Enzymatic Synthesis of Citric Acid

ENZYMATIC SYNTHESIS OF CITRIC ACID

IV. PYRUVATE AS ACETYL DONOR*

BY SEYMOUR KORKES, ALICE DEL CAMPILLO, I. C. GUNSALUS,t AND SEVER0 OCHOA

(From the Department of Pharmacology, New York University College of Medicine, New York, New York)

(Received for publication, July 30, 1951)

Evidence has been presented in Papers II and III (1, 2) that the synthesis of citric acid, catalyzed by the condensing enzyme, occurs through a reaction between acetyl CoA and oxalacetate to yield citrate and C0A.l In the oxidation of foodstuffs through the Krebs tricarboxylic acid cycle, acetyl groups for citrate synthesis are generated by oxidation of pyruvate and of fatty acids (3), and it is now apparent that such oxidation must result in the formation of acetyl CoA. Acetyl CoA must also be formed by breakdown of /3-keto fatty acids, which are known to yield acetyl groups for citrate synthesis (3, 4). The manner in which pyruvate par- ticipates in the synthesis of citric acid has now been elucidated.

As reported in a preliminary note (5), soluble enzyme preparations from Escherichia coli and Streptococcus jaecalis catalyze the dismutation of 2 molecules of pyruvate to acetyl phosphate, carbon dioxide, and lactate when orthophosphate is present. The system is inactive in the absence of added DPN. The dismutation can be formulated as follows:

(I) Pyruvate + phosphate + DPN + acetyl phosphate + COs + DPNH, (2) Pyruvate + DPNH:! + lactate + DPN (lactic dehydrogenase)

(3) Sum, 2 pyruvate + phosphate -+ acetyl phosphate + CO2 + lactate

Both the E. wli and S. jam&s extracts contain lactic dehydrogenase. In the absence of phosphate, the reaction rate is sharply reduced and no acetyl phosphate is formed. However, if condensing enzyme and oxalacetate are added, the dismutation proceeds at the same or higher rate as in the presence of phosphate and citrate is formed instead of acetyl phosphate (5). The reaction taking place under these conditions is Reaction

* Aided by grants from the United States Public Health Service, the American Cancer Society (recommended by the Committee on Growth of the National Re- search Council), the Office of Naval Research, the Rockefeller Foundation, and the Lederle Laboratories Division, American Cyanamid Company.

t Fellow of the John Simon Guggenheim Memorial Foundation. Permanent address, Department of Bacteriology, University of Illinois, Urbana, Illinois.

1 The abbreviations used are the same as in Paper III (2).

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722 ENZYMATIC SYNTHESIS OF CITRIC ACID. IV

4. These results proved that oxidation of pyruvate can yield directly

(4) 2 pyruvate + oxalacetate -+ citrate + CO2 + lactate

acetyl groups for citrate synthesis without the intermediary formation of acetyl phosphate and that orthophosphate is not an obligatory reactant in the oxidation of pyruvate.

The pyruvate oxidation system of E. COG has now been partially purified. Two enzyme fractions, referred to as Fractions A and B, have been isolated which, in the presence of DPN and CoA, catalyze the conversion of pyruvate either to acetyl phosphate, provided transacetylase and orthophosphate are added, or to citrate, provided condensing enzyme and oxalacetate are present (3, 6). There is no reaction in the absence of either acetyl acceptor system (i.e., transacetylase-orthophosphate or condensing enzyme-oxalacetate). The system also requires diphosphothiamine. In view of our knowledge of the mechanism of the reactions catalyzed by transacetylase and by the condensing enzyme (2), it can be concluded that the enzyme Fractions A and B from E. coli catalyze the over-all Reaction 5.

(5) Pyruvate + DPN + CoA + acetyl CoA + CO2 + DPNHz

Soluble enzyme preparations have recently been obtained from pig heart with which the results obtained with the E. coli preparations have been duplicated.2 Since animal tissues contain no transacetylase and acetyl phosphate does not occur in them as a free metabolic intermediate, it is of some interest that the pig heart system can convert pyruvate + phosphate to acetyl phosphate on addition of bacterial transacetylase. These results prove the general validity of the results obtained with the E. coli enzymes.

Pyruvate Dismutation in Bacterial Extracts-Table I illustrates typical results obtained with extracts of E. coli and S. faecalis prepared and dialyzed as previously described (5). Balance experiments showed that in the case of System 1 approximately 1 mole each of acetyl phosphate, carbon dioxide, and lactate was formed for 2 moles of pyruvate disappear- ing, thus fulfilling Reaction 3. The reaction shows a strict dependence on the presence of orthophosphate and DPN. The S. faecalis extracts also contain a very active enzyme system catalyzing the conversion of 2 molecules of pyruvate to 1 of acetylmethylcarbinol and 2 of carbon dioxide.3 For this reason the production of carbon dioxide is not a measure of the dismutation alone in this case and has been omitted from Table I. It is evident that the presence of orthophosphate is not necessary for citrate

2 Korkes, S., de1 Campillo, A., and Ochoa, S., unpublished experiments. BDolin, M. I., andeGunsalus, I. C., personal communication.


