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Formation of succinic acid in baker's yeast through the citric acid cycle
ARCHIVES
OF
BIOCHEMISTRY
AND
76, 463464 (1958)
BIOPHYSICS
Formation of Succinic Acid in Baker’s Yeast Through The Citric Acid Cycle A. 0. M. Stoppani, Susana L. S. de Favelukes and Lucfa Conches From the Department of Biochemistry, Buenos Aires, and the Laboratory Atomic Energy Commission, Received
School of Medicine, University Cell Metabolism, National Buenos Aires, Argentina
of
for
September
23, 1957
As is known, baker’s yeast (Saccharomycesceretisiae) forms succinic acid. In animal tissues succinate can be formed through the citric acid cycle, and evidence has been brought forward in support of a similar mechanism in baker’s yeast (1, 2). In this organism, however, the metabolic role of the Krebs cycle is still under discussion as some important effects dependent on the cycle operation cannot be easily demonstrated. In fact, (a) during the oxidation of acetate or pyruvate, there is no accumulation of succinate (3, 4) even in the presence of malonate (5); (b) the inhibition of succinic dehydrogenase with malonate does not prevent cold-treated yeast from oxidizing acetate at a fast rate (5) ; (c) the radioactive succinate formed by oxidation of acetate-C4 does not equilibrate with exogenous succinate although the latter can be simultaneously oxidized by the yeast preparation (5) ; (d) sue&ate and I-malate do not promote the oxidation of acetate by respiring mitochondria from baker’s yeast (6), and (e) fumarate and I-malate do not affect the malonate inhibition of acetate oxidation by yeast mitochondria (6) or yeast cells (5). The operation of the citric acid cycle implies (a) a flow of carbon through definite positions of the cycle intermediates (including succinate) and (5) an increase of the reservoir of succinic acid when the cycle is partially inhibited (e.g., by malonate) at the level of succinic dehydrogenase. In regard to the first point, it can be shown with acetate-C4 that the carboxyl carbon of acetate appears in the carboxyl groups of succinic acid whereas the methyl carbon appears in both the methylene 463
454
STOPPANI, DE FAVELUKES AND CONCHES
and the carboxyl groups but preferentially in the first. An ideal steadystate flow of Cl4 should label the methylene carbons twice as much as the carboxyl carbons (7), but the simultaneous oxidation of unlabeled (endogenous) acetate, the shortness of the incubation time, or the inhibition of the cycle operation delays relatively the incorporation of Cl4 into the carboxyl groups of succinic acid and maintains the ratio of the specific activities of the methylene to the carboxyl carbons above 2. The experiments reported in this paper show that these assumptions are valid in baker’s yeast in spite of the negative arguments quoted above. MATERIALS
AND METHODS
Sodium acetate-l-C’*, sodium acetate-2-Cr4, and pyruvamide-2-P were obtained from the Radiochemical Centre, Amersham, England (specific activities: 1 mc. per 0.24, 0.55, and 1.1 mmoles, respectively). The radioactive pyruvamide was hydrolyzed with 2 N HCl, and pyruvic acid-2-C!” was extracted with ethyl ether; the ether was evaporated at low temperature, and the acid was dissolved in water, neutralized, and kept frozen at -16”. The labeled compounds were diluted with nonisotopic acetate or pyruvate, respectively, to give the specific activities stated in each case. The yeast used in most of the experiments was starch-free commercial baker’s yeast, washed with distilled water and aereated for 15-20 hr. at 20”, under sterile conditions. The microscopic control did not show any significant contamination, and other experiments carried out with pure cultures of S. cerevisiae grown in the laboratory gave the same results. The concentration of the yeast suspensions was measured turbidimetrically with a photoelectric calorimeter and compared with a standard suspension whose concentration was established by the weight of the residue dried at 100”. The yeast suspensions were incubated with the additions stated in each case: (a) in stoppered 1%ml. Erlenmeyer flasks fixed on an electric shaker; (5) in a closed flask connected to a volume compensator like that used before for dark C1402 fixation experiments (8); and (c) in Warburg manometers to measure the oxygen consumption by Warburg’s direct method. Qo, values (~1. O%/hr./mg. yeast) are shown in some experiments.
Succinic Acid Isolation and Determination The yeast suspensions were deproteinized with sodium tungstate and sulfuric acid (9) and spun off; succinic acid was continuously extracted with ethyl ether from the supernatants, to which were previously added sodium bisulfite and phosphoric acid (10). The ether was evaporated, and the residue was taken up in water, oxidized with acid permanganate (9)) and re-extracted with ether. The oxidation with permanganate was required because of the addition of malonate to the yeast suspensions, and the acid bisulfite step because of the formation of succinic acid from the a-ketoglutaric and glutamic acids contained by the yeast extracts, if
SUCCINIC ACID FORMATION
455
these were oxidized with acid permanganate (11). The likelihood of this possibility is shown by the higher amounts of succinic acid obtained when the treatment with acid bisulfite was omitted. The succinic acid present in the second ethereal extract was either transferred to 0.04 2M phosphate buffer, pH 7.3 for manometric determination or further purified for degradation, by paper chromatography with the butanol-propionic acid-water mixture [cf. (12)]. On the other hand in one experiment (Table II) succinic acid was isolated from methanol extracts of the yeast cells by bidimensional chromatography and radioautography, carried out as described previously (8).
Acetic Acid Acetic acid was distilled and titrated with 0.013 N NaOH (13) ; thymol blue was used a.s internal indicator. From the titer of the distilled acid was subtracted the blank values and, when necessary, the titer of t,he volatile acid formed during the distillation by thermal decomposition of malonic acid which is proportional to t,he amount of malonic acid heated.
Fumaric and Pyruvic Acids These were determined according to Straub (14) and Friedemann and Haugen (15), respectively.
