pyrophosphoryl-enzyme. Other enzymes very likely have pyrophos- E-P E-P + PPi + AMP (le) phoryl forms as intermediates, and P-enolpyruvate synthase of E. coli, which catalyzes reaction 2, could be one example. AMP This reaction is quite similar to that of pyruvate, phosphate dikinase, and Cooper and Kornberg (11) have postulated This reaction would explain Andrews and Hatch's finding that it involves a pyrophosphoryl-enzyme via the following that the exchange of [14C]AMP into ATP did not occur in three partial reactions: the absence of Pi, and that Pi ;. PPi exchange occurred with ATP present but not when P-enolpyruvate was substituted Enzyme + ATP t enzyme-P-O-P + AMP (2a) for ATP. Perhaps in the reaction catalyzed by the plant Enzyme-P-O-P + H20 ± enzyme-P + Pi (2b) enzyme the phosphorolysis of the pyrophosphoryl-enzyme always occurs before dissociation of the AMP from the Enzyme-P + pyruvate 2 enzyme enzyme and, thus, the free pyrophosphoryl-enzyme is never + P-enolpyruvate (2c) an intermediate. Nevertheless, it should be possible to isolate The difference in the reactions by the two enzymes is hydrol- the enzyme-PP complex starting with ATP if Pi is excluded ysis in reaction 2b and phosphorolysis in reaction lb. Cooper from the reaction. and Kornberg observed exchange of ['4C]AMP into ATP, Reeves et al. (13) have shown with pyruvate, phosphate but a high concentration of Pi was required. However, the dikinase from Bacteriodes symbiosus that the distribution of other exchange reactions and the distribution of labeled 32p 32p from various labeled substrates into products is that from various labeled substrates into the products were in expected from reactions la, b, c. In preliminary studies with agreement with the proposed sequence. Cooper and Kornberg this enzyme, we have shown that it catalyzes the expected (12) isolated the phosphoryl-enzyme, but an attempt to isolate exchange reactions in accord with the mechanism of Evans the pyrophosphoryl-enzyme failed, probably because the and Wood. Hence, it is most probable that this enzyme oc- enzyme catalyzes the hydrolysis of the pyrophosphoryl group. curs as a free pyrophosphoryl intermediate. Berman and Cohn (3) attempted to overcome this difficulty Another reaction that may involve a pyrophosphoryl- by use of ATP analogues in which the oxygen bridge between enzyme is that catalyzed by 5-phosphoribosylpyrophosphate the ,B and y phosphorous was replaced with carbon or nitrogen; synthetase. It catalyzes the following reaction: however, they found no evidence of' the formation of an ana- Ribose-5-P + ATP >± P-O-P-ribosyl-5-P + ATP (3) logue of the postulated pyrophosphoryl-enzyme, but a very small rate of AMP T± APPCH2P exchange was observed. Switzer (14), using [-y-32P]ATP, found the enzyme from They also compared the rate of the H2180 : Pi exchanges Salmonella typhimurium acquired IT, probably as E-P IT, Downloaded by guest on September 29, 2021 2468 Biochemistry: Milner and Wood Proc. Nat. Acad. Sci. USA 69 (1972) but the 32p was not transferred to substrate acceptors; thus, 1. Evans, H. J. & Wood, H. G. (1968) Proc. Nat. Acad. Sci. its role in the catalysis remains in question. In addition, by USA 61, 1448-1453. kinetic analysis with phosphoribosylpyrophosphate syn- 2. Andrews, T. J. & Hatch, M. D. (1969) Biochem. J. 114, 117- thetase from human erythrocytes, Fox and Kelley (15) have 125. 3. Berman, K. M. & Cohn, M. (1970) J. Biol. Chem. 245, shown that the reaction occurs by a sequential bi, bi mech- 5309-5325. anism; this result makes it unlikely that a free pyrophos- 4. Milner, Y. & Wood, H. G. (1972) Fed. Proc. Abstr. 31, 452 phoryl-enzyme is a direct intermediate. Abstr. In addition, Switzer (16) found that catalysis of the AMP 5. Zamenhof, S. (1957) in Methods in Enzymology, eds. Colo- >± ATP exchange by this enzyme requires the presence of wick, S. P. & Kaplan, N. 0. (Academic Press, New York), low concentrations of Pi. Since PPi is not a product of the Vol. III, p.702. reaction, it is not likely that an explanation similar to that 6. Yagil, G. & Hoberman, H. D. (1969) Biochemistry 8, 352- of reaction 2d applies in this case. A possible explanation 360. 7. Evans, H. J. & Wood, H. G. (1971) Biochemistry 10, 721- of the phosphate requirement is that it acts as a secondary 729. modifier of the enzyrne and that this modification is necessary 8. Hanes, C. S. & Isherwood, F. A. (1949) Nature 164, 1107- for catalysis of the AMP ;± ATP exchange. The same ex- 1112. planation might apply to P-enolpyruvate synthase of E. 9. Boyer, P. D., Deluca, M., Ebner, K. E., Hultquist, D. E. coli and pyruvate, phosphate dikinase of plants. & Peter, J. B. (1962) J. Biol. Chem. 237, PC3306-3308. The demonstration and isolation of the three enzyme forms 10. Bell, R. M. & Koshland, D. E., Jr. (1970) Science 172, 1253- of pyruvate, phosphate dikinase confirms the tri (uni,uni) 1256. ping-pong mechanisms observed by steady-state kinetics 11. Cooper, R. A. & Kornberg H. L. (1967) Biochim. Biophys. (4) and the Evans-Wood mechanism (1). Acta 141, 211-213. The fact that the pyrophosphoryl form of pyruvate, phos- 12. Cooper, R. A. & Kornberg, H. L. (1967) Biochem. J. 105, phate dikinase from P. shermanji is stable should make it 49C-50C. 13. Reeves, R. E., Menzies, R. A. & Hsu, D. S. (1968) J. Biol. possible to characterize its structure. These findings may Chem. 243, 5486-5491. facilitate the understanding of other pyrophosphoryl-trans- 14. Switzer, R. L. (1968) Biochem. Biophys. Res. Commun. 32, ferring enzymes, such as P-enolpyruvate synthase, phos- 320-325. phoribosylpyrophosphate synthetase, thiamine pyrophos- 15. Fox, I. H. & Kelley, W. N. (1972) J. Biol. Chem. 247, phokinase (17), and pyruvate, phosphate dikinase from 2126-2131. plants. 16. Switzer, R. L. (1970) J. Biol. Chem. 245, 483-495. This research was supported by contract AT(11-1)-1783 from 17. Shimazono, N., Mano, Y., Tanaka, R. & Kazino, Y. (1959) the Atomic Energy Commission. J. Biochem. (Tokyo), 46, 959-963. Downloaded by guest on September 29, 2021