JOURNAL OF BACTERIOLOGYVol. 88, No. 6, p. 1678-1687 December, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

OXALOACETATE DECARBOXYLATION AND OXALOACETATE-CARBON DIOXIDE EXCHANGE IN ACETOBACTER XYLINUM

MOSHE BENZIMAN AND N. HELLER

Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel

Received for publication 12 August 1964

ABSTRACT

BENZIMAN, MOSHE (The Hebrew University ofJerusalem, Jerusalem, Israel), AND N. HELLER.Oxaloacetate decarboxylation and oxaloacetate-carbon dioxide exchange in Acetobacter xylinum.J. Bacteriol. 88:1678-1687. 1964.-Extracts ofAcetobacter xylinum, prepared by sonic treatment,were shown to catalyze the decarboxylation ofoxaloacetate (OAA) to pyruvate and C02, andthe exchange of C14-carbon dioxide into the p3-car-boxyl of OAA. Fractionation of the extracts withammonium sulfate resulted in a 10-fold increaseof the specific activity of the enzyme system cata-lyzing the C02 exchange and OAA decarboxylationreactions. The purified preparation catalyzed theexchange of pyruvate-3-C14 into OAA. SimilarpH curves with a pH optimum of 5.6 were ob-tained for the C02 exchange and OAA decarboxyl-ation reactions. Both reactions require the pres-ence of Mn2+ or Mg2+ ions. OAA decarboxylationwas more strongly inhibited than the exchange ofC02 by dialysis or metal-chelating agents. Avidindid not inhibit either reaction. Adenosine triphos-phate (ATP), adenosine diphosphate (ADP),guanosine triphosphate (GTP), guanosine di-phosphate (GDP), pyrophosphate, or inorganicphosphate did not promote OAA decarboxylationand the C02-exchange reaction catalyzed by thepurified preparation. The purified preparationfailed to catalyze the carboxylation of phospho-enolpyruvate in the presence of GDP, ADP, orinorganic phosphate, and that of pyruvate in thepresence of ATP or GTP, even when supplementedwith an OAA-trapping system. A scheme for OAAdecarboxylation which could account for the ob-served exchange reactions and for the failure toobtain net fixation of C02 is proposed. The rela-tion between the exchange reaction and the syn-thesis of cellulose from pyruvate by A. xylinumis discussed.

Exogenous C02 failed to serve as a carbonsource for cellulose synthesized by succinate-grown cells of Acetobacter xylinum in the presenceof pyruvate (Benziman and Burger-Rachamimov,1962). These cells, however, catalyze a rapid

exchange of C'402 into oxaloacetate (OAA)(Benziman and Abeliovitz, 1964).The purpose of this investigation was to

study the nature of the exchange reaction andits relationship to the mechanism of pyruvateconversion to cellulose. A preliminary report ofthis work has appeared (Heller and Benziman,1964).

MATERIALS AND METHODS

Cells and extracts. Succinate-grown cells of A.xylinum were grown and harvested, and extractswere prepared as previously described (Benzimanand Abeliovitz, 1964). A fraction rich in OAAdecarboxylation and OAA-CO2 exchange ac-tivities was obtained by fractional precipita-tion of crude extracts (containing 8 to 15 mg ofprotein per ml) with crystalline ammoniumsulfate. Fraction AS 60-90, precipitating between0.6 and 0.9 saturation, showed a 10-fold increasein the specific activity for both reactions, andcontained 4% of the protein and 40% of bothactivities, relative to the crude extract. Thefraction was stored in the frozen state, and wasdialyzed before use for 3 hr against 3,000 volumesof 0.04 M phosphate buffer (pH 6.0), unlessotherwise indicated.OAA decarboxylation. A unit of OAA decar-

boxylase activity corresponds to the amount ofenzyme required to decarboxylate 1 ,umole ofOAA in 5 min under standard conditions. Specificactivity is defined as units per milligram ofprotein. The standard reaction mixture (1 ml)contained: 0.2 M acetate buffer (pH 5.6), 3 mmMnC12, 0.01 M glutathione, and enzyme (0.05 to0.5 mg of protein, AS 60-90). Reaction wasstarted by addition of OAA (final concentration0.05 M), and the mixture was incubated at 30 Cfor 5 to 10 min as indicated. Enzyme activity wasmeasured by determining the amount of OAAconsumed. With more purified preparations, thereaction was carried out in Warburg double side-arm flasks, and the reaction was terminated by