Degradation of Succinic Acid Duplicate or triplicate samples of the purified radioactive succinic acid were diluted with carrier succinic acid and decarboxylated (16) or combusted (17). The carbon dioxide was precipitated as barium carbonate; this was washed, filt.ered, and dried. The radioactivity was measured with a mica end-window coun ter and corrected for self-absorption, in order to obtain the specific activity (counts/min./mg. BaCOa) at infinite thinness. The activity of 04 in the carboxyl groups was obtained directly from the carbon dioxide liberated by the Schmidt reaction (16), and the activity in the methylene groups by difference between the activity of the carboxyl groups and the total activity. As the distribution of CIA in succinic acid was deemed more significant than the total specific activity, in some experiments the active succinic acid was not quantitatively recovered from the chromatograms, and therefore total specific activities of succinic acid are not comparable. On the other hand, when succinic acid was transferred quantitatively to the chromatography paper, the total Cl* incorporated was directly counted on the chromatogram with a Geiger-Muller tube of the Scott t,ype. The activity, conveniently standardized and corrected, is expressed as counts/min. RESULTS
Formation of Succinic Acid from Acetate-C? and Pyruvate-W-C14 Baker’s yeast forms carboxyl-labeled succinic acid by oxidation of acetate-l-U4 (Table I). The Cl4 incorporated into succinic acid is
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TABLE I and Acetate-b-Cl4 Oxidation by Malonate. Distribution of Cl4 in Succinic Acid Expt. 1: 80 mg. of yeast in 20 ml. of 0.05 M phosphate buffer pH 2.8; incubation for 100 min. at 30’; acetate-l-C 14: 233 rmoles (6.7 X lo6 counts/min.); 0.011 M malonate. Expt. 2: similar experimental conditions; 390 mg. of yeast; incubation for 90 min. at 25”; acetate-2-U4: 143 rmoles (5.0 X lo6 counts/min.) ; 0.06 M malonate. Oxygen uptake measured independently on 2.0 ml. suspension. The succinic acid formed in the 20 ml. suspension was extracted with ether as described in the text. The ether solution was quantitatively evaporated on paper, and the succinic acid was chromatographed, radioautographed, and counted on the chromatogram (Expt. 1). Samples were diluted with 9.93 mg. nonisotopic succinic acid and degraded. Specific activities in counts/min./mg. BaCOs . Inhibition
Additions
Expt
of Acetate-i-P
Qo,
.&ate consumption
3 in succiic acid
CWOH
__
-
Specific activity of succinic acid carbon atoms @Ha
cts x t+ir.
1 1 1 1
Acetate Acetate + malonate None Malonate
60.5 17.6
Acetate Acetate + malonate
-
18.3 6.5
206 57
C”OOH/ (C’aHz + CWOH)
%
102 48.7 f 30 30.6 f
0.2” 0.5 f 0.6 -1.6 f
0.4” 99 f 0.6 105 f
3.2 f 2.9 f
0.02 7.9 f 0.02 13.1 f
0.2 2.5 f 0.1 4.5 f
1.2” 2.2
-10 -10 C”‘H&WOH
2 2 -
-
255 143
-
-
0.07 0.05
QProbable error of the mean.
proportional to the amount of acetate metabolized, as shown by the similar effect of malonate on the yeast consumption of acetate (69 % inhibition) and oxygen (74% inhibition) and, on the other hand, on the incorporation of Cl4 into succinic acid (70 % inhibition). The oxidation of acetate-2-Cl4 leads to the formation of succinic acid labeled in both the carboxyl and the methylene groups, but preferentially in the latter. In Expt. 2 of Table I, in the absence of malonate, the relationship of C1*HJC400H specific activities was 2.5. The relative labeling of the carboxyl groups was proportional to the amount of acetate consumed, as shown by the increase of the value of the ratio CY4HJU400H when the oxidation of acetate was diminished by mal-
SUCCINIC
ACID
457
FORMATIOS
TABLE II Eflect of the Oxidation Time on the Distribution of Cl4 in Succinic Acid Formed from Acetate-l-P Yeast, 185 mg. in 10 ml. of 0.05 M phosphate buffer, pH 2.8; 148rmoles acetate2-C14 (5.0 X 106 counts/min.); 25”. Experiment was carried out in a closed flask connected to a volume compensator (8). One-milliliter yeast suspension samples were taken at the times shown and thoroughly mixed with 9 vol. methanol. The Cl4 fixed by the suspension was measured; the succinic acid present in the methanol-water phase was isolated by bidimensional paper chromatography (Benson et al.), eluted, and degraded as in Table I. Time of acetate oxidation min.
5 10 20 30 60 75
C 14 fixed
by the yeast
cts./min. mg.cclls x 103 0.6 1.6 3.1 11.2 17.3 15.6
Specitic;cct$$ 1
0.30 0.33 1.22 2.30 4.40 4.13
f f f zk f *
of succinic
acid,ccmn
atoms 2
0.03” 0.03 0.04 0.02 0.2 0.03
1.80 1.36 4.24 7.51 12.70 8.07
f dz rt: f f f
0.055 0.04 0.06 0.05 0.21 0.04
6.0 4.1 3.5 3.3 2.9 2.0
f 0.63 f 0.45 f 0.13 f 0.03 f 0.14 f 0.02
a Probable error of the mean.
(Table I) or by shortening the time of incubation inhibition (Table II). These results do not appear incompatible with a direct condensation of acetyl groups, according to the Thunberg reaction. To check this possibility, the distribution of Cl4 from acetate-l-Cl4 was followed through the incubation with the same technique used previously (8, 22) to study the kinetics of Cl402 fixation, Fumaric acid was one of the latest to become radioactive, and this makes it unlikely that the “tail to tail” condensation of acetyl groups can be an important source of succinic acid in baker’s yeast. The point will be considered elsewhere with more detail. Radioactive succinic acid was also formed from pyruvate-2-C’*. The activity appeared about in the same proportion in the carboxyl and methylene carbons (Table III) of succinic acid, which shows that besides oxidative decarboxylation of pyruvate-2-C’* to coenzyme A-linked acetyl-l-Cl4 there is a carboxylation leading to oxalacetate-2-C14,otherwise Cl4 should locate only in the carboxyl groups of succinic acid, as with acetate-l-Cl4 (Table I). The carboxylation reaction is further confirmed by the more effective labeling of succinic acid methylene carbons in the presence of malonate (Table III), which by inhibiting pyruvate oxidation left more substrate available for carboxylation. onate
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TABLE III Inhibition of Pyruvate-2-C14
Oxidation by Malonate. in Succinic Acid
Distribution
of Cl4
Yeast, 360 mg, in 20 ml. 0.05 M phosphate buffer pH 2.8; incubation for 60 min., at 30”; pyruvate-2-W: 375 pmoles (5.2 X lo5 counts/min.); malonate 0.011 M. Pyruvate formation in the controls, 0. The succinic acid formed in the 20 ml. suspension was extracted with ether as described in the text. The ether solutions were quantitatively evaporated on paper, chromatographed, and radioautographed. Succinic acid was eluted from the paper, and the chromatography and radioautography were repeated. The Cl4 present in the second chromatogram was counted, corresponding 9%99’% to succinic acid. Samples of purified radioactive succinic acid were diluted with 9.93 mg. nonisotopic succinic acid and degraded as in Table I. Additions
