STUDIES ON THE MECHANISM OF ACETATE OXIDATION BY ESCHERICHIA COLI BY SAMUEL J. AJL AND MARTIN D. KAMEN (From the Department of Bacteriology and Immunology and the Mallinckrodt Insfitute of Radiology, Washington University School of Medicine, St. Louis, Missouri) (Received for publication, September 19, 1950) Considerable data have been accumulated from metabolic studies of various microorganisms, as well as mammalian tissues, which imply a tri- carboxylic acid cycle as a major pathway for acetate oxidation (1). How- ever, evidence bearing on acetate oxidation in bacteria has remained for the most part fragmentary and indirect. In this paper data on acetate oxidation by “adapted” Escherichia coli (2) will be presented. In the experiments to be described, Cr4-labeled acetate was oxidized simultaneously with cr-ketoglutarate, succinate, fumarate, malate, oxal- acetate, and pyruvate, singly or in combination. The fact that all sub- strates were metabolized at comparable rates was taken to indicate that no complications arose owing to differential permeability. From chemical analysis of COZ, residual acids and cell material, as well as manometric data on O2uptake, it was possible to show that, when an appreciable frac- tion of acetate and other substrates was metabolized, there was incorpora- tion and distribution of labeled carbon into all substrates with the excep- tion of cr-ketoglutarate. Some preliminary results on cell-free extracts are also reported. Fi- nally, these data are assessed on the basis of various schemes proposed for acetate oxidation. Materials and Methods E. coli (strain E-26) was grown for 48 hours, with constant aeration at 30” in the usual medium (2) containing 1.5 per cent sodium acetate as substrate. The handling of the cells and the manometric procedures were identical with those described previously (2). Cell-free extracts were prepared by the method of Kalnitsky, Utter, and Werkman (3). Reactions were allowed to continue until approximately 25 to 50 per cent of the substrates present were utilized. At the end of the incubation period, the various products were separated and the distribution of radio- activity was determined. The reaction mixture was first acidified either with 12 N H&Z+04 or with frichIoroacetic acid, the evoIved carbon dioxide being trapped in the alkali present in the center well of the Warburg cup. It was then con- 845 by guest on May 17, 2020 http://www.jbc.org/ Downloaded from
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STUDIES ON THE MECHANISM OF ACETATE OXIDATION BY ESCHERICHIA COLI
BY SAMUEL J. AJL AND MARTIN D. KAMEN
(From the Department of Bacteriology and Immunology and the Mallinckrodt Insfitute of Radiology, Washington University School of Medicine, St. Louis,
Missouri)
(Received for publication, September 19, 1950)
Considerable data have been accumulated from metabolic studies of various microorganisms, as well as mammalian tissues, which imply a tri- carboxylic acid cycle as a major pathway for acetate oxidation (1). How- ever, evidence bearing on acetate oxidation in bacteria has remained for the most part fragmentary and indirect. In this paper data on acetate oxidation by “adapted” Escherichia coli (2) will be presented.
In the experiments to be described, Cr4-labeled acetate was oxidized simultaneously with cr-ketoglutarate, succinate, fumarate, malate, oxal- acetate, and pyruvate, singly or in combination. The fact that all sub- strates were metabolized at comparable rates was taken to indicate that no complications arose owing to differential permeability. From chemical analysis of COZ, residual acids and cell material, as well as manometric data on O2 uptake, it was possible to show that, when an appreciable frac- tion of acetate and other substrates was metabolized, there was incorpora- tion and distribution of labeled carbon into all substrates with the excep- tion of cr-ketoglutarate.
Some preliminary results on cell-free extracts are also reported. Fi- nally, these data are assessed on the basis of various schemes proposed for acetate oxidation.
Materials and Methods
E. coli (strain E-26) was grown for 48 hours, with constant aeration at 30” in the usual medium (2) containing 1.5 per cent sodium acetate as substrate. The handling of the cells and the manometric procedures were identical with those described previously (2). Cell-free extracts were prepared by the method of Kalnitsky, Utter, and Werkman (3).
Reactions were allowed to continue until approximately 25 to 50 per cent of the substrates present were utilized. At the end of the incubation period, the various products were separated and the distribution of radio- activity was determined.