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KORKES, DEL CAMPILLO, GUNSALUS, AND OCHOA 723

synthesis when the system is supplemented with oxalacetate and condensing enzyme (System 2). Actually the rate of citrate synthesis was greater in the absence than in the presence of orthophosphate. Since transacetylase is present in both bacterial extracts, this result is probably due to competition between phosphate and oxalacetate for acetyl groups from pyruvate (cf. Fig. 2). System 2 also shows a strict DPN dependence. The S. faecalis extracts were completely free of condensing enzyme and no

TABLE I Pyruvate As Acetyl Donor for Acetyl Phosphate and Citrate Synthesis in Bacterial

Extracts

The complete System 1 contained per cc. either 50 PM of potassium phosphate buffer, pH 7.4, or 20 PM of tris(hydroxymethyl)aminomethane buffer of the same pH, 2.4 PM of MgC12, 1.6 FM of MnCL, 20 PM of L-cysteine, 0.15 PM of DPN, 0.1 PM of diphosphothiamine, 5 units of CoA, 50 HM of pyruvate, and either dialyzed E. coli (strain 4157) extract with 6 mg. of protein or dialyzed S. faecalis extract with 7 mg. of protein. The complete System 2 contained, in addition, 20 /.&M of oxalacetate and 50 y of crystalline condensing enzyme. Gas, nitrogen; temperature, 25. Incubation time, System 1,40 minutes; System 2, as indicated. Values given in micromoles per cc. of reaction mixture.

Extract components

system 1 system 2

co2 A$;? Citrate synthesis evolu-

tion phate synthesis 5 min. 10 min. 20 min. 40 min. ------

E. coli Complete 2.9 1.6 0.61 1.42 3.02 No phosphate* 0.6 0.1 0.64 1.51 3.38 DPN 0.2 0.1 phosphate, no DPN 0.09

S. faecalis Complete 3.2 0.47 0.88 1.82 3.34 No phosphate* 0.3 0.66 1.21 2.30 4.65 condensing enzyme 0

* Orthophosphate present in reaction mixture, 0.06 to 0.08 PM per cc.

citrate synthesis occurred unless this enzyme was added. Balance experiments showed that 1 mole of lactate was formed per mole of citrate synthesized, in agreement with Reaction 4. Because of the presence of oxalacetate, which is decarboxylated to pyruvate and carbon dioxide by the bacterial extracts, evolution of carbon dioxide and pyruvate disappear- ance were not determined in this case.

When E. coli extracts are dialyzed against neutral solutions, no addition of diphosphothiamine is required for optimum activity of the pyruvate dismutation system. When they are dialyzed against pyrophosphate buffer at pH 8.6 and subsequently against a neutral salt solution


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to remove the pyrophosphate, there is little or no activity unless diphosphothiamine is added along with the other components of the system. As shown in Table II, this is true of both the carbon dioxide evolution due to Reaction 3 and the citrate synthesis reaction.

Isolation of Pyruvate Oxidation Fractions from E. coli-Lyophilized cells of E. co.& (strain 4157), grown as previously described (4), were used throughout this work. The dry powder was ground in a mortar at room temperature with twice its weight of alumina4 and 15 to 16 volumes of 0.066 M potassium phosphate buffer, pH 7.0; the mixture was then centrifuged at 3-4 and 13,000 r.p.m. for 30 minutes or longer and the super-

TABLE II

Diphosphothiamine Dependence

The complete System 1 contained 100 PM of potassium phosphate buffer, PH 7.4, 2.5 PM of MnC12, 6.4 pM of L-cysteine, 0.15 PM of DPN, 0.2 PM of diphosphothiamine, 4 units of CoA, 50 PM of pyruvate, lactic dehydrogenase (1600 units), transacetylase (5 units), and 1.0 cc. of E. coli extract* with 12.5 mg. of protein. System 2 differed from System 1 in that it contained 4 PM of MgClz besides MnC12, 12.8 PM of L-cysteine, 30 PM of oxalacetate, 38 y of crystalline condensing enzyme, and no transacetylase. Final volume, 2.0 cc.; gas, nitrogen; incubation, 60 minutes at 25. Values given in micromoles.

