THE JOURNAL OP Bromorca~ CHE~STRY Vol. 241, No. 10, Issue of May 25, pp. 2405-2412, 1966

Printed in U.S.A.

The Enzymatic Carboxylation of Phosphoenolpyruvate

I. PURIFICATION AND PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYLASE*

(Received for publication, November 19, 1965)

HITOSHI MARUYAMA, RICHARD L. EASTERDAY, HUEI-CHE CHANG, AND M. DANIEL LANES

From the Department of Biochemistry, New York University School of Medicine, New York, New York 10016

SUMMARY

Phosphoenolpyruvate carboxylase has been isolated from germinating peanut cotyledons and purified 2700-fold. The purified enzyme is approximately 80% pure as judged from sedimentation patterns, has a sedimentation coefficient (szo,,J of 13.9 S, and catalyzes the carboxylation of 50 pmoles of phosphoenolpyruvate per mm per mg of protein (refracto- metrically determined) at 30’. Enzymatic carboxylation oc- curs optimally at pH 8.0 to 8.2. The K,,, values determined for HCOI-, phosphoenolpyruvate, and Mg2+ are 3.1 X 10p4, 5 to 6 x 10e4, and 3 to 4 x 10e4 M, respectively, at pH 7.9. Studies on the effect of pH on the K, values for phospho- enolpyruvate and Mgz+ were conducted. Evaluation of plots of -log K,,, (Mg 2+ and phosphoenolpyruvate) against pH indicate that a dissociable group (or groups) of the car- boxylase having a pK of 7.3, presumably an hnidazole group, is involved in Mg*+ and phosphoenolpyruvate binding. Other evidence supporting this view is presented.

During the enzymatic carboxylation of phosphoenolpyru- vate, 180 from substrate HC1803- is incorporated into the orthophosphate and oxalacetate reaction products in a ratio of 1:2. This indicates that the HCOI- (or COa2-) anion rather than COZ is the active species in the carboxylation reaction. A mechanism is proposed which is consistent with this and other observations on the phosphoenolpyruvate carboxylase-catalyzed reaction.

Three distinct enzymatic carboxylation reactions have been described (l-13) in which phosphoenolpyruvate is carboxylated to form oxalacetate. These reactions differ with respect to the mechanism of disposition of the enol phosphoryl group. The least complicated reaction (Reaction 1) which is catalyzed by P-enolpyruvate carboxylase (EC 4.1.1.31), the subject of this report, leads to the irreversible formation of oxalacetate and orthophosphate.

CHz=C*(OPOa*-)+COz’- + HCOa’- Mgt+

(1) ‘-OZC*CH~*CO*CO? + Pi*

* This work was supported by Research Grant AM 69177 from the National Institutes of Health, United States Public Health Service.

$ Career Development Awardee of the United States Public Health Service (Research Career Program Award K3-AM-18487).

This enzyme, which is widely distributed in plant tissues and microorganisms, including spinach leaves (l), wheat germ (3), peanut cotyledons (4), Thicbacillus thiooxidans (5), and Escher- ichiu coli (6), has not been found in animal tissues. The other two carboxylation reactions (Reactions 2 and 3) catalyzed by P-enolpyruvate carboxykinase (EC 4.1.1.32) (7-l 1) and P-enol- pyruvate carboxytransphosphorylase (EC 4.1.1.3) (12, 13), respectively, appear mechanistically similar.

CH~=C.(OPO?-).CO~l- + HCOal-

Md+ + GDP”(or IDP) + HI+ m (2)

r-OnC.CH,.C0.C021- + GTP+ (or ITP) + HOH

CHz=C . (OPOa2-). Cot’- + HCOa’-

Mnz+ or Mgs+ + Pi*- + H+ \ . (3)

‘-02C*CH2*CO*C02’ + PPi* + HOH

Carboxylation of P-enolpyruvate, accompanied by transfer of the enol phosphoryl group to either GDP (or IDP) or Pi, results in the formation of a new pyrophosphate bond.

It is conceivable that all of the P-enolpyruvate carboxylation reactions occur by the same basic mechanism or at least have certain mechanistic features in common. The objective of this investigation and those to follow in this series is the elucidation of the mechanism by which these enzymatic carboxylations take place. More specifically, the present report describes the isola- tion and purification of P-enolpyruvate carboxylase from peanut cotyledons, some of its properties, and studies on its reaction mechanism. A preliminary communication on part of this work has appeared (14).

EXPERIMENTAL PROCEDURE

P-enolpyruvate (basic sodium salt), 2-phosphoglycerate (bar- ium salt), 3-phosphoglycerate (barium salt), heart muscle malate dehydrogenase, and GSH were obtained from Calbiochem; NADH from Pabst Laboratories; NaHi4COa from New England Nuclear Corporation, and K&rsOa (59 to 72% l*O) from YEDA Research and Development Company, Ltd. (Rehovoth, Israel). DEAE (type 20)- and Ecteola (type 20)-cellulose were products of Schleicher and Schuell. Large seeded, Virginia-type peanuts (Arachis hypogeae) were obtained from the Peanut Growers Co- operative, Franklin, Virginia. Peanuts were stored in the cold room and treated with Arasan 50-Red (E. I. du Pont de Nemours and Company, Inc.) prior to germination. Hydroxylapatite was either prepared by the method of Tiselius, Hjert&r, and

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2406 Enzymatic Carboxylation of Phosphoenolpyruvate. I Vol. 241, No. 10

Levin (15) or purchased from Bio-Bad Laboratories. Sephadex G-299, obtained from Pharmacia, was equilibrated for 5 or 6 days at 4” with the elution buffer prior to packing columns (1.5 X 100 cm, Pharmacia). Liquid scintillator was composed of 0.25 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene (POPOP), 10 g of 2,5- diphenyloxazole (PPO), and 100 g of recrystallized napthalene per liter of dioxane.