Pyruvate Pyruvate + malonate
Pyruvate consumption @noles
Cl” in succinic acid
Specific activity of succinic acid carbon atoms 0” OOH @Hz
%
m% x 103
181 141
38 41
C”OOH C”H, + CWOH
69 f 18 f
3” 1
67 f 107 f
3a 1
51 zlz 2.25 14 f 0.8
0 Probable error of the mean.
E$ect of Malonate on Succinic Acid Formation The action of malonate has been studied with the following substrates: pyruvate, fumarate, I-malate, and acetate. The formation of succinic acid from pyruvate, fumarate, and I-malate was significantly increased by malonate (Tables IV and V). This result is consistent with the observations of Barron et al, (2) but at variance with the findings of Krebs et al. (5). It is noteworthy (Table IV) that when the extraction in the presence of acid bisulfite was omitted, larger amounts of succinic acid were found. This must not be attributed to losses of succinic acid during the double-extraction procedure as the same experimental conditions allowed the quantitative separation of a mixture of 14 rmoles of succinic and cr-ketoglutaric acids, that is, amounts far higher than those extracted from the yeast suspensions. The extra succinic must arise therefore during the permanganate oxidation of the deproteinized yeast extracts, from precursors that in the acid bisulfite medium become insoluble in ether. The succinic acid formation from acetate was not significantly affected by malonate (Table VI). In Expt. 2 there was a small extra formation of succinic acid which could be ascribed to the presence of malonate,
SUCCINIC
ACID
TABLE Efect
of Malonate
on Succinic
459
FORMATION
IV Acid Formation
from Pyruvate
Yeast, 635 mg. (Expt. 1) or 856 mg. (Expt. 2), in 20 ml. of 0.05 M phosphate buffer; pH 2.8. 26”; 2 hr. (Expt. 1) or 1 hr. (Expt. 2) incubation. In parentheses, succinic acid found when the acid bisulfite step was omitted from the extraction procedure. Pyruvate Succinic acid consumption Expt 1 Expt 2 pmoles pmoles
Additions
F’yruvate P.vruvate Xone l\lalonate
0.022 M 0.022 M + malonate
0.044 M
0.044 111
TABLE Effect of Malonate
412 204 0 0
formatmn Expt 1 pnroles
400 177 0 0
9.8 16.4 7.8 6.7
Expt 2 pnoles
11.5(32.6) 23.4(42.0) 3.6(26.0) 7.0(19.0)
V
on Succinic Acid Formation and I-Malate
from Fumarate
Yeast,, 80 mg. (Expt. 1) or 14 mg. (Expt. 2), in 2.0 ml. of 0.05 M phosphat,e buffer, pH 2.8. 30”. Incubation in Warburg manometers for 2 hr. Substrate consumption Expt. 1 /.&m&s
Additions
Pumarate 0.012 111 Fumarate 0.012 M + malonate 0.044 M I-Malate 0.012 M I-Malate 0.012 M + malonate 0.044 M None Malonate 0.044 M
TABLE Effect
of Malonate
on Succinic
Succinic acid formation Expt. 1 Expt. 2 pmoles /lmoles
Oxygen uptake Expt. 2 4
32.4 14.7 0 0
251 131 267 114 79 24
Acid Formation
jrom
6.5 9.5 3.8 7.9 3.8 5.7
0.6 1.3 0.2 0.6 0.1 0.0
VI Acetate
Yeast, 588 mg. (Expt. 1). 635 mg. (Expt. 2), or 600 mg. (Expt. 3), in 20 ml. of 0.05 M phosphate buffer, pH 2.8. 30’. 2 hr. (Expts. 1 and 2) or 1 hr. (Expt. 3) incubation. In parentheses, succinic acid obtained when the extraction in the presence of bisulfite was omitted. Additions
Acetate 0.02 M Acetate 0.02 M + malonate 0.044 M None Malonate 0.044 M
Acetat;ecysyptmn Expt. 1 jmoles pmoles
370 160 -10 -10
330 50 -10 -10
Expt. 3 /moles
402 187 -10 -10
Succinic acid Expt. 1 Expt. 2 pm&s pmoles
formation Expt. 3 pmoles
10.1 8.1
9.1 10.6
3.0(7.2) l.l(l8.0)
9.7 10.5
7.8 6.7
0.0(8.8) 0.6(4.1)
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TABLE VII Comparison of Malonate Action on Acetate and Pyruvate Oxidation Yeast, 20 mg. (Expt. l), and 11.5 mg. (Expt. 2), in 2.0 ml. of 0.05 M phosphate buffer incubated in Warburg manometers for 90 min. 30”. pH 4.6 (Expt. 1) or pH 2.8 (Expt. 2). Acetate and pyruvate 0.023 M (Expt. 1) or 0.015 IM (Expt. 2). The figures have been subtracted for the respective controls. In parentheses, percentage inhibition. Expt.
Additions Substrate:
1 None 1 Malonate 0.05 M 2 None 2 Malonate 0.011 M 2 Malonate 0.005 M
Acetate Oxygen uptfcecmsumptmn rl.
pmoles
607 214 (64.7) 756 74(90.2) 257(65.9)
27.0 10.5 30.8 3.0 7.1
Pyruvate Oxygen uptake consumption Pymvate Jmolcs 11.
693 817(-32.3) 718 388(45.9) 650(-4.4)
26.4 36.4 30.4 11.4 24.3
but in the other experiments the opposite effect is observed. This agrees with the observations of Krebs et al. (5). The omission of the acid bisulfite step in the extraction procedure (Expt. 3) gave, as with pyruvate, higher amounts of succinic acid which eventually stimulated an increase of the succinic acid formation by malonate. This could explain Lynen’s results (18), for in his experiments very concentrated yeast suspensions were used and no mention is made of addition of acid bisulfite to the deproteinized yeast extracts before the extraction of succinic acid. Malonate has a different action on succinic acid formation from acetate, on the one hand, and from pyruvate, I-malate, and fumarate on the other. This peculiar effect in regard to acetate is not unique as TABLE VIII Effect of Malonate on Acetate and Pyruvate Oxidation at Different Times of Incubation Yeast, 11.5 mg. in 2.5 ml. of 0.05 M phosphate buffer, pH 2.8. 30”. Incubation in Warburg manometers for 90 min.; acetate or pyruvate, 0.015 M; malonate 0.0088 M. The figures represent Qo, . In ~01s. B, the A values less the respective controls. In parentheses, percentage inhibition. Additions
Acetate Acetate + malonate Pyruvate Pyruvate + malonate None Malonate
O-30 A
45.8 10.8 52.0 15.8 7.6 4.0
Oxidation 6WJO A
51.2 7.4 47.0 39.6 5.4 2.1
period, min. c-30 B
WO B
38.2 6.8(82.1) 44.4 11.8(73.1) -
45.8 5.3(88.1) 41.6 37.5(9.8) -
SUCCINIC
ACID
461
FORMATIOK
TABLE IX Inhibition of Acetate Oxidation by Malonate in the Presence of Pumarule Same as in Table VIII. Acetate, 0.012 M; fumarate 0.024 M; malonate, 0.017 M. Additions
Acetate Acetate + None Malonate Acetate + Acetate + Fumarate Fumarate
malonate fumarate fumarate + malonate + malonate
Oxygen
uptake
Acetate A
consumption
A Pl.
B d.