Components System 1

CO2 evolution system 2

Citrate synthesis

Complete. . 1.35 2.12 No diphosphothiamine.. . 0.13 0

* Extract dialyzed at 3-4 for 24 hours against 0.025 M sodium pyrophosphate, pH 8.6, and then overnight against 0.9.per cent potassium chloride containing 0.005 M L-cysteine, pH 7.0.

natant extract was dialyzed at 3-4 against 0.9 per cent potassium chloride containing 0.005 M L-cysteine. As judged by the low ratio of light absorption at the wave-length 280 rnp to that at 260 rnp (7), these extracts contain large amounts of nucleic acid which have been found to interfere with the subsequent fractionation. Treatment of the extracts with 0.05 their volume of 1.0 M manganous chloride precipitates a fairly large amount of nucleic acid and makes possible the fractionation of the pyruvate oxidation enzymes in the supernatant solution. Unfortunately, this treatment results in a large loss of the pyruvate enzymes.

The assay was based on the rate of CO2 evolution from pyruvate at pH 6.0 and 25 in the dismutation system of Reaction 3. The volume of COZ produced was calculated by correcting the vessel constant for the small COZ ret,ention at pH 6.0. The reaction mixtures contained 100 PM

4 A-301, -325 mesh (Aluminum Company of America).


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of potassium phosphate buffer, pH 6.0, 2.5 PM of MnCl*, 6.4 PM of L- cysteine, 0.15 PM of DPN, 0.2 PM of diphosphothiamine, 5 units of CoA, 50 PM of pyruvate, lactic dehydrogenase (2000 units), and enzyme; final volume, 1.5 cc.; nitrogen in the gas phase. The reaction was started by tipping in the pyruvate from the side bulb of the Warburg vessels after temperature equilibration. 1 unit was taken as the amount of enzyme causing an evolution of 1 cmm. of COz per hour under the above conditions.

Separation of Two Fractions with Ammonium Sulfate-The supernatant solution from the manganous chloride precipitation was dialyzed as before, cooled to 0, and adjusted to pH 6.0 with dilute acetic acid. It was then fractionated with solid ammonium sulfate with mechanical stirring. Three

TABLE III Fractionation of E. coli Extract

40 gm. of lyophilized E. coli (strain 4157) extracted with 630 cc. of 0.066 M potassium phosphate buffer, pH 7.0, and 80 gm. of alumina.

step

Extract . . . . . . . . 485 122,000 9700 Dialyzed extract.. 512 140,000 7260 Mn++ supernatant (dialyzed). . 500 22,200 3900 (NHI)zSOl (O-O.45 saturation). 34 13,000 830

(0.6-1.0 I ). 45.5 300 386 I fractions combined 79.5 25,400 1216

-

7 Volume

CC.

-

Units

-

rotein I

m.