Methods Employed in IsO Experiments

Orthophosphate was isolated from reaction mixtures as the monobasic sodium salt (Experiments 1 and 2) by the method of Cohn (16), as the barium salt (Experiment 3), or as the silver salt. In Experiments 1 to 3, carrier Pi (160 pmoles) was added followed by 299 pmoles of MgClz and 1900 pmoles of NHdOH. The precipitated MgNHdPOd was recovered by filtration, redis- solved in 2 N HCl, and the solution taken to dryness several times to remove HCl. After dissolving the residue in water and ad- justing the pH to 4.5 with NaOH, the solution was passed through a Dowex 50-X4 column. In Experiments 1 and 2, the column eluate was adjusted to pH 4.5 with carbonate-free NaOH and taken to dryness in a vacuum several times between inter- mittent additions of water. The isolated NaHzPOa was then analyzed for l*O. In Experiment 3, Pi in the column eluate was precipitated as Bas(PO& at pH 10 in a COz-free atmosphere by addition of NaOH, NH&l, and BaClz. Following filtration and drying in a COz-free atmosphere, the isolated barium phosphate was subjected to I80 analysis. The preferred method which involved isolation of Pi as AgsP04 was used in Experiment 4. The enzymatic reactions were terminated at 0” by the addition of sufficient 70% HClOa to produce a final concentration of 3%. After addition of carrier Pi (105 pmoles), 750 mg of acid-washed Norit A were added and the mixture was filtered. This proce- dure was repeated with the filtrate with the use of 406 mg of Norit and was followed by washing the combined Norit portions with 3% HClOt. To the pooled filtrates, brought to pH 10 or 11 with 15 N NHdOH, were added 1.0 ml of magnesia solution (17) and sufficient 15 N NHdOH to produce a final concentration of 1.5 N. Following overnight precipitation at O’, the precipitate was recovered by filtration, washed with cold 1.5 M NHIOH (2 ml), and dissolved in 5.0 ml of 0.1 N HClOd. The solution was brought to the faint yellow end point (pH 6.0 to 6.5) of methyl red with 0.5 N carbonate-free NaOH, followed by the addition of 709 bmoles of AgClOd in the dark. All of the subsequent steps were conducted in a darkened room. The solution was brought to the methyl red end point as before; precipitated Ag,P04 was recovered by centrifugation, redissolved in 0.1 N HClO+ and reprecipitated by the same procedure. After washing the pre- cipitate three times with cold, CO%-free Hz0 it was dried in a vacuum over PzOa and then subjected to I*0 analysis.

Malic acid was isolated by silicic acid chromatography as de- scribed by Bruno and Moore (18). The reaction mixture, to which 160 pmoles of malic acid carrier had been added, was lyo- philized, redissolved in 2.0 ml of 1 N HaSOd, and applied to the silicic acid column. Gradient elution was effected with 70 ml of benzene in the mixing chamber and 70 ml of l-butanol-chloro- form (50:50, v/v) in the reservoir (both solvents were saturated with 1 N H&?OJ. After the reservoir emptied, more l-butanol- chloroform mixture was added until the malic acid was quantita- tively eluted. Fractions containing malic acid were collected, brought to dryness by lyophilization, and subjected to 180 analysis.

The methods of 180 analysis described by Anbar and Guttman (19) and by Samuel (20) were employed. The total oxygen of NaHzPO+ Baa(POJz, Ag,POd, and malic acid was converted to COZ by pyrolysis at 400” for 2 hours with a mixture of mercuric chloride and cyanide. The CO, was purified by heating at 299’

Mg Cl 2 ADDED 0.8 -

i PROTEIN

0.5

0.6 -

ABSORBANCY 1.0

AT 334 mp

0.4 1.5

-

2.0

0.2 - 2.5

,C 0 I 2 3 4

INCUBATION TIME, MINUTES

FIG. 1. Kinetics of P-enolpyruvate carboxylation at varying enzyme concentrations. Standard spectrophotometric carboxyl- ation assay conditions were employed with hydroxylapatite chro- matographically purified enzyme (specific activity, 18 units per mg of protein; Stage 7 of Table I).

0.05

0.04

0.03

0.02

0.01

FIG. 2. pH optimum of P-enolpyruvate carboxylase. The standard spectrophotometric carboxylation assay was employed and the pH of each reaction mixture was determined with a Beck- man model G pH meter. X-X, 0.1 M potassium phosphate buffer; O- - -0, 0.1 M Tris (Cll-) buffer. Hydroxylapatite chro- matographically purified enzyme (specific activity, 16 units per mg of protein; Stage 7 of Table I) was used.

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Issue of May 25, 1966 Maruyama, Easterday, Chang, and Lane 2407

for 2 hours with saturated zinc amalgam. The 180 analyses were conducted on the purified CO* by YEDA Research and Develop- ment Company, Ltd., with a CRC model MS 204 mass spec- trometer.

Phosphoenolpyruvate Carboxylase Assay

The Mg2+-dependent carboxylation of P-enolpyruvate results in the formation of oxalacetate and orthophosphate (see Reac- tion 1). The reaction rate in the presence of NADH and malic dehydrogenase is determined by following either the rate of in- corporation of H14C03 into malate (acid-stable 1°C activity) or the rate of NADH oxidation spectrophotometrically. The H14COa-fixation carboxylation assay reaction mixture contains the following components (in micromoles except as indicated): Tris (Cl-) buffer, pH 7.8, 80; KH14C0a (approximately lo6 cpm per pmole), 10; P-enolpyruvate, 2.0; MgC&, 2.0; GSH, 5.0; NADH, 2.0; malate dehydrogenase, 14 units; and P-enolpyruvate carboxylase, up to 0.004 unit in a total volume of 1.0 ml. The final pH is 7.9. After a 15-min incubation at 30”, the reaction is terminated by addition of 1 ml of 2 N HCl. A 0.5-ml aliquot is taken to dryness in a scintillation counting vial at 85” for 60 min in a forced draft oven. After addition of 1 ml of Hz0 to the vial, then 10 ml of liquid scintillator, acid-stable ‘4C activity (as I%!-malate) are determined with a liquid scintillation spec- trometer. Initial velocity follows zero order kinetics for 15 min and is proportional to enzyme concentration up to a level of 0.004 unit of P-enolpyruvate carboxylase.