The reaction mixture was first acidified either with 12 N H&Z+04 or with frichIoroacetic acid, the evoIved carbon dioxide being trapped in the alkali present in the center well of the Warburg cup. It was then con-
verted to barium carbonate, washed with alcohol, and weighed, and the Cl4 content was determined.
The cells were recovered by centrifugation and then washed several times with distilled water, as well as at least once with unlabeled acetate. Finally they were dried at 100” and their radioactivity was measured (see below).
The supernatant containing the water-soluble components of the reaction mixture was steam-distilled to remove residual acetic acid, and the dis- tillate titrated. Duclaux distillation showed the acid recovered to be at least 95 per cent acetic. An aliquot of the neutralized acetate was assayed for Cl4 content by direct evaporation on stainless steel disks (see below). In a control experiment it was shown that 200 PM of 2-U4-acetic acid containing 216,000 c.p.m. were removed completely from 100 PM
each of unlabeled a-ketoglutarate and succinate by steam distillation. The non-volatile acid-soluble fraction was extracted continuously for
48 to 72 hours in the presence of excess sodium bisulfite at pH 2 to 3 to separate the CJ-dicarboxylic acids from keto acids (4, 5). When pyruvate was the carrier acid employed, it was necessary to begin with the ether extraction rather than the steam distillation, because of the marked volatil- ity of the pyruvate. In this case, the acetate was recovered later from the ether layer by steam distillation.
The ether-soluble material containing Cd-dicarboxylic acids was sub- jected to one more steam distillation to remove all traces of acetate. A neutral aliquot of the residual liquor was assayed for Cl4 content (see below). Another aliquot was boiled with acid permangante; CO2 formed in this way was collected in 4 N COa-free NaOH and converted to BaC03 for Cl4 assay. Volatile aldehyde was collected in 3 per cent sodium bisulfite for Cl4 assay (see below). In no case was appreciable radio- activity obtained in these fractions unless large amounts of appropriate carrier, such as fumarate or malate, had been used in the experiment to trap acetate carbon.
The residue from the above reaction (containing manganese dioxide) was filtered and reduced in volume, and succinate was recovered by ether extraction. After neutralization, a portion was taken for radioassay. The remainder of the solution was converted to a mixture of fumarate and malate by a succinoxidase preparation from beef heart in the usual way (6). The oxygen uptake was used to estimate succinate.
The mixture was acidified to remove protein, filtered, and again oxidized with acid permanganate. The resulting CO2 and acetaldehyde corres- ponded to the carboxyl and methylene carbon atoms of succinic acid, respectively. These fractions supplied the data used in calculating the original distribution of 04 activity in succinate (4). The fact that con-
siderable activity appeared in acetaldehyde and CO2 only after dehydro- genation with succinoxidase was the basis for assurance that the Cl4 content observed in these fractions was truly indicative of that originally in the auccinate.
The bisulfite which was bound by the acetaldehyde was released by adding 2 gm. of sodium bicarbonate. The freed biaulfite was then titrated with standard iodine solution. In early experiments a portion of the aldehyde-biaulfite complex was evaporated under a vacuum at room temper- ature on a standard stainless steel disk plate and the Cl4 content deter- mined. However, this procedure was found sometimes to result in high losses, probably because of inadvertent failure to bring the solutions exactly to neutral pH. Hence, in later experiments, the aldehyde was de- graded with alkaline hypoiodite to iodoform and formate, and the activities found in these products were added to determine initial aldehyde activity.
Treatment of the water layer depended on whether pyruvate or OL- ketoglutarate was the carrier acid. First, the solution was freed of sulfite by acidifying and boiling, after which it was oxidized with ceric sulfate. The COZ evolved was used to estimate the keto acid present. In no case was sufficient organic matter, other than carrier keto acid, present to invalidate this procedure. When pyruvate was present, the acetate formed was removed by steam distillation and titrated. Both COZ and acetate were assayed in the usual way for Cl4 content. When or-ketoglutarate was present, the CO2 formed by the ceric sulfate oxidation was collected and used to estimate keto acid present. The succinate formed was freed of any contaminating acetate (resulting from traces of labeled pyruvate arising during metabolism) by steam distillation with carrier acetate, after which it was extracted with ether and characterized by the procedure described above. Again no organic matter other than succinate was present to invalidate the oxidation procedure. The COz, succinate, and degradation products of succinate were assayed for Cl4 content (see below).