Light bbsorp

tion ratiot

0.53 0.55 0.71 0.78 0.70

-

-

1

Specific activity Yield

--

~~~~n pn cm1

13 100 19 116 6 18

16 11 0.8 0.25

21 21

* 1 unit = 1.0 c.mm. of CO2 per hour at pH 6.0 and 25. t e8omplQoO*r.

fractions were obtained, Fraction I between 0 and 0.45, Fraction II between 0.45 and 0.60, and Fraction III between 0.60 and 1.0 ammonium sulfate saturation. The precipitates were dissolved in 0.02 M potassium phosphate buffer, pH 7.0, and dialyzed overnight against the same buffer at 34. When assayed separately, Fraction I was found to have about half of the original activity, whereas the activity of Fraction III was very low. The activity of Fraction II was intermediate. Both Fractions I and II contained transacetylase. Fraction III was almost free from transacetylase. When Fractions I and III were combined, the activity was about twice as high as the sum of the activities of the separate fractions. Thus two enzymes of the pyruvate oxidation system were partially sepa- rated. The results of a typical fractionation are illustrated in Table III. The procedure has been repeated a number of times with fairly uniform results.


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Further Purification of Ammonium Sulfate Fractions-The activity per mg. of protein of Fraction I, when assayed in the presence of an excess of Fraction III, was about 30. The specific activity was raised to a value between 60 and 100 by heat denaturation, followed by refractionation with ammonium sulfate. Heating completely destroys the transacetylase present in the ammonium sulfate fractions.

8The following describes a typical preparation. The solution of Fraction I, containing 24.4 mg. of protein per cc., was brought to pH 6.0 with dilute acetic acid and heated for 10 minutes at 50. The copious protein precipitate was removed by centrifugation. To the supernatant solution, containing 8.3 mg. of protein per cc., solid ammonium sulfate was added to 0.45 saturation; the precipitate was dissolved in 0.02 M potassium phosphate buffer, pH 7.0, and the solution dialyzed overnight at 3-4 against the same buffer. This will be referred to as Fraction A. The solution contained 8.3 mg. of protein per cc. and the light absorption ratio (see Table III) was 1.3. It was free from transacetylase. The solution of Fraction III, containing 8.5 mg. of protein.per cc., was adjusted to pH 6.0 and 0.07 volume of a solution of 2 per cent protamine sulfate adjusted to pH 6.0 was added. The precipitate was centrifuged and to the supernatant solution was added solid ammonium sulfate to 0.7 saturation. The solution of the precipitate (containing 19 mg. of protein per cc.; light absorption ratio, 1.4) was adjusted to pH 6.0 and heated for 5 minutes at 60. The large protein precipitate was centrifuged, yielding a solution containing 10 mg. of protein per cc. with a light absorption ratio of 1.3. This fraction, which was also free of transacetylase, will be referred to as Fraction B. Fractions A and B kept their activity for at least 2 weeks when stored in the refrigerator at 3-4, and for much longer periods if stored at -18.

When assayed in the standard test system, in the presence of added transacetylase, Fraction A has some activity which is undoubtedly due to slight contamination with Fraction B. Fraction B has practically no activity by itself. Similar results are obtained in the case of citrate synthesis when oxalacetate and condensing enzyme are substituted for orthophosphate and transacetylase. Both Fractions A and B must be present together for activity. When one of the fractions is in excess, the rate of reaction is proportional to the amount of the other fraction present. This is illustrated in Fig. 1. Curve 1 gives the rate of CO2 evolution in the standard test system (with added transacetylase) as a function of the concentration of Fraction B in the presence of an excess of Fraction A. Curve 2 gives the rate of citrate synthesis, on substitution of condensing enzyme (and oxalacetate) for transacetylase, as a function of the concentration of Fraction A in the presence of an excess of Fraction B.


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KORKES, DEL CAMPILLO, GTJNSALUS, AND OCHOA 727

Mechanism of Pyruvate Oxidation-Experiments wit. the partially purified Fractions A and B of E. coli showed that pyruvate requires CoA for

cc. FRACTION B cc. FRACTION A FIG. 1. Pyruvate dismutation as a function of the concentration of enzyme Frac-

tions A and B. The samples of Curve 1 contained 100 PM of potassium phosphate buffer, pH 6.0, 2.5 PM of MnClz, 6.4 PM of n-cysteine, 0.15 PM of DPN, 0.2 PM of diphosphothiamine, 6 units of CoA, 50 PM of pyruvate, lactic dehydrogenase (2000 units), transacetylase (7 units), Fraction A with 10 mg. of protein, and variable amounts of Fraction B containing 25.8 mg. of protein per cc. The samples of Curve 2 differed from those of Curve 1 in that they contained 30 PM of potassium phosphate buffer, pH 7.4, 4.0 PM of MgClz (besides MnClt), 30 PM of oxalacetate, 75 y of crystalline condensing enzyme, Fraction B with 4.15 mg. of protein, va.riable amounts of Fraction A, containing 8.8 mg. of protein per cc., and no transacetylase. Final volume, 1.7 cc. Gas, nitrogen; incubation, 30 minutes at 25.