A spectrophotometric carboxylation assay is generally used for P-enolpyruvate carboxylase preparations carried beyond Step 3 (0 to 55% saturated (NH&SO4 fraction) of the purifica- tion procedure. The reaction mixture and conditions are modi- fied from those described for the H*4COs-fixation carboxylation assay (above) to include, unlabeled, instead of 14C-bicarbonate and less NADH (0.15 pmole). The rate of NADH oxidation is

TABLE I Purijkation of phosphoenolpyruvate carboqllase

Stage of purification - .

1. Initial extractc.. 2. Aged extract.............. 3. 0 to 55% saturated

(NHI),SOI fraction 4. 32 to 42% saturated

(NH1)*SOI fraction. 5. Ecteola-cellulose eluate 6. DEAE-cellulose chromate

graphic fraction. 7. Hydroxylapatite chromate

graphic fraction 8. Sephadex G-200 gel filtrate

, -

, -

‘_

-

Protein”

g

598 207

45.3

10.4 7.00

0.28f 5

0.0% O.Ol(

- Total

a&vi+ Specific activity

unilr units/mg

6230 0.0104 6820 0.033

4960 0.109

3720 0.357 3020 0.432

1730 6.08

305 15.2 292 2S.ld

-

- Yield

%

100 109

80

60 48

28

4.9 4.7

a Determined spectrophotometrically according to the method of Warburg and Christian as described by Layne (21).

b Determined with the spectrophotometric P-enolpyruvate carboxylase assay method with exception of initial and aged extracts; Hl4COa fixation assay was used for the latter extracts.

c Initial extract was obtained from 6 kg of peanuts after ger- mination and homogenization.

d Specific activit,y based on refractometrically determined protein is 49.6 units per mg (see text for details).

followed at 334 rnp (l-cm light path; 30’) for 2 to 3 min after initiating the reaction with P-enolpyruvate. The carboxylation rate follows zero order kinetics for at least 2 min and is propor- tional to enzyme concentration up to a level of 0.05 unit of car- boxylase as shown in Fig. 1. A unit of P-enolpyruvate carboxyl- ase is defined as that amount of enzyme which catalyzes the carboxylation of 1.0 pmole of P-enolpyruvate per min under the assay conditions described. The requirements and stoichiom- etry of the P-enolpyruvate carboxylase-catalyzed reaction have been described (4,14). The pH optimum (see Fig. 2) for P-enol- pyruvate carboxylase determined with the spectrophotometric carboxylation assay is approximately 7.9. It is apparent from the points of the activity curve for phosphate buffer on the pH optimum plot (Fig. 2) that the Pi dianion is inhibitory. This is not surprising since the Pi dianion might be expected to compete with the phosphoryl dianion of P-enolpyruvate for its enzyme-binding site. Orthophosphate inhibition with Tris (Cl-), pH 7.8, as buffer is apparently competitive with P-enol- pyruvate since the inhibition can be reversed by increasing the P-enolpyruvate concentration. Exposure of the enzyme to the pH values in the range tested in Fig. 2 for 3 or 4 min, (approxi- mate time necessary for the spectrophotometric carboxylase assay) caused essentially no loss of carboxylase activity.

Puri,fcation of Phosphoenolpyruvate Carboxylase

All of the procedures are carried out at 4' unless otherwise specified. The results of the purification procedure are sum- marized in Table I.

Initial Extract and Aging Treatment-Shelled peanuts (drachis hypogeae), 6 kg, are dusted with 8 g of Arasan (active ingredient, tetramethylthiuram disulfide) and placed between several layers of paper toweling in Pyrex baking dishes covered with perforated aluminum foil. The peanuts are watered with 0.2% sodium propionate in tap water and are germinated for 4 days in the dark at 28-30”. Additional sodium propionate solution is added as needed. Cotyledons are removed, washed several times with distilled water, and then homogenized in 2 volumes (w/v) of 0.05 M phosphate buffer, pH 7.0 (2 X 10M4 M EDTA and 5 x 10-3 M 2-mercaptoethanol), for four 30-set periods at top speed in a Waring l3lendor. The homogenate is filtered through cheesecloth, centrifuged for 1 hour at 14,000 x g, the supernatant solution decanted, and filtered again through cheesecloth. To the resulting extract, referred to as “initial extract” are added sufficient neutralized GSH to produce a concentration of 5 x

1O-4 M and a few drops of toluene. This extract is aged for 25 hours at 30” in a water bath, stored overnight at 4”, and then centrifuged at 14,000 x g for 50 min.

Ammonium Sulfate Fractionation-The clear yellow superna- tant solution from the previous step is brought to 55% saturation

with solid ammonium sulfate’ by gradual addition of the salt (0.351 g per ml) with gentle stirring. After standing overnight, the suspension is centrifuged, the precipitate is dissolved in 300

ml of 0.02 M phosphate buffer, pH 6.5, (10’ M 2-mercaptoethanol and 2 x 10e4 M EDTA), and the enzyme solution is dialyzed against 8 liters of the same buffer for 8 hours. After dialysis, the enzyme solution is centrifuged to remove the voluminous precipitate (globulins) formed during dialysis. The supernatant solution (60 to 80 mg of protein per ml) is diluted with the dialysis

* All of the “percentage of ammonium sulfate saturation” fig- ures obtained with solid ammonium sulfate refer to percentage of saturation at 25’.

