THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 245, No. 11, Issue of June 10, pp. 2937-2945, 19Xl

Printed in U.S.A.

Citrate Synthase

A REGULATORY ENZYME FROM RHODOPXEUDOMONAX CAPXULATA”

LEON EIDELS~ AND JACK PREISS

(Received for publication, January 12, 1970)

From the Department oj Biochemistry and Biophysics, University of California, Davis, California 95616

SUMMARY

Citrate synthase (citrate oxaloacetate-lyase (CoA-acetylat- ing), EC 4.1.3.7) is very active in Rhodopseudomonas capsula~a, a nonsulfur purple bacterium. This activity varies greatly from carbon source to carbon source. The highest specific activity for this enzyme was obtained in acetate and acetate-bicarbonate-grown cells (1.7 to 1.8 pmoles per mg of protein-min). Citrate synthase activity was Z-fold greater in cells grown aerobically in the dark than in cells grown semianaerobically in the light, when either malate or pyruvate was the carbon source. The specific activities of this enzyme from glucose dark-grown and light-grown cells, and from pyruvate dark-grown cells were similar, and they were about one-half the specific activity of the enzyme from acetate- grown cells.

Citrate synthase in this organism was found to be a regula- tory enzyme. DPNH was a strong inhibitor of the enzyme and AMP was found to relieve this inhibition. DPNH, at 0.1 mM, increased the So.5 value and the fi value (Hill constant) for acetyl-CoA. It also increased the S0.s value but did not change the 5 value for oxalacetate. Increasing amounts of DPNH increased the 6 value for AMP, the “de- inhibitor”. AMP, 0.46 mM, in the presence of DPNH, restored the So.5 and the ti values for acetyl-CoA to those obtained in the absence of effector molecules. AMP (0.46 mM) alone did not affect these values. Thus, AMP acts strictly as a deinhibitor of the DPNH effect with respect to acetyl-CoA. AMP, in the presence or absence of DPNH, decreased the S0.s value and the iz value for oxalacetate acting here both as a deinhibitor and as an activator. These results suggest that citrate synthase activity in R. capsulata is modulated both by the “energy charge” and the “reducing state” of the cell.

The presence of citrate synthase (citrate oxaloacetate-lyase

* This research was supported in part by United States Public Health Service Grant AI 05520 from the National Institutes of Health.

$ Present address, Department of Microbiology, University of Connecticut School of Medicine, Farmington, Connecticut 06032. The material presented herein was submitted as part of a thesis in partial fulfillment of the requirements for the degree of Doctor of Philosophy in December 1969 (1).

(CoA-acetylating), EC 4.1.3.7) in a large number of bacteria has been reported (2). The enzymes from bovine heart, bovine liver, and Escherichiu coli were reported by Jangaard, Unkeless, and Atkinson (3) to be inhibited by ATP. However, Weitzman (4) reported that citrate synthase from E. coli was rather in- sensitive to ATP inhibition but was quite sensitive to DPNH inhibition (0.1 mM produced 90% inhibition). Subsequently, he reported that inhibition by I mM DPNH was completely over- come by 0.3 mM AMP (5). More recently, Weitzman and Jones (2) studied the effect of DPNH on the citrate synthase activity of a variety of bacteria. They found that the enzyme from gram-positive organisms was not inhibited by DPNH while the enzyme from gram-negative organisms was. More- over, the gram-negative organisms could be subdivided into two groups; in one group (strict aerobes) the inhibition of citrate synthase by DPNH (0.8 mM) was relieved by AMP (0.5 n@ while in the other group (facultative anaerobes) AMP had no effect.

Rhodopseudomonas capsulata is a photosynthetic, nonsulfur, purple bacterium (Athiorhoduceae) capable of photoautotrophic and photoheterotrophic growth. It is also considered to be a facultative aerobe and can grow in the dark in the presence of organic compounds and oxygen (6). R. capsulata has tradi- tionally been grown both in the light and in the dark in a min- imal salts medium with malate as the sole carbon source (7). It has also been grown on glucose or pyruvate both in the light and in the dark, on acetate or acetate-bicarbonate in the dark, and on hydrogen-carbon dioxide in the light’ (7, 8). Recently we have shown that this organism metabolizes glucose almost exclusively via an inducible Entner-Doudoroff pathway.1 More- over, very low levels of phosphofructokinase and of B-phospho- gluconate dehydrogenase were found in R. capsdata suggesting that both the Embden-Meyerhof pathway and the hexosemono- phosphate shunt are of minor physiological importance in this organism. Although the presence of citrate synthase in R. cupsuZufa has been reported (9), the regulation of the enzyme in this organism had not been studied.