g PYRUVATE + DPN + CoA+ -1 + CO, + DPNH2

f t-ok

& ACETYL-P + Co A CITRATE + CoA 2

FIG. 2. Pyruvate oxidation and its coupling with acetyl phosphate and citrate synthesis. The over-all Reaction a is catalyzed by enzyme Fractions A and B. Reaction b is catalyzed by transacetylase, Reaction c by the condensing enzyme. P = orthophosphate, OAA = oxalacetate, acetyl P = acetyl phosphate.

reaction and strongly suggested that, in the presence of CoA and DPN, pyruvate is oxidized to yield acetyl CoA, COZ, and DPNHz (Fig. 2, Re- action a). When CoA is present in catalytic amounts, as is the case in our experiments as well as in the cell, the reaction cannot proceed to any significant extent unless the acetyl group is passed on to another acceptor,


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thus releasing CoA for further reaction. This can occur in several ways. In the presence of orthophosphate and transacetylase, acetyl CoA is split by phosphate to yield acetyl phosphate and CoA (Reaction b). In the presence of oxalacetate and condensing enzyme, acetyl CoA is split by oxalacetate to yield citrate-and CoA (Reaction c). Under our conditions, the DPNHz formed through Reaction a is reoxidized by pyruvate in the presence of lactic dehydrogenase (Reaction 2) to yield DPN and lactate.

Evidence for the above mechanism is given in Tables IV and V. In the presence of transacetylase and orthophosphate (Table IV), the complete

TABLE IV Pyruvate Dismutation (Acetyl Phosphate Synthesis) with Purified Enzyme Fractions

A and B

The complete system contained 100 PM of potassium phosphate buffer, pH 7.4, 2.5 pM of MnCL, 6.4 pM of n-cysteine, 0.15 lrhr of DPN, 0.2 pM of diphosphothiamine, 5 units of CoA, 40 pM of pyruvate, lactic dehydrogenase (2000 units), transacetylase (15 units), and enzyme Fractions (Table III) A (1.6 mg. of protein) and B (1.9 mg. of protein). Final volume, 1.5 cc.; gas, nitrogen; incubation, 30 minutes at 25. Values given in micromoles.

Additions

Complete. No Fraction A.. B transacetylase DPN . .

TPN instead of DPN. No CoA. . . .

-

A

Pyruvate

-13.7 -0.2

-0.6

-1.6

Lactate

+5.7 +0.1 +1.2 +0.3

+o.s

AC&y1 phosphate

+4.9

0

+0.2 +0.2

+0.3

CO2

+6.0 0

+0.1 +0.1 +0.5 +0.5 +0.7

system catalyzed a dismutation according to Reaction 3. The presence of both Fractions A and B and, in accordance with Fig. 2, Reactions a and b, the presence of transacetylase, CoA, and DPN was essential for activity. TPN could not replace DPN. The dependence on orthophosphate and diphosphothiamine has already been mentioned: In the presence of oxalacetate and condensing enzyme (Table V), the complete system formed about equimolecular amounts of citrate and lactate, in agreement with Reaction 4. The presence of Fractions A and B and, in accordance with Fig. 2, Reactions a and c, the presence of condensing enzyme and CoA was essential for activity. The need of DPN and diphosphothiamine for this reaction has already been demonstrated in experiments with the bacterial extracts (Tables I and II). It will be seen in Table V that the synthesis of citrate was somewhat decreased on addition of transacetylase.


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Since orthophosphate was present, the decreased citrate synthesis, as already discussed, was probably due to competition between phosphate and oxalacetate for the acetyl groups from pyruvate (cf. Fig. 2).

As already mentioned, the E. coli extracts contain a DPN-specific lactic dehydrogenase. This enzyme still contaminates the partially purified Fractions A and B and hence dependence of the activity of the system. on addition of lactic dehydrogenase was only partial. This dependence was

TABLE V Pyruvate Dismutation (Citrate Synthesis) with Purified Enzyme Fractions A and B

The complete system contained 30 PM of potassium phosphate buffer, pH 7.4, 4.0 PM of MgC12, 2.5 PM of MnCl2, 6.4 PM of L-cysteine, 0.15 PM of DPN, 0.2 PM of diphosphothiamine, 5 units of CoA, 40 PM of pyruvate, 30 ELM of oxalacetate, lactic dehydrogenase (2000 units), 75 y of crystalline condensing enzyme, and enzyme fractions (in mg. of protein) as follows: Experiment 1, Fraction A, 1.6; Fraction B, 1.9; Experiment 2, Fraction A, 0.67; Fraction B, 1.23; Experiment 3, Fraction A, 0.5; Fraction B, 1.9. The fractions used in Experiment 1 were those described in Table III and the same as in Table IV. Final volume, 1.6 cc.; gas, nitrogen; incubation at 25; Experiment 1, 30 minutes; Experiments 2 and 3, 60 minutes. Values given in micromoles. The figures in parentheses (Experiment 1) are from a separate run with the same enzyme fractions.

I Experiment 1 I A citrate

Additions ( A citrate 1 A lactate 1 ,,,;i; 1 zzi

Complete*. . No Fraction A.. I B ( condensing enzyme.. Ii CoA. .

Complete + transacetylase units)........................

. . . .

;i

+4.5 (f5.3) -to.11 (+o.l) +0.72 (+0.76) +0.50 +0.40

+4.0