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2408 Enzymatic Carboxylation of Phosphoenolpyruvate. I Vol. 241, lKo. 10

buffer to produce a protein concentration of 40 mg per ml. Satu- rated ammonium sulfate,2 pH 7.5, (0.471 mg per ml of enzyme solution), is chilled to 4” and the enzyme solution is added to it with gentle mixing. The resulting 32% saturated ammonium sulfate solution is allowed to stand for 30 min and is then cen- trifuged at 15,000 x g for 15 min. The supernatant solution is added to sufficient chilled saturated ammonium sulfate, pH 7.5 (0.172 ml per ml of supernatant solution), to produce a 42% saturated ammonium sulfate solution. After standing for 8 to 10 hours, the suspension is centrifuged as before, the precipitate dissolved in 225 ml of 0.02 M phosphate buffer, pH 6.5 (lop2 M 2- mercaptoethanol and 5 x lop4 M EDTA), and then dialyzed against 8 liters of the same buffer for 8 hours.

E&da- and DEAE-cellulose Chromatography-The dialyzed enzyme solution from the previous step is applied to an Ecteola- cellulose column (4.5 cm diameter; 11.2 g of dry Ecteola-cellulose per g of protein; exchange capacity approximately 0.3 meq per g) previously equilibrated with 0.02 M phosphate buffer, pH 6.5 (lo+ M 2-mercaptoethanol and 5 X lo-’ M EDTA) . The enzyme is eluted with the same buffer and appears in the breakthrough peak. The column effluent is continuously monitored at 253 rnp (LKB Uvicord Absorptiometer) and carboxylase activity is located with the spectrophotometric assay method. The pooled carboxylase-containing fractions (approximately 280 ml, 8 g of protein, and 80% of the carboxylase activity applied) are imme- diately applied to a DEAE-cellulose column (4.5 x 45 cm) (exchange capacity 0.8 meq per g) previously equilibrated with 0.02 M phosphate buffer, pH 6.5 (1O-2 M 2-mercaptoethanol and 5 x 10m4 M EDTA). Stepwise, gradient elution is accomplished by placing 950 ml of the same buffer in a mixing chamber at- tached to the column and introducing the following phosphate buffers, all pH 6.5, into a separatory funnel attached to the mixing chamber: 2 liters of 0.2 M and 2 liters of 0.4 M containing lo”1 M 2-mercaptoethanol and 5 X 10m4 M EDTA. The effluent is continuously monitored for protein, collected fractionally, and fractions assayed for P-enolpyruvate carboxylase activity. Enzyme activity appears in the eluate after about 2400 ml have been collected. The most “active” fractions (approximately 600 ml) containing 50 or 60% of the carboxylase activity applied to the column are pooled, placed in dialysis bags, and then dia- lyzed against an a.mmonium sulfate solution, pH 6.5 (5 x 1O-4 M

EDTA and 1w2 M 2-mercaptoethanol), of sufficient concentra- tion to reach 6O70 saturation at equilibrium.

Hydroxylapatite Chromatography-The precipitated protein (250 to 350 mg) recovered from the preceding step by centrifuga- tion is dissolved in 15 ml of 0.02 M phosphate buffer, pH 6.5 (10e2 M 2-mercaptoethanol), and applied to a hydroxylapatite column (2 x 15 cm) previously equilibrated with the same buffer. Stepwise elution is carried out under pressure (2 to 4 p.s.i.) with 10 ml of 0.02 M, 80 ml of 0.1 M, 100 ml of 0.2 M, 100 ml of 0.3 M,

and 100 ml of 0.5 M phosphate buffers, pH 6.5, containing 10m2 M

2-mercaptoethanol. P-enolpyruvate carboxylase activity and protein concentration in the eluted fractions (3 ml) are deter- mined as described earlier. Elution of peak enzyme activity occurs during the addition of the 0.3 M buffer. These fractions are pooled (70 to 80 ml) and precipitated at 600% ammonium sulfate saturation (pH 6.5) in the presence of lo-2 M 2-mercapto- ethanol and 5 x 10m4 M EDTA by the dialysis technique de- scribed earlier.

2 Ammonium sulfate is saturated at room temperature and neu- tralized with NHdOH so that when diluted 5-fold the pH is that indicated.

Sephadex G-%‘@.I Gel Filtration-The enzyme suspension (10 to 20 mg of protein) from hydroxylapatite chromatography is cen- trifuged, the precipitate redissolved in 1 ml of 0.02 M phosphate buffer, pH 6.5 (containing 5 x lo-’ M EDTA, 5 X lo-’ M GSH, and low4 M P-enolpyruvate), and the solution applied to a Sepha- dex G-200 column (1.5 x 85 cm) previously equilibrated with the same buffer. Sephadex G-200 columns of this dimension have a flow rate of about 5 ml per hour under a hydrostatic head of 20 cm of buffer. The enzyme is eluted with the equilibrating buffer and appears in the eluate after approximately 45 ml have been collected. Enzymatic activity and protein concentration in the eluted fractions are determined as described previously. The V,/VO ratio (where V, = elution volume and V0 = void volume) was found to be 1.36. This value corresponds to a molecular weight of 3 to 3.5 x lo6 when related to a plot of log molecular weight against V,/VO obtained from Sephadex G-200 elution data for a series of proteins of known molecular weight. Fractions containing maximum carboxylase specific activity are pooled and precipitated at 60% ammonium sulfate saturation, pH 6.5 (5 X lo-’ M EDTA, 5 X 10e4 M GSH, and low4 M P-enol- pyruvate), by the dialysis technique described earlier.

As indicated in Table I, a 2800-fold purification of P-enolpyru- vate carboxylase from the initial extract is achieved in 5% yield with the procedure outlined. When stored as a suspension under 60% saturated ammonium sulfate (containing 5 x 10m4 M EDTA and GSH) at 4’ the enzyme is stable for at least 2 or 3 months. In dilute solution at low ionic strength (5 x lo+ M Tris (Cl-)) and pH 7.5, carboxylase activity is lost at about 10 to 15% per hour. The absorbance ratio (A,o/A& of the purified carboxyl- ase preparation is approximately 1.8.