&moles
B pm&s
948 124 127 62 817 291 270 201
821 62 (92.5) 547 gO(83.5) 143 139(2.8)
30.0 3.9 0.0 -1.0 26.4 2.8 -1.3 -2.0
30.0 4.9(83.6) 27.7 4.8(83.5) -
(a) acetate oxidation was comparatively the most sensitive to malonate (Tables VII and IX); (b) the inhibition of acetate oxidation by malonate hardly varied through the incubation, whereas the inhibition of pyruvate oxidation nearly disappeared (Table VIII) ; and (c) at variance with what could be expected from the Krebs cycle model, fumarate did not vary the inhibition of acetate oxidation by malonate (Table IX). Similar results were obtained with I-malate. This confirms Krebs et al. results (5) notwithstanding other workers (1, 2) claims. It must be noted that, the relative insensitivity of pyruvnte oxidation to malonate cannot be accounted for by the action of yeast carboxylase, at least at, pH 4.6, since at this pH the enzyme is not active on exogenous pyruvate (19, 2, 20). DISCUSSION
The citric acid cycle is the mechanism that gives a better fit for the distribution of Cl* in succinic acid formed by baker’s yeast from acetateI-C14, acetate-2-C14, and pyruvate-2-C’*, as well as for the distribution of Cl4 from Cl402 fixed during acetaldehyde, acetate, pyruvate, or glucose oxidation (21, 22). The “glyoxylate bypass” (23) whose existence in baker’s yeast is made likely by the presence of isocitritase (24) and other evidence (25) can be ruled out as a main path of substrate oxidation on account of the strong labeling of glutamic acid after the fixation of C’*02 (21, 22) or the oxidation of acetate-C’* [(26) and our own unpublished observations]. Further, glyoxylic acid does not, appear in significant proportions in the chromatograms of yeast extracts after fixation of Cl*02 (22) or the oxidation of acetate-C’* (unpublished observations) .
462
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The competitive inhibition of yeast succinic dehydrogenase by malonate (l&27) explains, according to the cycle model, the two actions of malonate on the oxidation of pyruvate, fumarate, and E-malate, namely, inhibition of the substrate oxidation and accumulation of succinic acid. It is noteworthy that the lack of action of malonate on the oxidation of fumarate reported in Table IX is quite compatible with the inhibition shown in Table VI, as in this case the malonate concentration was higher. The formation of succinic acid from fumarate and I-malate implies, according to the cycle model, oxidation to oxalacetate, successive decarboxylation of the latter to pyruvate and acetyl-CoA, synthesis of citrate from acetyl-CoA and a second molecule of oxalacetate, and oxidation of citrate up to succinate. The enzymes catalyzing the respective reactions are known in baker’s yeast, including oxalacetate carboxylase (28). With pyruvate a similar set of reactions is in operation and, in addition, the carboxylation of pyruvate to oxalacetate. The latter reaction is proved by (a) the significant labeling of the 2-carbon atoms of succinic acid formed from pyruvate-2-C14; (b) the labeling of the 4-carbon atom of aspartic acid after fixation of C1402 (22) ; and (c) the presence of phosphopyruvic carboxylase and pyruvic phosphokinase in cell-free extracts of baker’s yeast (observations with J. Cannata to be published). The action of malonate on acetate oxidation can be also explained by competitive inhibition of succinic dehydrogenase, for, according to the cycle model, the inhibition of this enzyme prevents the regeneration of the C4 acetyl acceptor. Since endogenous oxalacetate can be decomposed by oxalacetate carboxylase, and no exogenous precursor of oxalacetate is available, comparatively smaller concentrations of malonate can block acetate oxidation and consequently inhibit the formation of succinic acid. However, the inhibition of succinic dehydrogenase may not be the only mechanism of malonate effect. The lack of action of fumarate and Z-malate on yeast preparations inhibited by malonate (Krebs et al., Linnane and Still, and our own observations) leads us to assume that malonate may inhibit acetate oxidation at a site different from succinic dehydrogenase, e.g., acetate activation. In connection with this, it may be mentioned that in Pseudomonas JEuorescens (29) malonate is activated to malonyl-CoA by an enzyme similar to yeast acetokinase. If this reaction takes place in baker’s yeast, the lack of a malonyl-CoA decarboxylase should prevent, indirectly, the liberation of CoA required to activate acetate, and in this way acetate oxidation should be further inhibited by malonate.
SUCCINIC ACID FORMATION
463
ACKNOWLEDGMENTS We are grateful to Drs. A. S. Actis, F. L. Sacerdote, for their assistance in some of the experiments.
E. Rnmos, and M. Pigretti
SUMMARY 1. The distribution of Cl4 in succinic acid formed by baker’s yeast from acetate-l-W, acetate-2-C’*, and pyruvate-2-Cl4 fits the citric acid cycle model. 2. According to the cycle theory, malonate increases the synthesis of succinic acid from pyruvate, fumarate, and I-malate. 3. Malonate is most effective as inhibitor with acetate, but does not increase succinic acid formation from acetate. 4. The different action of malonate with pyruvate, fumarate, and I-malate on the one hand, and with acetate on the other, is due mainly to the possibility of the first forming oxalacetate. Evidence supporting the carboxylation of pyruvate is set forth.
REFERENCES 1. MARTIUS, C., AND LYNEN, F., Advances in Enzymol. 10, 167 (1950). 2. BARRON, E. S. G., ARDAO, M. I., AND HEARON, M., J. Gen. Physiol. 34, 211 (1950). 3. KLEINZELLER, A., Biochem. J. 36, 495 (1941). 4. WEINHOUSE, S., AND MILLINGTON, R. H., J. Anl. Chem. Sot. 69, 3089 (1947). 5. KREBS, H. A., GURIN, S., AND EGGLESTON, L. V., Biochem. J. 61, 614 (1952). 6. LINNANE, A. W., AND STILL, J. L., Arch. Biochem. Biophys. 69, 383 (1955). 7. STRISOWER, E. H., KOHLER, G. D., AND CHAIKOFF, I. L., J. Biol. Chew 198, 115 (1952). 8. STOPPANI, A. 0. M., FULLER, R. C., ANI) CALVIN, M., J. Racferiol. 69, 491 (1955). 9. KREBS, H. A., AND EGGLESTON, L. V., Biochem. J. 34, 442 (1940). 10. WIXL-MALHERBE, H., Biochem. J. 31, 299 (1937). 11. KREBS, H. A., AND EGGLESTON, L. V., Biochem. J. 43, 17 (1948). 12. BENSON, A. A., BASSHAM, J. A., CALVIN, M., GOODALE, T. C., HAAS, V. A., ANU STEPKA, W., J. Am. Chew Sot. 72, 1710 (1950). 13. LELOIR, 1~. F., AND Munoz, J. M., Biochem. J. 33, 734 (1939). II. STRAUA, F. B., 2. physiol. Chem. 236, 43 (1935). 15. FRIEUEI\IANN, T. E., ANU HAUGEN, G. E., J. Riol. Chem. 147, 415 (1943). 16. &ARES, E. F., AND LONG, RI., J. Am. Chem. Sot. 77, 2556 (1955). 17. LINDENBAUW, A., SCHUBERT, J., AND ARMSTRONG, W. I)., /Inal. Chem. 20, 1120 (1948). 18. LYNEN, F'., Ann. 664,40 (1943). 19. SMYTHE, C. V., J. Biol. Chem. 126, 635 (1938). 20. STOPPANI, A. 0. M., Nature 167, 653 (1951).