+4.4 +0.14 $0.84

+0.37

+6.3 +0.15 +0.75

+7.2 +0.11

+4.2

* The complete system contained no transacetylase.

more marked at pH 6.0 than at pH 7.4 and is illustrated in Table VI by two experiments carried out at the lower pH in the presence of transacetylase. The reaction at pH 6.0 is about half as fast as at pH 7.4. These experiments demonstrate the correctness of the formulation of Re- action 3 as a DPN-linked dismutation through coupling of Reactions 1 and 2. Similarly, Reaction 4 must be a dismutation resulting from the coupling of Reaction 6 with Reaction 2. Reaction 6 (the net result of Reactions a and c, Fig. 2) is catalyzed by enzyme Fractions A and B and

0% Pyruvate + oxalacetate + DPN --+ citrate + COS + DPNHl

condensing enzyme in the presence of CoA. Direct spectrophotometric


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determination of DPN reduction through either Reaction 1 or Reaction 6 has not yet been possible; because of the contamination of Fractions A and B with lactic dehydrogenase, DPNH2 is rapidly oxidized to DPN in the presence of pyruvate. It should be mentioned that the E. coli fractions catalyze neither the decarboxylation of pyruvate nor the oxidation of acetaldehyde.

The requirement of the pyruvate oxidation system for CoA is paralleled by its requirement in the pyruvate-formate exchange reaction recently demonstrated by Chantrenne and Lipmann (8) (cf. also Strecker (9)). Whether the pyruvate oxidation factor of OKane and Gunsalus (10) is involved in our system has not yet been established. The E. coli enzyme fractions have been found to contain the factor.

We have evidence that Reaction a (Fig. 2) can also be catalyzed by

TABLE VI

Dependence of Pyruvate Dismutation on Lactic Dehydrogenase Potassium phosphate buffer, pH 6.0; otherwise as in Table IV. Experiments 1

and 2 carried out with two different preparations of Fractions A and B.

Additions

Experiment 1

AC02 A acetyl phosphate

Complete. . . +2.2 +2.1 No transacetylase. . . +0.1 0 lactic dehydrogenase. . . +0.6 +0.1

Experiment 2

ACOa A acetyl phosphate

+2.1 +2.3

+0.9 +0.6

soluble enzyme preparations recently obtained from pig heart2 Ammo- nium sulfate fractionation of the heart extracts yields a fraction precipitating between 60 and 100 per cent saturation, which appears to be identical with Fraction B from E. coli and can be coupled with the E. coli Fraction A to reconstruct the pyruvate oxidation system when supplemented either with transacetylase-orthophosphate or with condensing enzyme-oxalacetate. The heart fraction precipitating between 0 and 45 per cent ammonium sulfate saturation has not yet been resolved into two distinct components; it appears to be contaminated with sufficient enzyme Fraction B to give maximum pyruvate oxidation rates without addition of the latter. In other respects the heart system is identical with that in E. di in its requirements for DPN, CoA, and an acetyl acceptor system as shown in Fig. 2.

While some bacteria oxidize pyruvate to acetyl phosphate and COZ, owing to the presence of transacetylase, animal tissues, which lack transacetylase but contain condensing enzyme, can oxidize pyruvate only by way of citric acid in the presence of oxalacetate. This explains why pyru-


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vate does not disappear aerobically in dialyzed heart homogenates unless a dicarboxylic acid (which can be oxidized to oxalacetate) is added (11). It should also be mentioned that E. coli, which contains both transacetylase and condensing enzyme, is capable of converting pyruvate either to acetyl phosphate or to citrate; it also can convert acetyl phosphate to citrate (cf. Fig. 2).

DISCUSSION

It is of interest that the system in C. kluyveri extracts which, in the presence of orthophosphate, oxidizes acetaldehyde to acetyl phosphate (12) is the counterpart of the pyruvate oxidation system described in this paper. When C. kluyveri extracts are dialyzed free of phosphate, acetaldehyde is not oxidized except on addition of phosphate or on supplementation with oxalacetate and condensing enzyme. In the latter case, no acetyl phosphate is formed, but citrate is synthesized in large amounts.6 Oxidation of acetaldehyde in C. kluyveri extracts requires the presence of both DPN and COA.~ The same is true of an acetddehyde oxidizing system present in E. coli (strain B) which seems to be identical with the C. kluyveri system? It thus appears that acetaldehyde oxidation by these systems in- volves at least two enzymes, a DPN-specific and CoA-dependent aldehyde dehydrogenase and either transacetylase, in the presence of phosphate, or condensing enzyme, in the presence of oxalacetate.

Cavallini (13) has recently found that when reduced glutathione (GSH) is oxidized by copper ions there occurs a coupled oxidative decarboxylation of pyruvate added to the system. This finding is of considerable significance in view of Lynen and Reicherts discovery (14) that the SH group in CoA (15, 16) is the active group of the coenzyme and of the participation of CoA in the enzymatic oxidation of pyruvate and acetaldehyde and in the pyruvate-formate exchange reaction? For his chemical model, Cavallini postulates the oxidation of a reduced glutathione-pyruvate addition compound, as formulated in Reaction 7. A similar addition product of CoA and pyruvate, or of CoA and an acetaldehyde derivative formed