In no case were both cr-ketoglutarate and pyruvate added together as carrier. Therefore no careful separation of the two keto acids was neces- sary. However, to remove small amounts of labeled pyruvate which might have formed during the experiment, carrier ac-ketoglutarate, added to the residue of ether-bisulfite extraction, was precipitated as the silver salt, dissolved in nitric acid, reextracted with ether for 24 hours, and neutralized, and the radioactivity was determined. Because it was shown early in this work that the Cg keto acid never contained appreciable U4, no precautions were taken in separating carrier pyruvate from possible small amounts of oc-ketoglutarate.
In experiments involving fumarate as carrier, residual fumarate in the ether layer was determined by the method of Stotz (7). An aliquot of the
ether layer was also degraded with acid permanganate, the resulting formate being recovered by steam distillation and its Cl4 content and acid titer determined. This formate provided a measure of Cl4 content of methylene carbon, as well as a check on residual fumarate. Finally, the purity of the fumarate was checked by paper chromatography (see below). The Cl4 content of residual fumarate was obtained in the following manner. The acid from the aliquot subjected to chromatographic separation was eluted completely from the paper, and its Cl4 content, and hence the total Cl4 content of the fumarate, was determined. From this value twice the Cl4 content of the formate was subtracted, giving the activity residing in the carboxyl groups.
In experiments involving malate as carrier, the acetaldehyde resulting from acid permanganate degradation of the ether layer was used as an index of residual malate. Instead of trapping aldehyde as the bisulfite complex, the oxidation was carried out with a micro distillation apparatus in which the aldehyde was distilled directly into a flask immersed in an ice-salt mixture. The aldehyde so collected was degraded further to iodoform and formate and assayed as described below. Paper chromato- graphy was used to check the purity of the residual malate, and the car- boxy1 Cl4 content was determined, as in the case of formate, by difference between total Cl4 content of malate, as obtained from the chromatographed aliquot, and the Cl4 content of the 2 central carbons, as given by the val- ues found for the iodoform and formate.
In experiments involving oxalacetate as carrier, the cell suspension was freed of cells by centrifugation, acidified with HzS04, and boiled for 30 minutes, thereby converting all oxalacetate remaining to pyruvate and COZ. The residual pyruvate which was a measure of original residual oxalacetate was treated as described above.
The paper chromatography procedures used were adapted with little change from those described in the literature (8). The chromatograms were calibrated by using a large variety of pure compounds, singly or in combination, The absolute RF values agreed satisfactorily with those cited in the literature, but for identification of labeled material, with or without addition of carrier, more reliance was placed on relative RF values. In many cases, control strips from the same paper stock were run with a solution of known content, side by side with strips or sheets containing the unknown, and bands were placed by direct comparison with those found on control strips. Bands corresponding to various compounds were cut out and eluted twice with boiling water. Final identification was ac- complished by rechromatographing eluted labeled compounds after ad- dition of a variety of unlabeled carriers. The solutions obtained were concentrated, if necessary, and assayed for Cl4 content. The components
which could be identified with certainty, if present, were succinate, (Y- ketoglutarate, citrate, malate, tartrate, fumarate, and lactate.
All radioassays were made with a conventional end window Geiger- Miiller t,ube counter connected to a Tracerlab autoscaler. Solutions were pipetted on recessed stainless steel disks after neutralization. Solids such as protein, cell material, and BaC03 were mixed in ethyl alcohol and plated evenly on the disks. Drying was conducted under an inf l-red lamp. Appropriate corrections for self-absorption and carbon content were ap- plied.
The C!14-methyl-labeled sodium acetate was obtained as a dry crystalline powder from Tracerlab, Inc., Boston, Massachusetts, and exhibited an initial activity of -4 X lo5 c.p.m. per mg. as assayed with the geometry used. This material was diluted with unlabeled acetate and subjected to Duclaux distillation. The Duclaux constants obtained for the active material showed it to be at least 95 per cent pure acetate.