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tipping in 0.2 ml of 5 N H2SO4 from the side arm.Activity was determined from the amount of CO2evolved. The results reported were corrected forspontaneous decarboxylation of OAA which didnot exceed 5% of total readings.OAA-C'402 exchange. Exchange activity was

measured by determining the radioactivity ofOAA after incubation of enzyme with OAA andNaHC'403. Reactions were carried out at 30 Cin stoppered side-arm tubes (10 X 150 mm).The standard reaction mixture (1 ml) contained:0.2 M acetate buffer (pH 5.6), 3 mm MnCl2,0.01 M glutathione, enzyme (0.05 to 0.2 mg ofAS 60-90 protein), and 0.05 M OAA. The sidearm contained 0.05 M NaHC'403 (10 to 20 pc).Reaction was terminated with 0.2 ml of 5 NHCl, and the mixture was centrifuged. Theradioactivity of the OAA was determined afterits conversion to the 2,4-dinitrophenylhydrazonederivative according to Maruyama and Lane(1962), modified as follows. To the protein-freereaction mixture, 4 ml of 2,4-dinitrophenylhy-drazine saturated in 2 N HCI were added, andthe mixture was allowed to stand at 30 C for 30min. Ethyl acetate (4 ml) was added, and themixture was shaken vigorously in a Vortextest-tube mixer; 0.5-ml samples of the ethylacetate phase were plated on aluminum planchetsand then counted in a gas-flow counter. Controlexperiments in the absence of enzyme gavereadings that did not exceed that of the back-ground. The enzyme concentration and theincubation periods were chosen so that the dilu-tion of radioactive CO2 by the CO2 arising fromOAA decarboxylation was lower than 10%.

Oxidation experiments were carried out at 30C in a Warburg apparatus by use of standardmanometric technique (Umbreit, Burris, andStauffer, 1957). Endogenous oxidation ratesaccounted for less than 5% of the total readings.The values reported were corrected for endoge-nous rates. Substrates used were in the form ofpotassium salts, and the solutions were freshlyprepared before each experiment.

Analytical methods. Protein was determinedaccording to Lowry et al. (1951), and inorganicphosphate according to Fiske and SubbaRow(1925). Total keto acids were determined by thedirect method of Friedman and Haugen (1943).OAA and pyruvate were determined enzymati-cally with malic dehydrogenase and lactic de-hydrogenase, respectively, as previously de-

scribed (Benziman and Abeliovitz, 1964).Acetaldehyde was determined according toBarker and Summerson (1941). Acetate was.measured as previously described (Benzimanand Abeliovitz, 1964). Pyruvate kinase activitywas assayed according to Cannata and Stoppani(1963a). All spectrophotometric determinationswere carried out in a Zeiss spectrophotometerwith cuvettes of 1-cm light path.

Radioactivity in the f3-carboxyl of OAA wasdetermined by decarboxylation with Al3+ (Krebsand Eggleston, 1945). The liberated CO2 wastrapped in KOH, precipitated as BaCO3, col-lected on Fiberglas discs, and counted.

Radioactivity of the nonvolatile acids wasdetermined by the method of Large, Peel, andQuayle (1962). Dinitrophenylhydrazone deriva-tives of OAA and pyruvate were prepared andseparated, and their radioactivity was deter-mined as described previously (Benziman andAbeliovitz, 1964).

Chemicals. Oxaloacetic acid, p-chloromercuri-benzoate, N-ethyl maleimide, o-phenanthroline,and aa' dipyridyl were purchased from SigmaChemical Co., St. Louis, Mo.; phosphoenol-pyruvic tricyclohexylammonium salt, malic de-hydrogenase (pig heart; specific activity, 36),lactic dehydrogenase (rabbit skeletal muscle;specific activity, 360), and reduced nicotinamideadenine dinucleotide phosphate (NADPH2) wereobtained from C. F. Boehringer & Soehne GmbH,Mannheim-Waldhof, Germany; avidin (2,500units per mg), a-ketoglutaric acid, adenosine tri-phosphate, and adenosine diphosphate from Nu-tritional Biochemicals Corp., Cleveland, Ohio;guanosine diphosphate and guanosine triphos-phate (sodium salts) from Pabst Laboratories,Milwaukee, Wis.; and Na2C1403 and pyruvate-3-C14 from the Radiochemical Centre, Amersham,England. The latter compound was purifiedaccording to Utter and Keech (1963) and usedimmediately. 2 ,4-Dinitrophenylhydrazine, ob-tained from the British Drug Houses, was re-crystallized twice from 2 N HCl before use.Potassium pyruvate was prepared according toRobertson (1942).