CHa CH,

(7) GSH + CO +GSH + 02

-+ GS-COH -

I I COOH COOH

GSSG + CHa-COOH + CO2 + H20

2 Stadtman, E. R., Stern, J. R., and Ochoa, S., unpublished experiments. 2 Stadtman, E. R., personal communication. 7 Racker, E., personal communication. * CoA has recently been found to be required for the oxidation of wketoglutarate

by a soluble enzyme system from heart muscle. This suggests oxidation to suc- cinyl CoA. Kaufman, S., and Ochoa, S., unpublished experiments.


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from pyruvate with the participation of diphosphothiamine, is also postulated by Lynen and Reichert (14) for the enzymatic oxidation of pyruvate to acetyl CoA (Reaction 8). The formation of an acetaldehyde-CoA

CHs CHa CH,

(8) H-C=0 + HS-R -+ H-C-S-R -2H

----+ C-S-R I II

OH 0

compound gains considerable support from Rackers finding (17) that one of the two enzymes of the glyoxalase system catalyzes a reaction between reduced glutathione and methylglyoxal, presumably to form a thiol-car- bony1 addition product, which undergoes further conversion to GSH and lactate through the action of the second enzyme.

Finally it may be pointed out that SH groups are probably involved in the oxidation of the carbonyl group of n-glyceraldehyde-S-phosphate by glyceraldehyde phosphate dehydrogenase in a similar manner as in pyruvate and acetaldehyde oxidation. Glyceraldehyde phosphate dehydrogenase requires SH groups for its action and, as postulated by Racker (17) the energy-rich phosphate bond of 1,3-diphosphoglyceric acid might be

0 II

formed by phosphorolysis of an R-S-C-R bond much as acetyl phosphate is generated by phosphorolysis of the corresponding bond in acetyl CoA through the action of transacetylase. The finding that crystalline glyceraldehyde phosphate dehydrogenase can slowly oxidize acetaldehyde to acetyl phosphate in the presence of phosphate (18) gives further support to the view that the various enzymes catalyzing the oxidation of carbonyl to carboxyl groups operate through basically identical mechanisms, with sulfhydryl as the catalytically active group.

Methods andPreparations

Pyruvate was determined by the method of Friedemann and Haugen (19) and occasionally spectrophotometrically with lactic dehydrogenase, as previously described (20). Lactate was determined by the method of Barker and Summerson (21). Acetyl phosphate and citrate were determined as in previous work (1).

The transacetylase used in this work was a highly purified preparation from Clostridium kluyveri kindly supplied by Dr. E. R. Stadtman.,

Lactic dehydrogenase was obtained as a crystalline fraction from rabbit muscle by the following procedure. An ammonium sulfate paste (fraction obtained between 0.52 and 0.72 saturation of aqueous rabbit muscle extract with ammoniacal ammonium sulfate, pH 8.0) was used after storage at 34 for 3 weeks or longer. The paste was dissolved in a minimum


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amount of water and made turbid at 0 by adding sufficient saturated ammoniacal ammonium sulfate. On standing at room temperature, crystals of aldolase formed and were removed by centrifugation. Repeated addition of saturated ammonium sulfate as above gave further crystalline fractions. Some of these were rich in lactic dehydrogenase. The specific activity of these preparations, assayed as previously described (22), was about 40,000. These fractions were used after dialysis at levels of 50 y per experimental vessel, corresponding to 2000 units.

Crystalline pot,assium pyruvate was prepared as follows: Pyruvic acid (Merck) from a previously unopened bottle was cooled to 0 and solid potassium carbonate was added slowly with vigorous stirring to about 90 per cent neutralization. Water was added in small amounts as needed to keep the potassium pyruvate in solution. The amount of water required did not exceed one-half of the volume of the pyruvic acid used. 4 volumes of alcohol were added to the solution at room temperature. Long, needle-shaped crystals of potassium pyruvate formed on standing at 0 for 2 to 3 hours and increased in amount overnight. The crystalline material was filtered with suction, washed with alcohol and ether, and dried in vacua over calcium chloride. Further crops of crystals were obtained by adding more alcohol to the mother liquor. The potassium pyruvate was about 97 per cent pure as assayed spectrophotometrically with lactic dehydrogenase (20). Oxalacetic acid was prepared as in previous work (4).