RESULTS

Purity and Sedkentation CoeJicient of Phosphoenolpyruvate Carboxylase-Several P-enolpyruvate carboxylase preparations, purified according to the procedure outlined, have been examined for purity in the analytical ultracentrifuge. Fig. 3 shows the sedimentation pattern for a preparation having a specific activity of 28.1 units per mg of protein (spectrophotometrically deter- mined). In order to ascertain which peak on schlieren patterns corresponds to P-enolpyruvate carboxylase, several highly puri- fied carboxylase preparations (specific activity, 20 to 25 units permgof protein) were subjected to centrifugation in the analyti- cal ultracentrifuge and to sucrose density gradient centrifugation according to the method of Martin and Ames (22). Following sucrose density gradient centrifugation (38,000 rpm, Spinco SW 39 rotor for 11 hours) and fractional collection of the gradient tube contents, each fraction was assayed for carboxylase activity and protein. The carboxylase preparations employed were purified either as described in the preceding section (“Purification of Phosphoenolpyruvate Carboxylase”) or by a modification thereof (Step 8, “Sephadex G-200 Gel Filtration,” was omitted and Step 7, “Hydroxylapatite Chromatography,” was repeated a second time). The modified procedure yielded carboxylase preparations which exhibited two peaks on schlieren patterns from the analytical ultracentrifuge. In a typical run, the fastest moving 13.8 S component comprised 68% of the total peak area (from comparator readings) and the slow moving 6.5 S component comprised 32%. The same preparation (specific activity, 21 units per mg of protein), when subjected to sucrose density gradient centrifugation, also exhibited two protein peaks. The most rapidly sedimenting and principal peak (65% of the total protein) contained all of the P-enolpyruvate carboxylase activity,

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Issue of May 25, 1966 Maruyama, Easterday, Chang, and Lane 2409

whereas, the slowly sedimenting protein peak (35% of the total protein) was devoid of carboxylase activity. Carboxylase prepa- rations prepared as described under “Purification of Phospho- enolpyruvate Carboxylase,” which includes a Sephadex G-200 gel filtration step, do not contain the 6.5 S component. As shown in Fig. 3, such preparations contain a major 13.9 S component and a less abundant, faster sedimenting component. Sucrose density gradient centrifugation experiments similar to those described above, revealed that carboxylase activity was asso- ciated only with the major (slowest sedimenting) component. These results coupled with the correspondence between enrich- ment of the 13.9 S peak and increase in specific activity during the course of purification, lead to the conclusion that the 13.9 S material is P-enolpyruvate carboxylase. The average sedimen- tation coefficient (a20,w, uncorrected for protein concentration) for P-enolpyruvat,e carboxylase based on analytical ultracentri- fuge runs on five highly purified carboxylase preparations is 13.9 i 0.13 S. The purity of the carboxylase preparation (spe- cific activity, 28 units per mg of protein) whose sedimentation pattern is illustrated in Fig. 3 was assessed from the area of the 13.9 S peak relative to total peak area (schlieren pattern 4 in Fig. 3). Areas were determined from comparator readings cor- rected for radial dilution. Based on this calculation, the purity

5

FIG. 3. Schlieren patterns of purified P-enolpyruvate carboxyl- ase obtained in the Spinco model E ultracentrifuge. P-enolpyru- vate carboxylase was centrifuged at 39,460 rpm at 5.8”. Photo- graphs 1 to 6 were taken at 20, 40, 64, 80, 96, and 128 min after initiation of the run. The protein concentration according to the method of Warburg and Christian as described by Layne (21) was 10.3 mg per ml and the specific activity was 28.4 units per mg of protein. The refractometrically determined protein concentra- tion (described in text) was 5.84 mg per ml. Centrifugation was conducted in the presence of 0.02 M Tris (Cl-) buffer, pH 7.5; 0.05 M NaCl; 2 X lo+ M EDTA; and 5 X UY3 M 2-merceptoethanol.

3 Standard deviation.

TABLE II

Kinetic constants for phosphoenolpyruvate carboxylase- catalyzed reaction

Substrate or cofactor’= KtTl Enzyme Assayb

I V/uPd

HC03-.. . . . 3.1 X 10-d 1.2 Spectrophotometric Phosphoenolpyru-

vate.. . . . 6.3 X 10-e 1.2 Hl4COa- fixation Phosphoenolpyru-

vate.. . . . . . . . . . . . . . 5.1 X lo+ 1.3 Spectrophotometric Mgz+, . . . . . . . . . . 4.0 X lo-4 1.1 HI*CO3- fixation Mg*+. . . . . . . . . . . . . . . 2.7 X lo-” 1.2 Spectrophotometric Mn2+. , . . . . . . . 3.1 X 10-b 0.25 Spectrophotometricc

a Range of concentration of each substrate or cofactor whose K,,, was being determined by Lineweaver-Burk analysis: HCOa-, 3.4 X 10-4 to 1.3 X lO+ M; P-enolpyruvate, 2.5 X 1iY4 to 8 X 10-a M (Ht*COa- fixation assay) and 5.0 X 10-e to 4.0 X 10-a M (spec- trophotometrie assay); Mgs+ (total), 5.0 X lo+ to lo-2 M (H”COa- fixation assay) and 2.5 X lo-’ to 1.5 X low3 M (spectrophotometric assay); and Mn2+ (total), 2.5 X 10-6 to 8 X 10-4 M. The con- centrations of all of the other components were as listed in “Ex- perimental Procedure.”

b Spectrophotometric and H**COa- fixation carboxylation assays conducted as described under “Experimental Procedure.” Final pH was 7.9.