RESULTS

End products of OAA degradation. In crudeextracts OAA is broken down to pyruvate,acetaldehyde, acetate, and C02, but in thepurified fraction only a single-step decarboxyla-

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TABLE 1. Stoichiometry of OAA degradation byAcetobacter xylinum extracts

System*

Crudeex-

tractNetchange

AS-60-90Netchange

Component measuredt

OAA Pyru-Ivate C02 02

-18.11+8.91+27.01-2.6

-8.11+8.0 +7.6 0.0

X

+3.9

0.0

Ace-tate

+5.31100

0.01 97

* Reaction mixtures (1 ml) in Warburg doubleside arm flasks contained in the main chamber:0.2 M phosphate buffer (pH 6.0), 3 mm MnCl2,0.01 M glutathione, and enzyme (crude extract,10 mg of protein; AS 60-90, 0.39 mg of protein).The side arm contained 0.02 M OAA. incubatedfor 10 min at 30 C. Reaction was terminated bytipping in 0.2 ml of 5 N H2SO4 from the secondside arm.

t Components were measured as described inMaterials and Methods; amounts are expressedin micromoles.

tion to pyruvate occurs (Table 1). The changesobserved in the crude extract were consistentwith a scheme in which OAA was decarboxylatedto pyruvate and CO2, and the pyruvate formedwas partly oxidized to acetate with acetaldehydeas an intermediary step. The proposed schemewas in line with earlier observations on pyruvatemetabolism in A. xylinum extracts, namely, thedecarboxylation of pyruvate to acetaldehyde andC02, the oxidation of acetaldehyde to acetate,and the inability of the extracts to oxidizeacetate further under the experimental condi-tions employed (Neeman and Benziman, 1962).It was further proven by incubating AS 60-90with pyruvate that the purified fraction isdevoid of activity toward pyruvate. We foundthat there was no change in the level of pyruvateafter 60 min of incubation. This observation was

in line with the previous finding that the pyruvicdecarboxylase of A. xylinum extracts precipi-tates below 50% saturation with ammoniumsulfate (Neeman and Benziman, 1962).When the crude extract and AS 60-90 were

assayed for their OAA decarboxylation activity

(Materials and Methods), it was found thatAS 60-90 had a specific activity 10 times that ofthe crude extract (1.8 and 18 ,umoles of OAAdecarboxylated in 5 min per mg of protein of thecrude extract and AS 60-90, respectively).Exchange of C02 into OAA. A. xylinum cels

were previously shown to catalyze the exchangeof C'402 into OAA (Benziman and Abeliovitz,1964). Crude extracts and fraction AS 60-90 weresimilarly tested for their ability to mediate theexchange reaction (Table 2). As seen from theresults, the OAA-CO2 exchange activity wasretained in the cell-free preparations. With bothpreparations practically all the labeling observedin the intact OAA molecule was found in thef-carboxyl, whereas the moiety of OAA contain-ing the a-carboxyl was unlabeled. A similardistribution of C14 in OAA was found when theexchange reaction was catalyzed by whole cells(Benziman and Abeliovitz, 1964).The dilution of radioactive CO2 by unlabeled

CO2 arising from the decarboxylation reactionswas negligible under the experimental conditionsused (less than 10%); therefore, the exchange ofCO2 could be calculated in absolute amountsfrom the specific radioactivity of the NaHC1403added to the reaction mixture. The values ob-tained by such calculations for the crude extractand AS 60-90 were, respectively, 0.018 and 0.18,ttmole of CO2 exchanged per mg of protein per 5min under standard conditions.The ratio between the CO2 exchange and OAA

decarboxylation activities observed in the crudeextract was 1:100. The same ratio was retainedin AS 60-90.

TABLE 2. Isotope distribution in OAA afterexchange of OAA and C1402 by

Acetobacter xylinumextracts

ActivityTotal Activity in in-CH2-

Extract* activity S-carboxyl -Co-in OAAt of OAAt -COOH of

OAAt

Crude extract 6,500 6,500 <10AS 60-90 6,700 6,750 <10

* Standard exchange reaction mixture as de-scribed in Materials and Methods. Crude extract,3.0 mg of protein. AS 60-90, 0.3 mg of protein.NaHC1403: 6 X 106 count/min. Reaction timewas 5 min.

t Expressed as counts per minute.

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VOL. 88, 1964 OXALOACETATE DECARBOXYLATION AND CO2 EXCHANGE

Influence of incubation time on CO2 exchangeand OAA decarboxylation activities. OAA de-carboxylation was linear with time, under thestandard conditions described for reaction periodsas long as 30 min (Fig. 1). The exchange activity,as measured by the rate of C0402 incorporationinto a pool of OAA, decreased with time. Thiscan be explained by the progressive dilution ofC402 with the nonisotopic carbon dioxide formedby decarboxylation of OAA. On this basis, thelabeling of OAA was taken as representative ofthe exchange activity only in the first minutesof incubation.