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21. DAVIS, J. W., CHELDELIN, V. H., CHRISTENSEN, B. E., AND WANG, C. H., Biochim. et Biophys. Acta 21, 101 (1956). 22. STOPPANI, A. 0. M., DE FAVELUKES, S. L. S., CONCHES, L., AND SACERDOTE, F. L., Biochim. et Biophys. Acta 26, 443 (1957). 23. KORNBERT, H. L., AND MADSEN, N. B., Biochim. et Biophye. Acta24,651 (1957). 24. OLSON, J. A., Nature 174, 695 (1954). 25. BOLCATO, V., DE BERNARD, B., AND LEWIERO, G., Arch. Biochem. Biophys. 69, 372 (1957). 26. LABBE, R. F., THOMAS, R. C., CHELDELIN, V. H., CHRISTENSEN, B. E., AND WANG, C. II., J. Biol. Chem. 197, 655 (1952). 27. SINQER, T. P., MASSEY, V., AND KEARNEY, E. B., Arch. Biochem. Biophys. 69, 405 (1957). 28. NOSSAL, P. M., HANSEN, I. A., AND LADD, J. N., Biochim. et Biophys. Acta 23, 103 (1957). 29. HAYAISHI, O., J. Biol. Chem. 216, 125 (1955).
OF
BIOCHEMISTRY
AND
76, 463464 (1958)
BIOPHYSICS
Formation of Succinic Acid in Baker’s Yeast Through The Citric Acid Cycle A. 0. M. Stoppani, Susana L. S. de Favelukes and Lucfa Conches From the Department of Biochemistry, Buenos Aires, and the Laboratory Atomic Energy Commission, Received
School of Medicine, University Cell Metabolism, National Buenos Aires, Argentina
of
for
September
23, 1957
As is known, baker’s yeast (Saccharomycesceretisiae) forms succinic acid. In animal tissues succinate can be formed through the citric acid cycle, and evidence has been brought forward in support of a similar mechanism in baker’s yeast (1, 2). In this organism, however, the metabolic role of the Krebs cycle is still under discussion as some important effects dependent on the cycle operation cannot be easily demonstrated. In fact, (a) during the oxidation of acetate or pyruvate, there is no accumulation of succinate (3, 4) even in the presence of malonate (5); (b) the inhibition of succinic dehydrogenase with malonate does not prevent cold-treated yeast from oxidizing acetate at a fast rate (5) ; (c) the radioactive succinate formed by oxidation of acetate-C4 does not equilibrate with exogenous succinate although the latter can be simultaneously oxidized by the yeast preparation (5) ; (d) sue&ate and I-malate do not promote the oxidation of acetate by respiring mitochondria from baker’s yeast (6), and (e) fumarate and I-malate do not affect the malonate inhibition of acetate oxidation by yeast mitochondria (6) or yeast cells (5). The operation of the citric acid cycle implies (a) a flow of carbon through definite positions of the cycle intermediates (including succinate) and (5) an increase of the reservoir of succinic acid when the cycle is partially inhibited (e.g., by malonate) at the level of succinic dehydrogenase. In regard to the first point, it can be shown with acetate-C4 that the carboxyl carbon of acetate appears in the carboxyl groups of succinic acid whereas the methyl carbon appears in both the methylene 463
454
STOPPANI, DE FAVELUKES AND CONCHES
and the carboxyl groups but preferentially in the first. An ideal steadystate flow of Cl4 should label the methylene carbons twice as much as the carboxyl carbons (7), but the simultaneous oxidation of unlabeled (endogenous) acetate, the shortness of the incubation time, or the inhibition of the cycle operation delays relatively the incorporation of Cl4 into the carboxyl groups of succinic acid and maintains the ratio of the specific activities of the methylene to the carboxyl carbons above 2. The experiments reported in this paper show that these assumptions are valid in baker’s yeast in spite of the negative arguments quoted above. MATERIALS
AND METHODS
Sodium acetate-l-C’*, sodium acetate-2-Cr4, and pyruvamide-2-P were obtained from the Radiochemical Centre, Amersham, England (specific activities: 1 mc. per 0.24, 0.55, and 1.1 mmoles, respectively). The radioactive pyruvamide was hydrolyzed with 2 N HCl, and pyruvic acid-2-C!” was extracted with ethyl ether; the ether was evaporated at low temperature, and the acid was dissolved in water, neutralized, and kept frozen at -16”. The labeled compounds were diluted with nonisotopic acetate or pyruvate, respectively, to give the specific activities stated in each case. The yeast used in most of the experiments was starch-free commercial baker’s yeast, washed with distilled water and aereated for 15-20 hr. at 20”, under sterile conditions. The microscopic control did not show any significant contamination, and other experiments carried out with pure cultures of S. cerevisiae grown in the laboratory gave the same results. The concentration of the yeast suspensions was measured turbidimetrically with a photoelectric calorimeter and compared with a standard suspension whose concentration was established by the weight of the residue dried at 100”. The yeast suspensions were incubated with the additions stated in each case: (a) in stoppered 1%ml. Erlenmeyer flasks fixed on an electric shaker; (5) in a closed flask connected to a volume compensator like that used before for dark C1402 fixation experiments (8); and (c) in Warburg manometers to measure the oxygen consumption by Warburg’s direct method. Qo, values (~1. O%/hr./mg. yeast) are shown in some experiments.
Succinic Acid Isolation and Determination The yeast suspensions were deproteinized with sodium tungstate and sulfuric acid (9) and spun off; succinic acid was continuously extracted with ethyl ether from the supernatants, to which were previously added sodium bisulfite and phosphoric acid (10). The ether was evaporated, and the residue was taken up in water, oxidized with acid permanganate (9)) and re-extracted with ether. The oxidation with permanganate was required because of the addition of malonate to the yeast suspensions, and the acid bisulfite step because of the formation of succinic acid from the a-ketoglutaric and glutamic acids contained by the yeast extracts, if
SUCCINIC ACID FORMATION
455
these were oxidized with acid permanganate (11). The likelihood of this possibility is shown by the higher amounts of succinic acid obtained when the treatment with acid bisulfite was omitted. The succinic acid present in the second ethereal extract was either transferred to 0.04 2M phosphate buffer, pH 7.3 for manometric determination or further purified for degradation, by paper chromatography with the butanol-propionic acid-water mixture [cf. (12)]. On the other hand in one experiment (Table II) succinic acid was isolated from methanol extracts of the yeast cells by bidimensional chromatography and radioautography, carried out as described previously (8).
Acetic Acid Acetic acid was distilled and titrated with 0.013 N NaOH (13) ; thymol blue was used a.s internal indicator. From the titer of the distilled acid was subtracted the blank values and, when necessary, the titer of t,he volatile acid formed during the distillation by thermal decomposition of malonic acid which is proportional to t,he amount of malonic acid heated.
Fumaric and Pyruvic Acids These were determined according to Straub (14) and Friedemann and Haugen (15), respectively.