0 Final pH was 7.5.

of this carboxylase preparation was 80%. The protein concen- tration of the same preparation was calculated from the total peak area (schlieren pattern 4 of Fig. 3), corrected to the menicus, with a factor of 1.862 x 10e4 for the specific refractive index increment (concentration in milligrams per ml). This factor is the average for several proteins studied by Perlmann and Longs- worth (23) and corrected to 20” and a wave length of 546 mF. The protein concentration determined by this refractometric method was 5.84 mg per ml which indicates that the protein con- centration determined by the spectrophotometric method (10.3 mg per ml; Reference 21) considerably overestimates the true value. The specific activity of the 80% pure carboxylase prepara- tion was 49.6 units per mg of protein (refractometrically deter- mined) which corresponds to a specific activity of 62 units per mg of protein (refractometrically determined) for the pure en- zyme. The estimated molecular weight of 350,000 for P-enol- pyruvate carboxylase, arrived at on the basis of Sephadex G-200 gel filtration experiments (see “Sephadex G-200 Gel Filtration”) is consistent with a sedimentation coefficient of 13.9 S. With the estimated molecular weight of 350,000, the calculated molecu- lar activity of the carboxylase is 22,000 moles of substrate car- boxylated per min per mole of enzyme under standard assay conditions.

Kinetic Con&w&--K, and V values for Mg++, HCOJ, and P-enolpyruvate were determined by the method of Lineweaver and Burk (24) as described by Dixon and Webb (25) with either or both the H14C03’--fixation and spectrophotometric carboxyla- tion assays. These kinetic constants are summarized in Table II. Despite the fact that essentially “CO,-free” solutions were employed in the K, determination for HCOa- the initial velocity in the absence of added HCOa- was approximately half-maximal. Therefore, the endogenous HCOs (and CO*) concentration of the reaction mixture was determined by conducting spectrophoto- metric carboxylase assays without added HC03- in the presence of excess P-enolpyruvate carboxylase. Correcting for the en-

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2410

-LOG

KM

-LOG

KY

I MCI 2 The pK, (7.3) observed indicates that the ionizable group in- volved is probably an imidazole. Mildvan and Cohn (32) have obtained evidence that two imidazole groups and one free amino group participate in divalent metal cation binding to bovine serum albumin. Other evidence which suggests the involvement of an imidazole (or free amino group) in the binding of P-enol- pyruvate is the inhibition caused by reaction of aromatic diazo- nium compounds with P-enolpyruvate carboxylase. Purified P-enolpyruvate carboxylase (specific activity, 18.4 units per mg) suffers a 70% loss of enzymatic activity after reaction with low6 M diazotized sulfanilic acid (approximately 100 times the molar

6.8 7.0 7.2 7.4 16 7.8 concentration of the enzyme, lo-’ M) in 6 X 10e3 M phosphate

8.0 buffer, pH 7.5, for 10 min at 0”.

PH It had been reported earlier by Maruyama and Lane (4) that

P-enolpyruvate carboxylase is reversibly inhibited by p-chloro- FIG. 4. Effect of pH on -log K, for P-enolpyruvate (PEP) mercuribenzoate.

and Mg2+. Reaction Mechanism Studies with 180-Bicarbona&-Since it has not been possible (24, 14) to show partial reactions (e.g. oxal-

dogenous HCOz- concentration (3.4 x lo-’ M), which is equal to acetate-14C-pyruvate or oxalacetate-Hi4C0a exchange reac-

the concentration of NADH oxidized, a K,,, value of 3.1 X 10-4 tions), intermediates, or reversibility of the P-enolpyruvate car- M for HCOa- (+COz) at pH 7.9 was obtained. Subsequent boxylase-catalyzed reaction, very little progress has been made experiments indicated that the species of COz involved in the toward the elucidation of the reaction mechanism. At the pH

enzymatic carboxylation of P-enolpyruvate is HCOz- (or CO$-) optimum (pH 8.0) of the carboxylase, the principal species of and not COa. The K, for HCOS- for this carboxylase is con- CO2 is bicarbonate; the ratio of HC03- to H2C03 being about 45.

siderably lower than those reported for most other carboxylases. The K, for CO2 calculated from data obtained in the bicarbonate The following K, values for HCO, (or HCOz- plus COa) have K,,, experiment is less than 7 X 10d6 M. The fact that this K,,,

been reported: P-enolpyruvate carboxylase from Thiobacillus seems too low and that there is precedent (33) for HCO, as th&xridans, 1.2 x 10-z (HCOs- plus COa) (5); P-enolpyruvate active species in an enzymatic carboxylation reaction led us to carboxytransphosphorylase from Propionibacterium shermanii, consider the possibility that HCOa- rather than molecular COz, 2.9 X 10-z M (HC03) (13); P-enolpyruvate carboxykinase from as visualized by others (34, 35), is the active species in the car-

pig liver, 2 X lo- M (HC03) (26) and from bakers’ yeast, boxylation of P-enolpyruvate. Nucleophilic attack by the bi- 5 X 10-z (HCOa-) (27); ribulose diphosphate carboxylase from carbonate anion on the phosphoryl phosphorus atom of P-enol- Spinucea &acea, 9 x 1W3 M (HCO;-) (28); and propionyl-CoA carboxylase, 2 x 10e3 M (HCOs-) (29, 30). The relatively low K,,, value for HCOa indicates that P-enolpyruvate carboxylase, which is able to catalyze a rapid carboxylation at low HCO;- concentrations, can serve as an effective HCOa- trap.

The K, values found for P-enolpyruvate and Mgz+ are in the same range. Investigations on the effect of pH on the K, values H C’eO- 3 Pi for P-enolpyruvate and Mg2+ are summarized in Fig. 4 in which -log K, (i.e. pK,, for P-enolpyruvate and Mg2+) is plotted

P-enolpyruvate Oxaloacetate

against pH. An extension of the linear portion of the slope as FIG. 5. Possible reaction mechanism for the enzymatic ear-

described by Dixon (31) intersects the plateau at a pH corre- boxylation of P-enolpyruvate.

sponding to the pK of an ionizable group of the substrate or 4 Results of magnetic resonance experiments on P-enolpyruvate enzyme which is involved in substrate binding to enzyme. Since carboxylase and carboxykinase conducted in collaboration with enzyme inactivation in this pH range during the assay was ruled Drs. A. Mildvan and M. Cohn (The Johnson Foundation, Uni-

out earlier and since none of the substrates have pK values in versity of Pennsylvania) will be reported later as part of this

this region, it appears that this pK is due to an ionizable group series.

on the enzvme which is involved in substrate or cation hindinp. 6 H. C. Chang and M. D. Lane, results of equilibrium dialysis

cI. experiments to be reported subsequently.

