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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
syn- thesis 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 oxal- acetate are added, the dismutation
proceeds at the same or higher rate as in the presence of phosphate
and citrate is formed instead of acetyl phos- phate (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).
721
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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
puri- fied. 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 ortho- phosphate are added, or to citrate,
provided condensing enzyme and oxal- acetate 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 + phos- phate 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 dia- lyzed 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
condens- ing enzyme (System 2). Actually the rate of citrate
synthesis was greater in the absence than in the presence of
orthophosphate. Since transacetyl- ase is present in both bacterial
extracts, this result is probably due to com- petition between
phosphate and oxalacetate for acetyl groups from py- ruvate (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 oxal- acetate 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
ex- periments 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
addi- tion of diphosphothiamine is required for optimum activity of
the py- ruvate dismutation system. When they are dialyzed against
pyrophos- phate buffer at pH 8.6 and subsequently against a neutral
salt solution
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721 ENZYMATIC SYNTHESIS OF CITRIC ACID. IV
to remove the pyrophosphate, there is little or no activity
unless diphos- phothiamine 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 cen- trifuged 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-cys- teine, 30 PM of oxalacetate, 38 y
of crystalline condensing enzyme, and no trans- acetylase. 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 ab- sorption 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
oxi- dation enzymes in the supernatant solution. Unfortunately,
this treat- ment 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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KORKES, DEL CAMPILLO, GUNSALUS, AND OCHOA 725
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 potas- sium 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
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7 Volume
CC.
-
Units
-
rotein I
m.
Light bbsorp
tion ratiot
0.53 0.55 0.71 0.78 0.70
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-
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 be- tween 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 trans- acetylase. 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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726 ENZYMATIC SYNTHESIS OF CITRIC ACID. IV
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 pre- cipitate 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 phos- phate 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 super- natant 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 syn- thesis when oxalacetate and condensing
enzyme are substituted for ortho- phosphate 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 con- centration 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
puri- fied 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 di- phosphothiamine, 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 crystal-
line 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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728 ENZYMATIC SYNTHESIS OF CITRIC ACID. IV
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 oxal- acetate 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 bac- terial
extracts (Tables I and II). It will be seen in Table V that the
syn- thesis of citrate was somewhat decreased on addition of
transacetylase.
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KORKES, DEL CAMPILLO, GUNSALUS, AND OCHOA 729
Since orthophosphate was present, the decreased citrate
synthesis, as al- ready 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; incu- bation
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 trans- acetylase. 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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730 ENZYMATIC SYNTHESIS OF CITRIC ACID. IV
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 frac- tions 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 precipi- tating between 60 and 100 per cent saturation,
which appears to be iden- tical with Fraction B from E. coli and
can be coupled with the E. coli Fraction A to reconstruct the
pyruvate oxidation system when supple- mented either with
transacetylase-orthophosphate or with condensing en-
zyme-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 trans- acetylase 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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KORKES, DEL CAMPILLO, GUNSALUS, AND OCHOA 731
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 phos- phate 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
sys- tem? 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 signifi- cance 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 participa- tion 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 addi- tion 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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732 ENZYMATIC SYNTHESIS OF CITRIC ACID. IV
from pyruvate with the participation of diphosphothiamine, is
also pos- tulated by Lynen and Reichert (14) for the enzymatic
oxidation of pyru- vate 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 pyru- vate and acetaldehyde
oxidation. Glyceraldehyde phosphate dehydro- genase 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 phos-
phate 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 deter- mined 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 ex- tract 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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KORKES, DEL CAMPILLO, GUNSALUS, AND OCHOA 733
amount of water and made turbid at 0 by adding sufficient
saturated ammoniacal ammonium sulfate. On standing at room
temperature, crys- tals 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 re- quired 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 ob-
tained by adding more alcohol to the mother liquor. The potassium
py- ruvate was about 97 per cent pure as assayed
spectrophotometrically with lactic dehydrogenase (20). Oxalacetic
acid was prepared as in pre- vious 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 min- utes 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 prepara- tion, as assayed by
the sulfanilamide acetylation method (23), was about 1 unit per mg.
For purposes other than enzyme assays, a CoA prepara- tion
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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734 ENZYMATIC SYNTHESIS OF CITRIC ACID. IV
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
dehydro- genase system, was 67 per cent.
The E. coli used in this and in previous work (1) was grown and
lyo- philized 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
ortho- phosphate, to acetyl phosphate, COZ, and lactate requires
two enzyme fractions (A and B), transacetylase, lactic
dehydrogenase, diphosphothia- mine, diphosphopyridine nucleotide,
and coenzyme A. Triphosphopyri- dine nucleotide is inactive. When
oxalacetate and condensing enzyme are substituted for
orthophosphate and transacetylase, citrate is formed in- stead of
acetyl phosphate, but there is no reaction in the absence of an
acetyl acceptor system. The enzyme Fractions A and B have been iso-
lated 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 re- action between pyruvate, coenzyme
A, and diphosphopyridine nucleotide to form acetyl coenzyme A, COZ,
and reduced diphosphopyridine nucleo- tide. 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
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KORKES, DEL CAMPILLO, GUNSALUS, AND OCHOA 735
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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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