Degradation of Succinic Acid Duplicate or triplicate samples of the purified radioactive succinic acid were diluted with carrier succinic acid and decarboxylated (16) or combusted (17). The carbon dioxide was precipitated as barium carbonate; this was washed, filt.ered, and dried. The radioactivity was measured with a mica end-window coun ter and corrected for self-absorption, in order to obtain the specific activity (counts/min./mg. BaCOa) at infinite thinness. The activity of 04 in the carboxyl groups was obtained directly from the carbon dioxide liberated by the Schmidt reaction (16), and the activity in the methylene groups by difference between the activity of the carboxyl groups and the total activity. As the distribution of CIA in succinic acid was deemed more significant than the total specific activity, in some experiments the active succinic acid was not quantitatively recovered from the chromatograms, and therefore total specific activities of succinic acid are not comparable. On the other hand, when succinic acid was transferred quantitatively to the chromatography paper, the total Cl* incorporated was directly counted on the chromatogram with a Geiger-Muller tube of the Scott t,ype. The activity, conveniently standardized and corrected, is expressed as counts/min. RESULTS
Formation of Succinic Acid from Acetate-C? and Pyruvate-W-C14 Baker’s yeast forms carboxyl-labeled succinic acid by oxidation of acetate-l-U4 (Table I). The Cl4 incorporated into succinic acid is
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TABLE I and Acetate-b-Cl4 Oxidation by Malonate. Distribution of Cl4 in Succinic Acid Expt. 1: 80 mg. of yeast in 20 ml. of 0.05 M phosphate buffer pH 2.8; incubation for 100 min. at 30’; acetate-l-C 14: 233 rmoles (6.7 X lo6 counts/min.); 0.011 M malonate. Expt. 2: similar experimental conditions; 390 mg. of yeast; incubation for 90 min. at 25”; acetate-2-U4: 143 rmoles (5.0 X lo6 counts/min.) ; 0.06 M malonate. Oxygen uptake measured independently on 2.0 ml. suspension. The succinic acid formed in the 20 ml. suspension was extracted with ether as described in the text. The ether solution was quantitatively evaporated on paper, and the succinic acid was chromatographed, radioautographed, and counted on the chromatogram (Expt. 1). Samples were diluted with 9.93 mg. nonisotopic succinic acid and degraded. Specific activities in counts/min./mg. BaCOs . Inhibition
Additions
Expt
of Acetate-i-P
Qo,
.&ate consumption
3 in succiic acid
CWOH
__
-
Specific activity of succinic acid carbon atoms @Ha
cts x t+ir.
1 1 1 1
Acetate Acetate + malonate None Malonate
60.5 17.6
Acetate Acetate + malonate
-
18.3 6.5
206 57
C”OOH/ (C’aHz + CWOH)
%
102 48.7 f 30 30.6 f
0.2” 0.5 f 0.6 -1.6 f
0.4” 99 f 0.6 105 f
3.2 f 2.9 f
0.02 7.9 f 0.02 13.1 f
0.2 2.5 f 0.1 4.5 f
1.2” 2.2
-10 -10 C”‘H&WOH
2 2 -
-
255 143
-
-
0.07 0.05
QProbable error of the mean.
proportional to the amount of acetate metabolized, as shown by the similar effect of malonate on the yeast consumption of acetate (69 % inhibition) and oxygen (74% inhibition) and, on the other hand, on the incorporation of Cl4 into succinic acid (70 % inhibition). The oxidation of acetate-2-Cl4 leads to the formation of succinic acid labeled in both the carboxyl and the methylene groups, but preferentially in the latter. In Expt. 2 of Table I, in the absence of malonate, the relationship of C1*HJC400H specific activities was 2.5. The relative labeling of the carboxyl groups was proportional to the amount of acetate consumed, as shown by the increase of the value of the ratio CY4HJU400H when the oxidation of acetate was diminished by mal-
SUCCINIC
ACID
457
FORMATIOS
TABLE II Eflect of the Oxidation Time on the Distribution of Cl4 in Succinic Acid Formed from Acetate-l-P Yeast, 185 mg. in 10 ml. of 0.05 M phosphate buffer, pH 2.8; 148rmoles acetate2-C14 (5.0 X 106 counts/min.); 25”. Experiment was carried out in a closed flask connected to a volume compensator (8). One-milliliter yeast suspension samples were taken at the times shown and thoroughly mixed with 9 vol. methanol. The Cl4 fixed by the suspension was measured; the succinic acid present in the methanol-water phase was isolated by bidimensional paper chromatography (Benson et al.), eluted, and degraded as in Table I. Time of acetate oxidation min.
5 10 20 30 60 75
C 14 fixed
by the yeast
cts./min. mg.cclls x 103 0.6 1.6 3.1 11.2 17.3 15.6
Specitic;cct$$ 1
0.30 0.33 1.22 2.30 4.40 4.13
f f f zk f *
of succinic
acid,ccmn
atoms 2
0.03” 0.03 0.04 0.02 0.2 0.03
1.80 1.36 4.24 7.51 12.70 8.07
f dz rt: f f f
0.055 0.04 0.06 0.05 0.21 0.04
6.0 4.1 3.5 3.3 2.9 2.0
f 0.63 f 0.45 f 0.13 f 0.03 f 0.14 f 0.02
a Probable error of the mean.
(Table I) or by shortening the time of incubation inhibition (Table II). These results do not appear incompatible with a direct condensation of acetyl groups, according to the Thunberg reaction. To check this possibility, the distribution of Cl4 from acetate-l-Cl4 was followed through the incubation with the same technique used previously (8, 22) to study the kinetics of Cl402 fixation, Fumaric acid was one of the latest to become radioactive, and this makes it unlikely that the “tail to tail” condensation of acetyl groups can be an important source of succinic acid in baker’s yeast. The point will be considered elsewhere with more detail. Radioactive succinic acid was also formed from pyruvate-2-C’*. The activity appeared about in the same proportion in the carboxyl and methylene carbons (Table III) of succinic acid, which shows that besides oxidative decarboxylation of pyruvate-2-C’* to coenzyme A-linked acetyl-l-Cl4 there is a carboxylation leading to oxalacetate-2-C14,otherwise Cl4 should locate only in the carboxyl groups of succinic acid, as with acetate-l-Cl4 (Table I). The carboxylation reaction is further confirmed by the more effective labeling of succinic acid methylene carbons in the presence of malonate (Table III), which by inhibiting pyruvate oxidation left more substrate available for carboxylation. onate
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TABLE III Inhibition of Pyruvate-2-C14
Oxidation by Malonate. in Succinic Acid
Distribution
of Cl4
Yeast, 360 mg, in 20 ml. 0.05 M phosphate buffer pH 2.8; incubation for 60 min., at 30”; pyruvate-2-W: 375 pmoles (5.2 X lo5 counts/min.); malonate 0.011 M. Pyruvate formation in the controls, 0. The succinic acid formed in the 20 ml. suspension was extracted with ether as described in the text. The ether solutions were quantitatively evaporated on paper, chromatographed, and radioautographed. Succinic acid was eluted from the paper, and the chromatography and radioautography were repeated. The Cl4 present in the second chromatogram was counted, corresponding 9%99’% to succinic acid. Samples of purified radioactive succinic acid were diluted with 9.93 mg. nonisotopic succinic acid and degraded as in Table I. Additions