Enzymatic carboxylation of Phosphoenolpyruvate. I Vol. 241, No. 10

It is significant that the pK, values observed with P-enolpyruvate or Mg++ were the same (i.e. 7.3). Experiments conducted in collaboration with Drs. Mildvan and Cohn4 indicate that both P-enolpyruvate carboxylase and carboxykinase bind divalent cation (Mnef) in the absence of P-enolpyruvate. Possibly a ternary (enzyme-metal-P-enolpyruvate) complex is formed in both reaction sequences in which the divalent metal cation serves as a bridge between enzyme and P-enolpyruvate. If, as is the case with P-enolpyruvate carboxykinase, P-enolpyruvate binding is dependent upon divalent cation,6 the ionizable group (or groups) on the enzyme responsible for divalent cation binding would also be indirectly essential for P-enolpyruvate binding.

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Issue of May 25, 1966 Maruyama, Easter-day, Chang, and Lane 2411

TABLE III

Incorporation of bicarbonateJ80-into reaction products

In Experiments I, II, and III the reaction mixtures contained (in micromoles except as indicated): Tris-Cl buffer, pH 8.0, 914; P-enolpyruvate, 40; KJY80a (59 atom ‘% excess), 103; MgC12, 45; GSH, 57; NADH, 60; malic dehydrogenase, 35 units; and purified P-enolpyruvate carboxylase, 4.1, 5.9, and 6.7 units, respectively, in a final volume of 6 ml. The reaction was initiated by the addi- tion of crystalline K,CisOa (immediately after carboxylase) to the cold reaction mixture followed by immediate mixing of tube contents at 35”. Tubes were then incubated (including thermal equilibration time) for 9, 6, and 5 min in Experiments I, II, and III, respectively. In Experiment IV the reaction mixture in- cluded the following components (in micromoles except as indi- cated) in a final volume of 12.5 ml: Tris-Cl buffer, pH 8.0, 1599; P-enolpyruvate, 80; K&i*0~ (72 atom $Y$ excess), 159; MgClz, 90; GSH, SO; NADH, 130; malic dehydrogenase, 118 units; and puri- fied P-enolpyruvate carboxylase, 18 units. The reaction was initiated as described above; tubes were incubated for 5 min at 30”. The final pH of all of the reaction mixtures was approxi- mately 8.3. Reactions were terminated, carrier added, reaction products isolated, and ‘80 analyses conducted as described in “Experimental Procedure.” Aliquots were taken at the be- ginning and end of the reaction for Pi and NADH determinations; these analyses indicated that in all of the cases the reaction had proceeded to within at least 95% of completion.

‘so content of substrate bicarbonate

Experiment I 59

Experiment II 59

Experiment III

59 (4 59 (b) 59 (4 59 (b)

Experiment IV 72 72 72

Reaction product

Compound

Pi

pi

pi Pi Malate Malate

pi pi pi

-

I

5 .-

I

-

&an- tity

onned

‘moles

40

40

40 40 40 40

80 80 80

-

--

-

6.9

5.8 5.7

12.5 10.7

12.8 12.7 10.7

-

2

il 4toms of bicarbonate-O ncorporate~ per molecp

of reactloon product

0.32

0.47

0.39 0.39 1.06 0.91

0.71 0.70 0.59

= Corrected for dilution by carrier Pi and malate (see “Experi- mental Procedure”). ‘80 content of Pi and malic acid isolated from negative control reaction (carboxylase omitted) contained no more than the natural abundance of ‘80, i.e. 0.21 to 0.22 atom

%. * Calculated from equation given below on the basis of no loss

of 180 from substrate bicarbonate through HCr*O*--H*r*O ex- change. The extent of this exchange is reflected in the deviation of values for “Atoms of bicarbonate-0 incorporated per molecule of reaction product” from theoretical values of 1.0 and 2.0 for Pi and malate, respectively. See text for further discussion.

excess gram atom ‘8O in reaction

Atoms of bicarbo- = product 109.

X nate-0 incorporated

moles of reaction atom y0 excess

per molecule of re- product ‘80 in sub-

action product strate bicar- bonate

e Theoretical value = 0.59.

pyruvate could result in the reaction mechanism depicted in Fig. 5. This mechanism was tested by using i*O-bicarbonate as substrate and then analysis of the reaction products, Pi and malate, for ‘80 after completion of the enzymatic carboxylation reaction. If correct, 1 atom of isO should be incorporated into orthophosphate for every 2 atoms of I*0 incorporated into oxal- acetate. An excess of purified P-enolpyruvate carboxylase was incubated with HCi*O*-, P-enolpyruvate, and the other essential components of the reaction mixture. The reaction was per- mitted to go to completion. In order to minimize loss of l*O- bicarbonate label due to nonenzymatic HC*O*--H*laO exchange, short incubation times (5 to 9 min) and a relatively alkaline reaction pH (pH 8.3) were employed. Due to the lability of oxalacetate, it was rapidly and quantitatively converted to malate by the inclusion of NADH and malate dehydrogenase in the reaction mixture. Orthophosphate and malate were then isolated from the reaction mixture and analyzed for inO as de- scribed in ‘(Experimental Procedure.” Other experimental de- tail is given in the legend for Table III. Support for the pro- posed mechanism (Fig. 5) was obtained as evidenced by the results of four experiments summarized in Table III. I*0 from HCY*O*- was incorporated into the Pi reaction product to the extent of 32 to 71 y0 of that theoretically possible; variations were due to differences in reaction conditions. Deviations from the theoretical degree of incorporation, due to nonenzymatic HCi*- 0*-H&*0 exchange, were anticipated even though conditions had been employed to minimize them. As shown in Experiment III of Table III, the ratio of ‘So incorporation into Pi relative to malate (0.37 to 0.43) is close to the theoretical value (0.50) which indicates that 2 atoms of bicarbonate oxygen are incorporated into malate per atom incorporated into Pi. The extent of loss of 180 from HC*O*- through HCY*O*--H*leO exchange varied from 28 to 68% depending upon reaction conditions. The shortest incubation time, lowest reaction temperature, and high- est carboxylase concentration resulted in the lowest percentage of 180 loss due to exchange (28% in Experiment IV). Other investigators (33) working with HC*O*- and the propionyl-CoA carboxylase-catalyzed reaction experienced similar losses due to HCY*O*--H***O exchange. 180 incorporation into orthophos- phate via H$*O could not have occurred to a significant extent, since any H*l*O generated through exchange would have under- gone tremendous dilution with H*i60. Water samples obtained at the end of the reaction contained no more than t,he natural abundance of 180 (i.e. 0.19, 0.21, and 0.21 at,om y0 isO).