Relationship between protein concentration andenzyme activity. The relationship between proteinconcentration and enzyme activity was linearunder the standard conditions described up to0.25 and 0.5 mg of protein (AS 60-90) for theCO2 exchange and OAA decarboxylation activi-ties, respectively (Fig. 2). The deviation fromlinearity observed in the exchange experimentwas explained as being due to the dilution ofradioactive CO2 by the CO2 arising from OAAdecarboxylation.

Effect of substrate concentration on enzymeactivity. The rate of OAA decarboxylation in-creased with increasing concentrations of OAA

14

12

10

2

o OAA DECARBOXYLATION

a OAA-CO2 EXCHANGE

{ Al

I I~~~~~~~~~~~~~~~~~

14

12

10 o0

,

xB >zA

C-,r4

6-0

4

2

5 10 15 20 25 30

MINUTES

FIG. 1. OAA-CO2 exchange and OAA decarboxyl-ation as a function of time. Standard reaction mix-tures for OAA decarboxylation and CO2 exchangeas described in Materials and Methods. Enzyme:0.13 mg of protein, AS 60-90. NaHC'403: 3 X 106count/min.

o OAA DECARBOXYLATION

A OAA-CO2 EXCHANGE

0.2 0.3mg PROTEIN

FIG. 2. OAA-CO2 exchange and OAA decarboxyl-ation as a function of protein concentration. Stand-ard reaction mixtures for OAA decarboxylation andC02 exchange as described in Materials and Meth-ods. NaHC'403: $ X 106 count/min. Protein (AS60-90) as indicated. Reaction time, 6 min.

(Fig. 3). The apparent Michaelis-Menten con-stant (Kin) of OAA (Fig. 3, insert), calculatedfrom a Lineweaver and Burk plot, was 2.8 X10-2 M. The OAA decarboxylases from Micro-coccus lysodeikticus (Herbert, 1951) and rat livermitochondria (Corwin, 1959) were reported tohave much lower Km values (2 X 10-s and 1.1 X10-4 M, respectively).The data summarized in Fig. 4 and 5 show the

dependence of the rate of OAA-CO2 exchange onthe concentrations of OAA and bicarbonate,respectively. When NaHCO3 was held constantat 50 mm, the enzyme system was saturated withOAA at about 2.5 X 10-2 M. Conversely, whenOAA was held constant at 50 mm the maximalrate of exchange was given by about 4.5 X 10-2M NaHCO3.

Optimal pH. The dependence of decarboxyl-ation and exchange activities on the pH is shownin Fig. 6. The CO2 exchange and OAA decarboxyl-ation activities had similar pH curves with maxi-mal activity given at pH 5.6. The pH optimumobtained was similar to that reported by Herbert(1955) for OAA decarboxylase of M. lysodeikticus.The OAA decarboxylase of rat liver mitochondriahas a much higher pH optimum of 7.5 (Corwin,

08

az0

06

041

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BENZIMAN AND HELLER

1959), whereas the optimal pH for the enzyme ofAzotobacter vinelandii ranged from 6.5 to 8.0(Plaut and Lardy, 1949).Requirement of divalent cations. CO2 exchange

C 40._

E

Il)

CnJJ 320

7-E

c 24z4x

wrv 160-

20 30 L0OAA(MxIO' )

FIG. 3. Effect of substrate concentration onoxaloacetate decarboxylation. Standard assay sys-tem for OAA decarboxylation, as described in Ma-terial and Methods. Enzyme: 0.7 mg of protein,AS 60-90. OAA concentration as indicated.

3.5r

FIG. 5. Effect of NaHCO3 concentration on rateof exchange. Standard assay system for OAA-CO2exchange as described in Materials and Methods.Enzyme: 0.15 mg of protein AS 60-90. NaHCO3added in concentrations as indicated. Reaction time,5 min. The values in the ordinate were calculatedfrom the average specific activity of NaHC'403 duringthe reaction period (initial specific activity, cor-rected for isotope dilution by OAA decarboxylation).

o

- 7

-I

10 20

OAA (

FIG. 4. Effect of OAA cexchange. Standard assay

exchange as described in AEnzyme: 0.15 mg of protein6 X 106 count/min. OAA co:Reaction time, 5 min.