The experiments designed to supply data for quantitative deductions regarding mechanisms were carefully checked with regard to recovery of CY4. Only experiments in which recovery of Cl4 in various fractions was greater than 85 per cent of the total, initially added as acetate, were used to supply necessary data on reaction mechanisms. Systematic errors, unless otherwise indicated, did not exceed f5 per cent of the values given.
EXPERIMENTAL
Experiments with Intact Cells-Preliminary experiments of a semiquanti- tative nature were performed, in each of which labeled acetate was partially oxidized in the presence of unlabeled pyruvate, or-ketoglutarate, or suc- cinate.
It was found that under conditions in which all three substrates were available to the bacteria, as evidenced by oxidative metabolism, acetate carbon was recovered only in succinate and pyruvate and not in a-keto- glutarate. These exploratory experiments revealed that oxidation of ap- proximately 200 pM of acetate simultaneously in the presence of approxi- mately 1 mM of each of three substrates trapped between 25 and 35 PM
of acetate carbon in succinat.e and pyruvate, respectively, while less than 0.5 PM of acet,ate carbon was trapped in ac-ketoglutarate.
The results obtained when experiments were performed to determine quantitatively the entrapment of acetate carbon in various postulated intermediates of the tricarboxylic acid cycle are best exemplified by con- sidering results in which labeled acetate was oxidized simultaneously with oc-ketoglutarate and succinate. The data obtained are summarized in Table I.
Qualitatively, the salient results were as follows: (1) the low Cl4 content
The complete system consisted of 1 ml. of 0.2 M phosphate buffer, pH 7.0, 1 ml. of a 10 per cent suspension of freshly harvested (acetate-grown) E. coli, substrates as indicated, and NaOH in the cent.er well. Total volume 10 ml., temperature 30”. Time of incubation, 4 hours.
Recovery data, total recovered, 98,410 c.p.m.; initial activity, 114,000 c.p.m., recovery 86 per cent. Manometric data, 02 observed with substrates, 3649 pl., without substrates, 1327 pl., calculated from substrates disappeared, 3650 ~1.
TABLE II
Distribution of Radioactivity in Products of Dissimilation of 2-C14-Acetate by E. coli’ in Presence of Inactive or-Ketoglutaric Acid
Fraction
Acetate...................... cu-Ketoglutarate.............. “C4 fraction” later deter-
mined to be succinate). Carbonate. Cells..
Chemical data Total activity Specific activity
Initial Final Initial Final Initial Final -- _____
I*Jf #M c.p.m. c.+.m. c&n. $67 IIM
224 188 200,000 90,000 890 250 203 0 360 0
200 215
Avera e 2 spea c
activity
c.@n. w wf
685 1.5
The complete system consisted of 2 ml. of a 10 per cent suspension of a week old acetate-grown E. coli, 2 ml. of 0.2 M phosphate buffer, pH 7.0. Substrates as in- dicated and NaOH in the center well. Total volume 12 ml.; temperature 30”. Time of incubation, 6 hours.
Recovery data, total recovered, 172,700 c.p.m.; initial activity of acetate, 200,000 c.p.m.; recovery, 86 per cent. Manometric data, total 02 uptake observed with substrates, 4239 pl., without substrates, 1179 J. Theoretical oxygen uptake as calculated from substrate disappearance, 4040 ~1.
of evolved carbonate and of acid-insoluble cell material, (2) the high CP content of succinate, and (3) the absence of detectable Cl4 in oc-ketoglu- tarate. In this experiment all substrates were metabolized; thus the inefficiency of a-ketoglutarate in trapping acetate carbon could only be ascribed to intracellular rather than extracellular factors. Quantitative consequences of these data will be considered in the next section.
It is of interest to include one experiment in which ar-ketoglutarate alone was oxidized simultaneously with labeled acetate; the results are shown in Table II. Here, it will be noted that the Cl4 content of evolved carbonate and acid-insoluble cell material was relatively much greater than that
Total volume of reactants 12 ml. The system consisted of 1 ml. of 20 per cent suspension of freshly harvested (acetate-grown) E. coli, 1 ml. of PO, buffer (0.2 M), pH 7.0, sodium acetate as indicated, alkali in center well, and distilled water to volume. Aerobic incubation, temperature 30”. Time of incubation, 5 hours.