DISCUSSION

A number of facts bearing on the mechanism of action of P- enolpyruvate carboxylase have already been established. The carboxylase-catalyzed reaction (Reaction 1) is essentially irrevers- ible (2, 4, 34) ; the AP calculated (34) for this reaction at 25” is -7.2 kcal per mole. Attempts in our laboratory to show P-enol- pyruvate formation by coupling to the pyruvate kinase and lactate dehydrogenase system and use of high concentrations of carboxylase (10 units), of oxalacetate (lo+ to lo+ M), and of Pi (up to 0.1 M) have been unsuccessful. Furthermore, high levels of carboxylase failed to support oxalacetate-Hi%O*- exchange in the presence or absence of Pi or PPi.

An enzymatic reaction mechanism (see &actions 4 and 5) similar to that for the acyl-CoA carboxylase-catalyzed reaction (30) in which a carboxylated enzyme intermediate is formed was ruled out earlier by Maruyama and Lane (4).

P-enolpyruvate + HCOa- + E e E-C02- + pyruvate + Pi (4)

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2412 Enzymatic Carboxylaticm of Phosphoenolpyruvate. I Vol. 241, No. 10

E-COi- + pyruvate = oxalacetate + E (5)

It was shown that i4C-pyruvate is not incorporated into oxal- acetate during the over-all forward reaction nor is oxalocetate- i4C-pyruvate exchange supported in the presence or absence of Pi. Insensitivity of P-enolpyruvate carboxylase action to avidin indicates (4) that biotin is not a prosthetic group for this enzyme.

The observation, in the present investigation (Table III), that 1 atom of 180 from substrate HCY803 is incorporated into Pi per 2 atoms of ‘60 incorporated into oxalacetate, sheds new light on the reaction mechanism. This indicates that the bicarbonate (or carbonate) anion rather than COz is the “active species” in the carboxylation reaction. Because of these results and the fact that it has not been possible to show partial reactions, a “con- certed reaction” shown in Fig. 5 is suggested. Nucleophilic attack by a bicarbonate oxygen on the enol phosphoryl phos- phorus atom is visualized as leading to the cyclic transition state (6-membered ring) shown. The simultaneous displacement at the bicarbonate carbon and phosphoryl phosphorus atoms lead to initial formation of the keto form of oxalacetate. This is con- sistent with the finding of Tchen, Loewus, and Vennesland (34) that the keto and not the enol form of oxalacetate is the primary carboxylation product of this reaction. Magnetic resonance (nuclear and electron paramagnetic) experiments4 conducted in collaboration with Drs. Mildvan and Cohn (Johnson Foundation, University of Pennsylvania) indicate that P-enolpyruvate car- boxylase (as well as P-enolpyruvate carboxykinase) form binary complexes with Mn2+ in the absence of other cofactors and sub- strates. Furthermore, P-enolpyruvate carboxylase interacts with Mn2+ causing an enhancement of its effect on the proton relaxation rate of water of the same magnitude as enolase (36,37) and P-enolpyruvate carboxykinase.4 The binding of P-enol- pyruyate (K8 = 3 x 1O-6 M) by the related enzyme, P-enolpyru- vate carboxykinase, has been found5 to be absolutely dependent upon the presence of divalent cation (Mn”+). Were P-enolpyru- vate carboxylase-bound Mn2+ a ligand for P-enolpyruvate bind- ing similar to that visualized for pyruvate kinase and enolase, enhancement of the positive character of the phosphoryl phos- phorus of P-enolpyruvate would be expected. This would im- prove its susceptibility to nucleophilic attack by HCO,- in the carboxylation reaction. Strong precedent for a mechanism of this type was provided by Kaziro et al. (33) in experiments with HC1803 in the propionyl-CoA carboxylase-catalyzed reaction. 180 from HC*803 was incorporated during the reaction into Pi, arising from the y-phosphate of ATP, and into the free carboxyl group of methyhnalonyl-CoA in a ratio of 1 to 2.

Experiments (Fig. 4) on the influence of pH on the pK, of Mg2+ and P-enolpyruvate indicate that an ionizable group (or groups) of the carboxylase with a pK (i.e. pK,) of about 7.3 is involved in the binding of both of these components. The fact that both pK, values are the same is consistent with the view that Mg2f acts as a bridge between the carboxylase binding site (pK, = 7.3) and P-enolpyruvate. The pK value of the Mgz+ binding site is tentatively assigned to an imidazole group. Imi- dazole groups have been implicated at the divalent cation (Mn2+ and Mg”+) binding sites of other proteins (32, 38).

Acknowledgments--We wish to express our appreciation to Dr. Robert C. Warner of this Department for conducting the sedi-

mentation velocity experiments on phosphoenolpyruvate car- boxylase. The skillful technical assistance of Miss Inge Grumm is gratefully acknowledged.

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Hitoshi Maruyama, Richard L. Easterday, Huei-Che Chang and M. Daniel LaneAND PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYLASEThe Enzymatic Carboxylation of Phosphoenolpyruvate: I. PURIFICATION

1966, 241:2405-2412.J. Biol. Chem. 

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