and OAA decarboxylating activities in the crudeextract were increased 40% by addition of Mn2+(Table 3). Mg2+ caused only a slight increase inthe activities. Dialysis reduced both activities,though to a different extent. WVhereas the de-carboxylating activity was 90% reduced, theexchange activity was reduced by only 35%.In both cases activity was restored by additionof Mn2+ or Mg2+, but not by Fe2+, Cd2+, Co2+, orCu2+. The optimal concentration for Mn2+ andMg2+ was approximately 3 X 10- M. These ob-servations differ from the cation requirementsreported for the OAA decarboxylation and OAA-CO2 exchange system of M. lysodeikticus. Withextracts of this organism, decarboxylation ofOAA was much more rapid with Mn2+ than withMg2+, though the OAA-C02 exchange was more

30 40 50 60 rapid in the presence of MgW+ than of Mn2+(Mx 10-3) (Utter, 1961). In addition, the OAA decarboxyl-

ase of M. lysodeikticus was activated by Co2+system for OAAtCeo and Cd2+ to the same extent as by Mn2+ (Her-

Vtaterials and Methods. bert, 1951). The exchange of C1402 with OAA, AS 60-90. NaHC'403: was much less affected by ethylenediaminetetra-ncentration as indicated. acetic acid (EDTA) than was OAA decarboxyla-

tion (Table 3). At 10-3 M, this metal-chelating

0o---o0 0

20 40 60 80NaHCO3 (MxJ103)

100

3.0~

c2.5

U')i6r-0x 2.0

a.2wCD 1.5zx

w 1.0

0.5

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VOL. 88, 1964 OXALOACETATE DECARBOXYLATION AND CO2 EXCHANGE

reagent completely inhibited OAA decarboxyla-tion without affecting the exchange activity atall. Addition of Mn2+ partially restored thedecarboxylation activity. Of other metal-chelat-ing agents tested, 8-hydroxyquinoline (10-3 M)inhibited the decarboxylation and exchangeactivities 100 and 60%, respectively. o-Phenan-throline, potassium fluoride, and aa' dipyridylhad no effect on either activity even at 10 mMconcentrations.

Inhibition of the reactions. Ammonium ions at10 mm concentration inhibited the OAA de-carboxylating activity of crude extracts approxi-mately 50% without affecting their exchangeactivity. Preincubation of crude extracts with10-3 M p-chloromercuribenzoate or N-ethyl-maleimide inhibited both the exchange anddecarboxylation reactions approximately 35%.Avidin (25 units), preincubated with the crudeextract or with AS 60-90, did not inhibit CO2exchange activity or OAA decarboxylation. Theexchange system of M. lysodeikticus was alsofound to be unaffected by avidin (Utter, 1961).In contrast to the OAA decarboxylase of A.vinelandii (Plaut and Lardy, 1949), malate didnot inhibit OAA decarboxylation by A. xylinumextracts.

Substrate specificity. Fraction AS 60-90 didnot decarboxylate pyruvate, a-ketoglutarate,

TABLE 3. Effect of divalent cations on CO2 exchangeand OAA decarboxylationa

Per cent ofcontrol

Enzyme treatment Cations addedb.E-Decar-

change Dboxyl-ce ation

None None 100 100Mn2+ 140 140Mg2+ 110 110

Dialysis c None 65 10Mn2+ 140 140Mg2+ 110 100Fe2 , Cd2, 65 10Co2+ orCu2+

EDTAd (10-3 M) None 100 0EDTA (10-3 M) Mn2+ (10-2 M)- 60EDTA (7 X 10-2 M) None 70

a Assayed as described in Materials and Meth-ods. Enzyme: AS 60-90 (0.4 and 0.15 mg of proteinfor decarboxylation and exchange experiments,respectively). Reaction time was 5 min.

bEnzyme was preincubated with cations givenas the chlorides at 10-3 M, for 10 min.

c Enzyme was dialyzed in the cold for 16 hragainst 3,000 volumes of 0.04 M phosphate buffer(pH 6.0).

d Enzyme was preincubated with ethylenedi-aminetetraacetic acid (EDTA) for 10 min.

9 5

v,

0x

3!

t,i

pH

IN

pH

FIG. 6. Effect of pH on C02 exchange and OAAdecarboxylation. Standard assay systems for OAAdecarboxylation and C02 exchange as described inMaterials and Methods, with 0.2 m phosphate or

acetate buffer at pH values as indicated; pH valuesin exchange experiments are after addition of thebicarbonate. Enzyme: 0.2 and 0.4 mg of protein,AS 60-90, for exchange and decarboxylation ex-

periments, respectively. NaHC1403 : 7.6 X 106counts per min. Reaction time, min.

acetoacetate, and dihydroxymaleate, nor did itexchange CO2 with these acids.