04 recovery data, total recovered, 111,360 c.p.m.; initial activity, 114,000 c.p.m.; recovery, 98 per cent. Manometric data, 02 uptake observed with acetate, 3620 pl., without acetate, 1700 ~1.
observed in the experiment of Table I. In fact, following the addition of a-ketoglutarate, the distribution of Cl4 in the various cell fractions did not change appreciably from that observed with labeled acetate alone (see Table III).
The major datum of Table III is the 3.2-fold dilution of acetate resulting from turnover with endogenous substances. This endogenous dilution prevented the use of data for acetate dilution, obtained from experiments on simultaneous oxidation of various substrates, to indicate unambiguously participation or non-participation of a citric acid cycle, as was possible in studies with yeast which exhibited neglible endogenous turnover (9).
Runs with labeled acetate and unlabeled pyruvate, fumarate, malate, and oxalacetate, singly or in combination, all yielded results similar to those obtained with succinate. However, in experiments with fumarate, malate, and oxalacetate, an unexpected complication arose. It was ob-
served that a large endogenous reduction of these compounds to succinate occurred, even though a rapid uptake of oxygen was noted and conditions were such that vigorous oxidation could proceed (with rapid shaking in
TABLE IV Oxidation of MP-Acetate in Presence of Either 8 mM of Malate or I mM of Fumarate
“CA fraction” other than suc- cinate or fuma- rate, possibly malate and ox- alacetate
Cells -
COlICelI- tration
- hi- .ial - L?d
7E Do(
C
76 DOC
C
-
- I Total counts Specific activity
Fi- nal - udd
21: 92!
7!
492 OO(
3t
Initial Final Initial --- c.p.m. C.$.rn. c.p.?n.
@Jr w 00,00087,300 1280
0 19,100 16,700
2,400
0 18,000
1,520 00,000 75,000 1240
0 1,500 1,100
400 0 1,300
0 14,200
765
Final
412
9.2/2 (4.6) per PM c
1.3/2 (0.6) per PM c
60 per NM C (as- suming uni- form distri- bution)
250
0.25 per ~iw[ C 0.10 per PM C 11 per pM C (as-
suming uni- form distri- bution)
-
A .verage pecific ctivity
c.$Q7z. >I?? pdd 846
745
In the experiment with malate as carrier, the complete system consisted of 2 ml. of 20 per cent suspension 20 days-old, acetate-grown E. COG, 1 ml. of phosphate buffer (0.2 M), pH 7.0. Substrates as indicated and NaOH in center well. Temperature, 30”. Time of incubation, 5 hours.
In the experiment with fumarate as carrier, the same conditions as above except that the time of incubation was 8 hours.
air). Thus, in one experiment beginning with 100 PM of labeled acetate and 125 PM each of unlabeled fumarate and cr-ketoglutarate, there remained 44 PM of acetate, 103 PM of ar-ketoglutarate, and less then 30 PM of fumarate.
104 PM of succinate appeared. Thus, it seemed that practically all the fumarate had been converted to succinate, possibly through the inter- vention of endogenous hydrogen donors. Insufficient hydrogen was avail- able from the acetate utilized to account for an appreciable fraction of the succinate formed. However, in early observations on E. coli it has been shown that fumarate can undergo a dismutation under anaerobic conditions, 7 molecules of fumarate forming 6 molecules of succinate and 4 molecules of carbonate (10). A similar reaction may have occurred ‘in this experi- ment, owing perhaps to the density of cell suspensions used, which may have resulted in maintaining cells much of the time under essentially anaerobic conditions, despite vigorous shaking in air.
Attempts to minimize this effect by using freshly harvested young cells (18 hours old) failed. It was found possible to demonstrate unambigu-
TABLE V
Oxygen Uptake by E. coli Extract with Various Substrates (7 Hour Experiment) in Phosphate Buffer, pH 7.0
Experiment No. Substrates Oa uptake
d.