Pyruvate Pyruvate + malonate
Pyruvate consumption @noles
Cl” in succinic acid
Specific activity of succinic acid carbon atoms 0” OOH @Hz
%
m% x 103
181 141
38 41
C”OOH C”H, + CWOH
69 f 18 f
3” 1
67 f 107 f
3a 1
51 zlz 2.25 14 f 0.8
0 Probable error of the mean.
E$ect of Malonate on Succinic Acid Formation The action of malonate has been studied with the following substrates: pyruvate, fumarate, I-malate, and acetate. The formation of succinic acid from pyruvate, fumarate, and I-malate was significantly increased by malonate (Tables IV and V). This result is consistent with the observations of Barron et al, (2) but at variance with the findings of Krebs et al. (5). It is noteworthy (Table IV) that when the extraction in the presence of acid bisulfite was omitted, larger amounts of succinic acid were found. This must not be attributed to losses of succinic acid during the double-extraction procedure as the same experimental conditions allowed the quantitative separation of a mixture of 14 rmoles of succinic and cr-ketoglutaric acids, that is, amounts far higher than those extracted from the yeast suspensions. The extra succinic must arise therefore during the permanganate oxidation of the deproteinized yeast extracts, from precursors that in the acid bisulfite medium become insoluble in ether. The succinic acid formation from acetate was not significantly affected by malonate (Table VI). In Expt. 2 there was a small extra formation of succinic acid which could be ascribed to the presence of malonate,
SUCCINIC
ACID
TABLE Efect
of Malonate
on Succinic
459
FORMATION
IV Acid Formation
from Pyruvate
Yeast, 635 mg. (Expt. 1) or 856 mg. (Expt. 2), in 20 ml. of 0.05 M phosphate buffer; pH 2.8. 26”; 2 hr. (Expt. 1) or 1 hr. (Expt. 2) incubation. In parentheses, succinic acid found when the acid bisulfite step was omitted from the extraction procedure. Pyruvate Succinic acid consumption Expt 1 Expt 2 pmoles pmoles
Additions
F’yruvate P.vruvate Xone l\lalonate
0.022 M 0.022 M + malonate
0.044 M
0.044 111
TABLE Effect of Malonate
412 204 0 0
formatmn Expt 1 pnroles
400 177 0 0
9.8 16.4 7.8 6.7
Expt 2 pnoles
11.5(32.6) 23.4(42.0) 3.6(26.0) 7.0(19.0)
V
on Succinic Acid Formation and I-Malate
from Fumarate
Yeast,, 80 mg. (Expt. 1) or 14 mg. (Expt. 2), in 2.0 ml. of 0.05 M phosphat,e buffer, pH 2.8. 30”. Incubation in Warburg manometers for 2 hr. Substrate consumption Expt. 1 /.&m&s
Additions
Pumarate 0.012 111 Fumarate 0.012 M + malonate 0.044 M I-Malate 0.012 M I-Malate 0.012 M + malonate 0.044 M None Malonate 0.044 M
TABLE Effect
of Malonate
on Succinic
Succinic acid formation Expt. 1 Expt. 2 pmoles /lmoles
Oxygen uptake Expt. 2 4
32.4 14.7 0 0
251 131 267 114 79 24
Acid Formation
jrom
6.5 9.5 3.8 7.9 3.8 5.7
0.6 1.3 0.2 0.6 0.1 0.0
VI Acetate
Yeast, 588 mg. (Expt. 1). 635 mg. (Expt. 2), or 600 mg. (Expt. 3), in 20 ml. of 0.05 M phosphate buffer, pH 2.8. 30’. 2 hr. (Expts. 1 and 2) or 1 hr. (Expt. 3) incubation. In parentheses, succinic acid obtained when the extraction in the presence of bisulfite was omitted. Additions
Acetate 0.02 M Acetate 0.02 M + malonate 0.044 M None Malonate 0.044 M
Acetat;ecysyptmn Expt. 1 jmoles pmoles
370 160 -10 -10
330 50 -10 -10
Expt. 3 /moles
402 187 -10 -10
Succinic acid Expt. 1 Expt. 2 pm&s pmoles
formation Expt. 3 pmoles
10.1 8.1
9.1 10.6
3.0(7.2) l.l(l8.0)
9.7 10.5
7.8 6.7
0.0(8.8) 0.6(4.1)
460
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TABLE VII Comparison of Malonate Action on Acetate and Pyruvate Oxidation Yeast, 20 mg. (Expt. l), and 11.5 mg. (Expt. 2), in 2.0 ml. of 0.05 M phosphate buffer incubated in Warburg manometers for 90 min. 30”. pH 4.6 (Expt. 1) or pH 2.8 (Expt. 2). Acetate and pyruvate 0.023 M (Expt. 1) or 0.015 IM (Expt. 2). The figures have been subtracted for the respective controls. In parentheses, percentage inhibition. Expt.
Additions Substrate:
1 None 1 Malonate 0.05 M 2 None 2 Malonate 0.011 M 2 Malonate 0.005 M
Acetate Oxygen uptfcecmsumptmn rl.
pmoles
607 214 (64.7) 756 74(90.2) 257(65.9)
27.0 10.5 30.8 3.0 7.1
Pyruvate Oxygen uptake consumption Pymvate Jmolcs 11.
693 817(-32.3) 718 388(45.9) 650(-4.4)
26.4 36.4 30.4 11.4 24.3
but in the other experiments the opposite effect is observed. This agrees with the observations of Krebs et al. (5). The omission of the acid bisulfite step in the extraction procedure (Expt. 3) gave, as with pyruvate, higher amounts of succinic acid which eventually stimulated an increase of the succinic acid formation by malonate. This could explain Lynen’s results (18), for in his experiments very concentrated yeast suspensions were used and no mention is made of addition of acid bisulfite to the deproteinized yeast extracts before the extraction of succinic acid. Malonate has a different action on succinic acid formation from acetate, on the one hand, and from pyruvate, I-malate, and fumarate on the other. This peculiar effect in regard to acetate is not unique as TABLE VIII Effect of Malonate on Acetate and Pyruvate Oxidation at Different Times of Incubation Yeast, 11.5 mg. in 2.5 ml. of 0.05 M phosphate buffer, pH 2.8. 30”. Incubation in Warburg manometers for 90 min.; acetate or pyruvate, 0.015 M; malonate 0.0088 M. The figures represent Qo, . In ~01s. B, the A values less the respective controls. In parentheses, percentage inhibition. Additions
Acetate Acetate + malonate Pyruvate Pyruvate + malonate None Malonate
O-30 A
45.8 10.8 52.0 15.8 7.6 4.0
Oxidation 6WJO A
51.2 7.4 47.0 39.6 5.4 2.1
period, min. c-30 B
WO B
38.2 6.8(82.1) 44.4 11.8(73.1) -
45.8 5.3(88.1) 41.6 37.5(9.8) -
SUCCINIC
ACID
461
FORMATIOK
TABLE IX Inhibition of Acetate Oxidation by Malonate in the Presence of Pumarule Same as in Table VIII. Acetate, 0.012 M; fumarate 0.024 M; malonate, 0.017 M. Additions
Acetate Acetate + None Malonate Acetate + Acetate + Fumarate Fumarate
malonate fumarate fumarate + malonate + malonate
Oxygen
uptake
Acetate A
consumption
A Pl.