Effect of nucleotides, pyrophosphate, and phos-phate on the decarboxylation and exchange reac-tions. To establish whether reactants other thanC02, pyruvate, and OAA participate in the ex-change and decarboxylation reactions, the effectsof various compounds known to participate inOAA decarboxylation systems on the activity ofa dialyzed AS 60-90 preparation were examined(Table 4). Long period dialysis did not diminishthe activity of the enzyme preparation for bothreactions when assayed in the presence of Mn +.The addition of adenosine triphosphate (ATP),adenosine diphosphate (ADP), guanosine tri-phosphate (GTP), inosine triphosphate (ITP),pyrophosphate (PP), or inorganic phosphate(Pi) did not stimulate OAA decarboxylation orthe exchange reaction. It was still possible thatATP did participate in these reactions, with

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phosphoenol pyruvate (PEP) as one of the prod-ucts, and that the observed lack of ATP stimula-tion was due to the activity of pyruvic kinase,generating ATP, which was present in smallamounts in the preparation. This possibilitywas examined by testing for pyruvic kinaseactivity in the dialyzed preparation, but no suchactivity was detected.

Noncarboxylation of pyruvate or PEP to OAA.Attempts to show carboxylation of pyruvate or

PEP to OAA in our system were carried out inseveral ways. In one set of experiments, AS 60-90was incubated with pyruvate or PEP, and highspecific activity C'402, in the presence of variousfactors known to participate in enzymaticsystems catalyzing the carboxylation of pyruvateor PEP to OAA (Wood and Stjernholm, 1962).At the end of the incubation period, carrier OAAwas added, the mixture was treated with 2,4-dinitrophenylhydrazine, and the radioactivity inthe hydrazone fraction was examined. In allsystems tested, practically no radioactivity couldbe detected in the hydrazone fraction (Table 5).On the other hand, under similar conditions, theexchange into OAA of 20 m,umoles of CO2 couldbe demonstrated. Under the experimental con-

ditions used, the presence of at least 0.25 m,u-moles of OAA arising from carboxylation couldhave been detected.

TABLE 4. Effect of various factors on C02 exchangeand OAA decarboxylation

OAAEnzyme prepn* Addition boxyl- exchange

ated

Jmoles ,umoles count/min

Undialyzed None 3.5 2,200

Dialyzed None 3.4 2,2501 ATP 3.1 2,3003ATP 3.1 2,2003 ADP + 3 Pi 3.0 2,2003 GTP 3.2 2,2003 ITP 3.5 2,3503 PP 3.2 2,2003 Pi 3.4 2,200

* Standard assay systems for OAA decarboxyla-tion and CO2 exchange, as described in Materialsand Methods. Enzyme: AS 60-90, 0.2 mg of pro-

tein. AS 60-90 was dialyzed in the cold against3,000 volumes of 0.04 M acetate buffer (pH 5.6).NaHC1403 : 3 X 106 counts per min. Reactiontime was 5 min.

TABLE 5. Carboxylation of pyruvate or PEP

Radioactivityof 2,4-dinitro-

System* phenylhy-drazonefraction

count/min

Pyruvate + C1402 .............. <50Pyruvate + ATP + C'402 ...... <50Pyruvate + GTP + C1402 ...... <50Pyruvate + ITP + C1402....... <50

PEP+GDP+ C1402 .......... <50PEP+ADP+ C1402 .......... <50PEP + IDP + C1402........... <50PEP+Pi+ C'402 ............. <50

OAA + C1402................... 40,000

*Reaction mixtures (1 ml) contained: 0.2 Macetate buffer (pH 5.6), 3 mm MnCl2, 10 mMglutathione, 0.01 M NaHC1403 (1.5 X 107 count/min), and 1 mg of AS 60-90 protein. Other addi-tions included: 3 mM pyruvate; PEP and OAA; 5mM ATP, GTP, ITP, GDP, ADP, IDP, and Pi.Incubation time was 5 min. Reaction was ter-minated with 0.2. ml of 5 N HCl, followed immedi-ately by addition of 25 ,umoles of OAA. Radio-activity was determined as in the standardexchange assay method (Materials and Methods).