1 56 MM acetate,* 1.2 mM succinate 2097 2 56 “ “ 1.2 “ “ 5 mg. adenosinetri- 3267
.- * Included 17.6 PM of 2-V-acetate containing 414,000 c.p.m.
ously entrapment of acetate carbon in malate and fumarate only when relatively enormous quantities (2 mM) of these acids were used (see Table IV).
Experiments with Cell-Free Extracts--It has been recorded that extracts of E. coli incubated anaerobically with unlabeled pyruvate and carboxyl- labeled acetate produce appreciable quantities of carboxyl-labeled suc- cinate (11). Attempts to extend these observations to aerobic dissimilation of labeled acetate in the presence of a variety of added unlabeled substrates will be summarized briefly.
Pooled juices could be prepared which invariably exhibited good oxygen uptake (cf. Table V) but which only occasionally utilized acetate carbon in synthetic reactions. Thus, as an example, all of the extracts in Experi- ments 1 to 3 (Table V) showed comparable oxygen uptake and substrate dissimilation. Yet, in Experiments 1 and 2, only 120 and 300 c.p.m., respectively, out of a total 414,000 c.p.m. as acetate, appeared as non- volatile material, while in Experiment 3, the non-volatile matter assayed
6000 c.p.m. Of these 6000 c.p.m., the major fraction (approximately 50 per cent) appeared to be citrate, but appreciable activity also appeared in succinate (600 c.p.m.), in lactate (900 c.p.m.), and in pyruvate (375 c.p.m.). No activity ((5 c.p.m.) was found in cr-ketoglutarate, although a very distinct band due to the carrier was present.
Similar results were obtained in all successful experiments; i.e., there was incorporation of acetate carbon in citrate, pyruvate, succinate, and lactate, and no significant incorporation in a-ketoglutarate.
No positive evidence for a Knoop-Thunberg type of condensation could be obtained in extracts prepared from adapted E. coli, as hoped, because of results typified above. The mere appearance of appreciable acetate carbon in succinate, while an encouraging sign, could not be interpreted solely as arising directly from condensation of acetate with itself, since only a small fraction (< 10 per cent) of the total activity incorporated into various compounds and originating from acetate found its way into suc- cinate.
DISCUSSION
No data on variation due to time in specific Cl4 content of various pro- posed intermediates and of evolved COZ during oxidation of labeled acetate have been presented to date, primarily because the rate of equilibration is very rapid, owing to the small amount of intermediary carbon used to maintain an oxidation cycle. In the absence of these data, the validity of the tricarboxylic acid cycle, or so called “citric acid” cycle, is inferred from measurements of equilibrium isotope distribution.
Other condensation reactions have been proposed to bring acetate into the cycle at a point other than citrate. The best known reaction of this type is the Knoop-Thunberg condensation, involving oxidative coupling of acetate with itself to form succinate, followed by a cycle involving reformation of acetate via the G-dicarboxylic acids and pyruvate. In addition, there are possible all the other condensation reactions which can be written involving acetate and any one of the citric acid cycle inter- mediates. Also, work on Axotobacter agilis indicates the possibility that acetate may be oxidized without participation of the citric acid cycle at all (12).
It has been shown that, in the oxidation of carboxyl-labeled barium or magnesium acetate by yeast, the citrate which accumulates exhibits the isotopic content to be expected if the citric acid cycle is the major oxidative pathway; moreover, the distribution of labeled carbon in the carboxyl groups leads to the inference that a Knoop-Thunberg condensation may function as an auxiliary reaction (9). More recently, evidence based on the equilibrium distribution of acetate carbon in CL-dicarboxylic acids,
during oxidation of labeled acetate by molds, has been presented and in- terpreted as establishing a C&-C2 type of condensation (i.e. Knoop-Thun- berg type of reaction) as a major pathway for acetate oxidation, as well as synthesis of Ca-dicarboxylic acids (13).
The data presented in this paper which indicate strongly that a cyclic mechanism is involved in acetate oxidation by E. coli are as follows: (1) nearly symmetrical distribution in succinate of carbon originally in the methyl group of acetate and (2) equivalence of Cl4 content of succinyl, carboxyl, and evolved CO2 (see Table I).