A crude preparation of CoA for routine assays was obtained from rabbit livers as follows: The livers were rapidly removed and chilled in ice. They were then homogenized briefly in a Waring blendor with a minimum of water, and the homogenate was added gradually with stirring to boiling water so that the temperature did not drop below 80. After 2 to 3 minutes the mixture was cooled to about 40 and pressed through three layers of cheesecloth. Saliva (about 3 cc. per 100 cc.) was added to the juice and the mixture was incubated for 3 hours at 25 to hydrolyze the glyco- gen. 12 per cent trichloroacetic acid was then added to about pH 3.0 and the mixture was centrifuged. The supernatant fluid was poured with stirring into 10 volumes of acetone at 0 and, after standing in the cold for 15 minutes, the precipitate was filtered with suction, washed with acetone and ether, and dried in vacua over calcium chloride. The material gave clear yehow solutions of about pH 6.0. The CoA potency of this preparation, as assayed by the sulfanilamide acetylation method (23), was about 1 unit per mg. For purposes other than enzyme assays, a CoA preparation containing 30 units per mg. was used. This preparation was kindly supplied by Dr. M. A. Mitz, Research Division, Armour and Company, and was free from DPN and diphosphothiamine.

Diphosphothiamine was generously supplied by Dr. R. A. Peterman,


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Merck and Company, Inc., and protamine sulfate by E. R. Squibb and Sons. DPN and n-cysteine were commercial preparations. The purity of the DPN, assayed spectrophotometrically with the alcohol dehydrogenase system, was 67 per cent.

The E. coli used in this and in previous work (1) was grown and lyophilized in the laboratories of the Medical Research Division, Sharp and Dohme, Inc., by Dr. J. R. Stern, Dr. A. K. Miller, and Dr. A. E. Wasser- man. We are greatly indebted to Dr. W. F. Verwey for the facilities placed at our disposal.

SUMMARY

The dismutation of 2 molecules of pyruvate, in the presence of orthophosphate, to acetyl phosphate, COZ, and lactate requires two enzyme fractions (A and B), transacetylase, lactic dehydrogenase, diphosphothiamine, diphosphopyridine nucleotide, and coenzyme A. Triphosphopyri- dine nucleotide is inactive. When oxalacetate and condensing enzyme are substituted for orthophosphate and transacetylase, citrate is formed instead of acetyl phosphate, but there is no reaction in the absence of an acetyl acceptor system. The enzyme Fractions A and B have been isolated from Escherichia wli. There is evidence for the occurrence of these enzymes in soluble preparations from pig heart.

From our present knowledge of the mechanism of the reactions catalyzed by transacetylase and the condensing enzyme and the results presented in this paper, it is concluded that enzyme Fractions A and B catalyze a reaction between pyruvate, coenzyme A, and diphosphopyridine nucleotide to form acetyl coenzyme A, COZ, and reduced diphosphopyridine nucleotide. The significance of these facts is discussed.

We wish to thank Dr. J. R. Stern, Mr. M. C. Schneider, and Mr. Tibor G. Farkas for help in part of this work.

BIBLIOGRAPHY

1. Ochoa, S., Stern, J. R., and Schneider, M. C., J. Biol. Chem., 193, 691 (1951). 2. Stern, J. R., Shapiro, B., Stadtman, E. R., and Ochoa, S., J. Biol. Chem., 193,

703 (1951). 3. Ochoa, S., Physiol. Rev., 31, 56 (1951). 4. Stern, J. R., and Ochoa, S., J. Biol. Chem., 191, 161 (1951). 5. Korkes, S., Stern, J. R., Gunsalus, I. C., and Ocboa, S., Nature, 166, 439 (1959). 6. Korkes, S., de1 Campillo, A., and Gunsalus, I. C., Federation hoc., 10,210 (1951). 7. Warburg, O., and Christian, W., Biochem. Z., 310, 384 (1941-42). 8. Chantrenne, H., and Lipmann, F., J. BioZ. Chem., 167,757 (1956). 9. Strecker, H. J., J. BioZ. Chem., 169,815 (1951).

10. OKane, D. J., and Gunsalus, I. C., J. Bact., 66,499 (1948). 11. Ochoa, S., J. BioZ. Chem., 166, 87 (1944).


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12. Stadtman, E. R., and Barker, H. A., J. Biol. Chem., 180, 1095 (1949). 13. Cavallini, D., Biochem. J., 49, 1 (1951). 14. Lynen, F., and Reichert, E., Angew. Chem., 63, 47 (1951). 15. DeVries, W. H., Govier, W. M., Evans, J. S., Gregory, J. D., Novelli, G. D.,

Soodak, M., and Lipmann, F., J. Am. Chem. Sot., 73, 4838 (1950). 16. Snell, E. E., Brown, G. M., Peters, V. J., Craig, J. A., Wittle, E. L., Moore, J.

A., McGlohon, V. M., and Bird, 0. D., J. Am. Chem. Sot., 72, 5349 (1950). 17. Racker, E., J. Biol. Chem., 190, 685 (1951). 18. Harting, J., Federation Proc., 10, 195 (1951). 19. Friedemann, T. E., and Haugen, G. E., J. Biol. Chem., 147, 415 (1943). 20. Ochoa, S., Mehler, A. H., and Kornberg, A., J. Biol. Chem., 174, 979 (1948). 21. Barker, S. B., and Summerson, W. H., J. BioZ. Chem., 138, 535 (1941). 22. Mehler, A. H., Kornberg, A., Grisolia, S., and Ochoa, S., J. BioZ. Chem., 174,

961 (1948). 23. Kaplan, N. O., and Lipmann, F., J. BioZ. Chem., 174, 37 (1948).


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Gunsalus and Severo OchoaSeymour Korkes, Alice del Campillo, I. C. DONORACID: IV. PYRUVATE AS ACETYL ENZYMATIC SYNTHESIS OF CITRICARTICLE:

1951, 193:721-735.J. Biol. Chem.

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Enzymatic Synthesis of Citric Acid

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