Fraction AS 60-90 used for the carboxylationexperiments has a strong OAA decarboxylatingactivity; therefore, it was possible that OAAformed by carboxylation could not be detected,because it was rapidly decarboxylated. Thus, inother experiments the carboxylation systemswere coupled to an OAA-trapping reaction,constituted by an excess of exogenous malicdehydrogenase and reduced nicotinamide adeninedinucleotide (NADH2). The carboxylation reac-tion mixtures (Table 5) were supplenmented with7.2 units of malic dehydrogenase and 5,umoles ofNADH2, and, at the end of the incubationperiod, the radioactivity of the nonvolatile-acidsfraction was determined (Materials andMethods). No labeling was detected in the non-volatile-acids fraction obtained from all reactionsystems. In still other experiments, the carboxyla-tion systems supplemented by male debydro-genase and NADH2 were incubated in spectro-photometric cuvettes, and changes in absorbancyat 340 m,u were determined spectrophotometri-cally. The results obtained did not show anymeasurable formation of nicotinamide adenine

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V"OL. 88, 1964 OXALOACETATE DECARBOXYLATION AND C02 EXCHANGE

dinucleotide (NAD), which would have indicatedsynthesis of OAA.

Exchange of pyruvate into OAA. Table 6 com-pares the exchange of pyruvate-3-CI4 with OAA,and the exchange of C'402 with OAA. The resultsdemonstrated that pyruvate exchange wascatalyzed at a rate which was of the same orderof magnitude as the rate of the C02 exchange.

DISCUSSION

Though OAA decarboxylation to pyruvate andCO2 has been observed with enzyme preparationsfrom various sources (Utter, 1961), it is only theMl. lysodeikticus system that also catalyzes,similar to our system, the exchange of C02and OAA. By comparing the rate of CO2 exchangewith that of OAA decarboxylation observed inour system, a ratio value of 0.01 was obtained.This value is approximately five times higherthan the respective value in the M. lysodeikticussystem as calculated from the data of McManus(1951).OAA decarboxylation and C02 exchange

activities were found to differ in their responseto dialysis and to the action of metal-chelatingagents (Table 3). Whereas dialysis of the en-zyme preparation resulted in almost completeloss of its decarboxylating activity, the loss ofexchange activity amounted only to 35%. Lowconcentration of EDTA completely inhibitedOAA decarboxylation, but had no effect on C02exchange. Addition of MAn2+ relieved the effect ofboth dialysis and EDTA. From these observa-tions, it can be concluded that the two activitiesdiffer in their requirements for this ion. A differ-ence in metal ion requirements for OAA de-carboxylation and for C02 exchange was alsonoted by Utter (1961) in the purified OAA de-carboxylase system of M. lysodeikticus. In thissystem, Mn2+ was more effective than Mg2+ inthe decarboxylation reaction; in the exchangereaction the reverse was true. The role of metalions in the decarboxylation and exchange reac-tions has been discussed by Utter (1961) in thelight of the model system provided by the chemi-cal studies of Steinberger and Westheimer (1951)on the metal ion-catalyzed decarboxylation ofOAA.

In the exchange reaction in A. xylinum, severalpathways are known which may lead to anexchange of C02 and OAA. Though both mam-malian and bacterial malic enzyme do not pro-

TABLE 6. Exchange of pyruvate into OAA

System* Activity of OAA

count/minOAA + pyruvate-S_C14............. 2,300OAA + C402 ..................... 3,800

* Reaction mixtures (1 ml) contained: 0.2 Macetate buffer (pH 5.6), 3 mm MnCl2, 10 mMglutathione, 0.05 M OAA, and 0.35 mg of AS 60-90protein. Other additions included: 0.05 M pyru-vate-S-C'4 (1.5 X 106 counts per min), or 0.05 MNaHC'403 (1.5 X 106 counts per min). Controlswithout OAA and without enzyme were includedfor each experiment. Incubation time was 5 min.For both exchange reactions, OAA was separatedchromatographically and its radioactivity wasdetermined (Materials and Methods).

mote an exchange of OAA and C02 (Salles et al.,1950; Korkes, del Campillo, and Ochoa, 1950),it was possible that the labeling in OAA origi-nated in malate which was subsequently oxidizedto OAA. This possibility seems to be excluded,because AS 60-90 has no malate-oxidizing activity(unpublished data). Even for the crude extractwhich oxidizes malate, the isotope distributiondata (Table 3) are not compatible with malate asthe labeled precursor of OAA, considering thehigh fumarase activity present in the extract.The other pathways to be considered include thecarboxylation of pyruvate to OAA by pyruvatecarboxylase (Utter and Keech, 1963; Seubertand Remberger, 1961), and carboxylation of PEPto OAA by pyruvate carboxykinase (Utter andKurahashi, 1954, Cannata and Stoppani, 1963b)or by PEP carboxytransphosphorylase (Siu andWood, 1962). These reactions are reversible andinclude as reactants compounds like ATP, ADP,GDP, ITP, pyrophosphate, and Pi. It waspossible that the exchange reaction in A. xylinumwas accomplished by one of these pathways andwas independent of the decarboxylation reaction.In this case, the presence of only catalyticamounts of some of the above-mentioned reac-tants, in addition to OAA and C02, would havebeen required for the exchange reaction to occur.Moreover, it could be visualized that the de-carboxylation reaction results from the couplingof pyruvate carboxylase with an adenosinetriphosphatase regenerating ADP and Pi, orfrom PEP carboxykinase coupled with pyruvickinase regenerating ATP, assuming the presenceof these cofactors in the enzyme preparation in