The salient fact which emerges from these researches is that ar-keto- glutarate is completely ineffective as a trapping agent for acetate carbon, under conditions in which acetate carbon is oxidized and trapped quanti- tatively in succinate and pyruvate. From the data of Table I, <0.5 PM of acetate carbon was trapped in a+ketoglutarate, while succinate trapped effectively all of the acetate carbon metabolized (-50 PM). The data of Table II show even more strikingly the inefficiency of oc-keto- glutarate as a trapping agent. Although the total Cd acid (including succinate) did not exceed 11 pM, and although 250 jtM of or-ketoglutarate were present, the oxidation of at least 36 pM of acetate resulted in the trapping of less than 0.002 PM in the keto acid, whereas succinate showed an equilibration with acetate at least 500 times greater.
If a citric acid cycle of the conventional type is assumed, then it is necessary to suppose that intracellular equilibration with extracellular ar-ketoglutarate is lower by at least three orders of magnitude than that for succinate or pyruvate. Alternatively, one may postulate that some Cg compound other than a-ketoglutarate is the actual intermediate. A sim- pler explanation is that the primary condensation product is either succin- ate or a Cd compound in equilibrium with it.
Good correlations can be obtained in 02 uptake, acetate dilution, and CP content of succinate from the data of Table I, as well as in many other experiments, by making appropriate calculations, assuming that the basic mechanism of acetate oxidation involves a Knoop-Thunberg type of con- densation. However, a number of assumptions requiring separate justi- fication are hidden in this type of calculation. Among others, these are that (1) no acetate arises from endogenous metabolism, and (2) the only products of succinate or a-ketoglutarate metabolism are acetate, COZ, and water.
As far as assumption (1) is concerned, it was found that the cells ex- hibited an endogenous O2 upt,ake of 500 Al., with formation of no acetate (<2 PM). Assumption (2) is in agreement with the observed R. Q. values for cr-ketoglutarate and succinate metabolism (a), but direct evidence on this point is still required.
The recent observations by Novelli and Lipmann (14) support a con- densation involving citrate as a product. These workers have shown that cell-free extracts of E. coli (the same strain used in these studies), when incubated with acetate, oxalacetate, and adenosinetriphosphate, accomplish a net synthesis of citrate. This result is confirmed in our researches, in that cell-free extracts incubated with labeled acetate and an oxidizable substrate (e.g. succinate) yield appreciable quantities of labeled citrate, as well as succinate, pyruvate, and lactate. So far, the attempt to demonstrate in such extracts a direct formation of succinate from acetate by way of a Knoop-Thunberg condensation has been unsuccessful because of the variety of synthetic reactions encountered.
We are indebted to Dr. Stanley L. Ranson for the chromatographic analyses and to Mr. Donald T. 0. Wong who participated in a number of the experiments.
SUMMARY
1. Simultaneous oxidation by Escherichia coli (strain E-26) of labeled acetate and unlabeled succinate, pyruvate, cr-ketoglutarate, fumarate, malate, and oxalacetate, singly or in combination, leads to incorporation of labeled carbon in all substrates with the exception of cu-ketoglutarate. The isotopic distribution in the carrier and in evolved COZ demonstrates equilibration of methyl carbon of acetate with carbon of substrates, but not with carbon of a-ketoglutarate, during acetate oxidation. All sub- strates are metabolized; therefore uncertainties in interpretation arising from differential permeability appear excluded.
2. A large endogenous reduction of all C&dicarboxylic acids tested with formation of succinate is observed under aerobic conditions and even when oxidation is proceeding vigorously, as evidenced by 02 uptake.
3. Isotopic distribution in succinate and other dicarboxylic acids, as well as in pyruvate and evolved COZ, reveals operation of a cyclic mechan- ism.
4. Incubation of cell-free extracts with labeled acetate and unlabeled citric acid cycle intermediates results in incorporation of acetate carbon mainly in citrate, succinate, pyruvate, and lactate, but not in cu-keto- glutarate.
5. The data presented are evaluated on the basis of various mechanisms proposed for the mechanism of acetate oxidation.
BIBLIOGRAPHY
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