B d.
&moles
B pm&s
948 124 127 62 817 291 270 201
821 62 (92.5) 547 gO(83.5) 143 139(2.8)
30.0 3.9 0.0 -1.0 26.4 2.8 -1.3 -2.0
30.0 4.9(83.6) 27.7 4.8(83.5) -
(a) acetate oxidation was comparatively the most sensitive to malonate (Tables VII and IX); (b) the inhibition of acetate oxidation by malonate hardly varied through the incubation, whereas the inhibition of pyruvate oxidation nearly disappeared (Table VIII) ; and (c) at variance with what could be expected from the Krebs cycle model, fumarate did not vary the inhibition of acetate oxidation by malonate (Table IX). Similar results were obtained with I-malate. This confirms Krebs et al. results (5) notwithstanding other workers (1, 2) claims. It must be noted that, the relative insensitivity of pyruvnte oxidation to malonate cannot be accounted for by the action of yeast carboxylase, at least at, pH 4.6, since at this pH the enzyme is not active on exogenous pyruvate (19, 2, 20). DISCUSSION
The citric acid cycle is the mechanism that gives a better fit for the distribution of Cl* in succinic acid formed by baker’s yeast from acetateI-C14, acetate-2-C14, and pyruvate-2-C’*, as well as for the distribution of Cl4 from Cl402 fixed during acetaldehyde, acetate, pyruvate, or glucose oxidation (21, 22). The “glyoxylate bypass” (23) whose existence in baker’s yeast is made likely by the presence of isocitritase (24) and other evidence (25) can be ruled out as a main path of substrate oxidation on account of the strong labeling of glutamic acid after the fixation of C’*02 (21, 22) or the oxidation of acetate-C’* [(26) and our own unpublished observations]. Further, glyoxylic acid does not, appear in significant proportions in the chromatograms of yeast extracts after fixation of Cl*02 (22) or the oxidation of acetate-C’* (unpublished observations) .
462
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The competitive inhibition of yeast succinic dehydrogenase by malonate (l&27) explains, according to the cycle model, the two actions of malonate on the oxidation of pyruvate, fumarate, and E-malate, namely, inhibition of the substrate oxidation and accumulation of succinic acid. It is noteworthy that the lack of action of malonate on the oxidation of fumarate reported in Table IX is quite compatible with the inhibition shown in Table VI, as in this case the malonate concentration was higher. The formation of succinic acid from fumarate and I-malate implies, according to the cycle model, oxidation to oxalacetate, successive decarboxylation of the latter to pyruvate and acetyl-CoA, synthesis of citrate from acetyl-CoA and a second molecule of oxalacetate, and oxidation of citrate up to succinate. The enzymes catalyzing the respective reactions are known in baker’s yeast, including oxalacetate carboxylase (28). With pyruvate a similar set of reactions is in operation and, in addition, the carboxylation of pyruvate to oxalacetate. The latter reaction is proved by (a) the significant labeling of the 2-carbon atoms of succinic acid formed from pyruvate-2-C14; (b) the labeling of the 4-carbon atom of aspartic acid after fixation of C1402 (22) ; and (c) the presence of phosphopyruvic carboxylase and pyruvic phosphokinase in cell-free extracts of baker’s yeast (observations with J. Cannata to be published). The action of malonate on acetate oxidation can be also explained by competitive inhibition of succinic dehydrogenase, for, according to the cycle model, the inhibition of this enzyme prevents the regeneration of the C4 acetyl acceptor. Since endogenous oxalacetate can be decomposed by oxalacetate carboxylase, and no exogenous precursor of oxalacetate is available, comparatively smaller concentrations of malonate can block acetate oxidation and consequently inhibit the formation of succinic acid. However, the inhibition of succinic dehydrogenase may not be the only mechanism of malonate effect. The lack of action of fumarate and Z-malate on yeast preparations inhibited by malonate (Krebs et al., Linnane and Still, and our own observations) leads us to assume that malonate may inhibit acetate oxidation at a site different from succinic dehydrogenase, e.g., acetate activation. In connection with this, it may be mentioned that in Pseudomonas JEuorescens (29) malonate is activated to malonyl-CoA by an enzyme similar to yeast acetokinase. If this reaction takes place in baker’s yeast, the lack of a malonyl-CoA decarboxylase should prevent, indirectly, the liberation of CoA required to activate acetate, and in this way acetate oxidation should be further inhibited by malonate.
SUCCINIC ACID FORMATION
463
ACKNOWLEDGMENTS We are grateful to Drs. A. S. Actis, F. L. Sacerdote, for their assistance in some of the experiments.
E. Rnmos, and M. Pigretti
SUMMARY 1. The distribution of Cl4 in succinic acid formed by baker’s yeast from acetate-l-W, acetate-2-C’*, and pyruvate-2-Cl4 fits the citric acid cycle model. 2. According to the cycle theory, malonate increases the synthesis of succinic acid from pyruvate, fumarate, and I-malate. 3. Malonate is most effective as inhibitor with acetate, but does not increase succinic acid formation from acetate. 4. The different action of malonate with pyruvate, fumarate, and I-malate on the one hand, and with acetate on the other, is due mainly to the possibility of the first forming oxalacetate. Evidence supporting the carboxylation of pyruvate is set forth.
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21. DAVIS, J. W., CHELDELIN, V. H., CHRISTENSEN, B. E., AND WANG, C. H., Biochim. et Biophys. Acta 21, 101 (1956). 22. STOPPANI, A. 0. M., DE FAVELUKES, S. L. S., CONCHES, L., AND SACERDOTE, F. L., Biochim. et Biophys. Acta 26, 443 (1957). 23. KORNBERT, H. L., AND MADSEN, N. B., Biochim. et Biophye. Acta24,651 (1957). 24. OLSON, J. A., Nature 174, 695 (1954). 25. BOLCATO, V., DE BERNARD, B., AND LEWIERO, G., Arch. Biochem. Biophys. 69, 372 (1957). 26. LABBE, R. F., THOMAS, R. C., CHELDELIN, V. H., CHRISTENSEN, B. E., AND WANG, C. II., J. Biol. Chem. 197, 655 (1952). 27. SINQER, T. P., MASSEY, V., AND KEARNEY, E. B., Arch. Biochem. Biophys. 69, 405 (1957). 28. NOSSAL, P. M., HANSEN, I. A., AND LADD, J. N., Biochim. et Biophys. Acta 23, 103 (1957). 29. HAYAISHI, O., J. Biol. Chem. 216, 125 (1955).