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BENZIMAN AND HELLER

catalytic amounts. However, the failure of nucleo-tides, inorganic phosphate, or pyrophosphate tostimulate the decarboxylation and exchangeactivities of dialyzed preparations (Table 4),in addition to the observed absence of adenosinetriphosphatase and pyruvic kinase activities insuch preparations, make it very unlikely thateither of these pathways plays a role in the de-carboxylation and exchange activities of oursystem. This conclusion is further strengthenedby the finding that enzyme preparations activein the exchange reaction fail to catalyze anycarboxylation of pyruvate or PEP to OAA, evenwhen supplemented by an OAA-trapping system(Table 5).

Failure to promote net synthesis of OAA wasalso noted with the OAA-CO2 exchange systemof M. lysodeikticus. Herbert (1951), Mehler et al.(1948), and Ochoa, Mehler, and Kornberg (1948)have tried to couple that system with malicdehydrogenase to obtain net fixation of CO2,but the results were negative. The thermody-namic considerations underlying these experi-ments were extensively discussed by Wood andStjernholm (1962), who accordingly proposed ascheme for the exchange of CO2 catalyzed byOAA decarboxylase. Inasmuch as the exchange ofCO2 and pyruvate into OAA in A. xylinum areparts of the decarboxylation reaction, our findingscould be explained by a mechanism of OAA de-carboxylation as outlined in Fig. 7. In thisscheme, the complex of the enzyme with both thepyruvate and ,B-carboxyl moieties of OAA maybe reversibly dissociated either to free pyruvateand enzyme-bound CO2, or to free CO2 andenzyme-bound pyruvate. According to the pro-posed reaction mechanism, formation of enzyme-bound pyruvate or enzyme-bound CO2 occursmuch faster from OAA than from free pyruvateor CO2, respectively. Thus, an exchange of CO2or pyruvate with OAA would be expected, butthe synthesis of OAA from these compounds

co,)- Enz-Pyruvate--_ Enz.Pyruvat.

Enz.OAA =Enz-OAA= -PyruvateEn EA-nz-OAA± Enz ,

Enz- CO, ---_ Enz. CO,Pyruvate

FIG. 7. Scheme for OAA decarboxylation andexchange of pyruvate and C02 into OAA. Dottedlines indicate very slow reactions.

would be very much slower. The difference be-tween the exchange and decarboxylation intheir response to EDTA (Table 3) could beexplained according to this scheme if the laststeps of the decarboxylation, in which the freeenzyme is released, are more sensitive thanprevious steps to the action of EDTA. Similarly,these steps may be those inhibited by ammoniumions, explaining the observation that these ionsinhibited OAA decarboxylation, but not CO2exchange.

Incorporation of CO2 carbon into carbohy-drates, synthesized from substrates at the levelof pyruvate or lactate, is generally believed toarise from the carboxylation of pyruvate to OAA,which is equilibrated with a symmetrical 4-car-bon compound, like fumarate, prior to its con-version to PEP (Shargo et al., 1963). In earlierwork (Benziman and Burger-Rachamimov,1962), however, it was observed that exogenousCO2 fails to serve as a carbon source for cellulose,synthesized by A. xylinum cells in the presenceof pyruvate. This observation may be explainedby the findings reported here on the inability ofA. xylinum extracts to carboxylate pyruvate orPEP (Bloom and Foster, 1962). However, it canstill be argued that pyruvate carboxylationactivity of intact cells was destroyed during theextraction procedures, and synthesis of cellulosefrom pyruvate, demonstrable only with wholecells, may still occur via a carboxylation mecha-nism. In this case, the C02-cellulose data couldbe explained, as suggested earlier (Benziman andAbeliovitz, 1964), as due to the inability of thecells to equilibrate OAA with fumarate as aresult of the irreversibility of the malate oxida-tion system present in A. xylinum.

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