At present there is no detailed report on the kinetics of a citrate synthase that is inhibited by DPNH and whose inhibi- tion is relieved by AMP.

The present communication reports the levels of citrate syn- thase from R. capaulata grown under a variety of conditions, as well as the detailed kinetic properties of this enzyme which is specifically inhibited by DPNH and deinhibited by AMP. The

1 L. Eidels and J. Preiss, unpublished results.

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2938 Regulation of Citrate Xynthase Vol. 245, No. 11

significance of these observations with respect to the regulation of the flow of carbon in this organism is discussed.

EXPERIMENTAL PROCEDURE

The organism was obtained from Dr. H. Gest of the Depart- ment of Bacteriology, Indiana University, and was maintained in the laboratory as stab cultures (10). Cultures were grown semianaerobically in the light or aerobically in the dark in New Brunswick fermentors in 12 liters of the synthetic medium of Ormerod, Ormerod, and Gest (7). The cultures were grown at 31 to 33” until they reached stationary phase, at which time they were harvested at 4” in a Sharples continuous flow centri- fuge. The cells were stored at -12”.

Grade I protamine sulfate, Tris, DPN+, and DPNH were obtained from Sigma. TPN+, acetyl-CoA, and HS-CoA2 were obtained from P-L Biochemicals. Oxalacetate, ADP, and ATP were obtained from Calbiochem. AMP was obtained from Schwarz BioResearch, and DTNB was obtained from Aldrich. DEAE-Cellulose (DE 52) was obtained from Whatman, and DEAE-Sephadex A-50 from Pharmacia. All other chemicals were obtained from commercial sources.

The following Solvent System A was used for thin layer chro- matography : water-saturated ether-formic acid (7 : 1).

Protein concentration was determined by the method of Lowry et al. (11). Nucleotides and pyridine nucleotides were assayed spectrally. DTNB solutions were prepared in 0.1 M

Tris-HCl (pH 8.0) and assayed spectrally (12). Acetyl-CoA was assayed both by its ultraviolet absorbance and by a coupled citrate synthase-DTNB assay, and oxalacetate concentration was determined with malate dehydrogenase and DPNH.

Assay of Citrate Synthase-Citrate synthase was assayed by measuring the rate of oxalacetate-dependent production of free HS-CoA from acetyl-CoA. The HS-CoA produced was coupled to DTNB reduction, and the progress of the reaction was fol- lowed by measuring the increase in absorbance at 412 rnp (13). The reaction mixture contained per ml: 100 pmoles of Tris- HCl (pH 8.0), 0.16 to 0.20 pmole of DTNB, 1 pmole of oxalace- tate, and 0.18 pmole of acetyl-CoA (this concentration of acetyl- CoA does not saturate the enzyme). A reaction mixture minus oxalacetate was always included to account for nonspecific “de- acylase” activity. One millimolar solution of reduced DTNB gives an absorbance change of 13.6 at 412 rnp (14). The activ- ity of the enzyme was calculated as micromoles of HS-CoA formed per mg of protein-min from the initial rate. All assays were carried out with a Cary model 14 recording spectrophotom- eter.

A two-step assay, in which the reaction mixture was first in- cubated for 10 min, in the absence of the chromogen, stopped by boiling for 1 min, and the HS-CoA formed detected with DTNB, was used to show that there is no inhibition of the enzyme by DTNB in the continuous assay.

RESULTS

Activity of Enzyme under Varying Growth Conditions

Citrate synthase has the highest specific activity of any en- zyme studied in R. capsu1ata.l The specific activity of citrate synthase varies greatly from carbon source to carbon source

2 The abbreviations used are: HS-CoA , reduced form of coen- zyme A, DTNB, 5,5’-dithiobis(2-nitrobenzoic acid).

(Table I). The highest specific activity for this enzyme is ob- tained in acetate-bicarbonate and acetate-grown cells. The specific activities of this enzyme from glucose, dark-grown and light-grown cells, and from pyruvate, dark-grown cells are sim- ilar, and they are about one-half the specific activity of the en- zyme from acetate-grown cells. The specific activity of this enzyme in pyruvate, light-grown cells is one-half of the value obtained for pyruvate, dark-grown cells. With malate as carbon source, the activity of this enzyme in dark-grown cells is 2-fold greater than the specific activity of this enzyme in the light- grown cells. Malate, light-grown cells, and hydrogen-carbon dioxide, light-grown cells have the lowest citrate synthase spe- cific activity.

PurQkation of Citrate Xynthase

Preparation of Cell-free Extract-Acetate-, aerobically dark- grown cells, 10 g, were resuspended in 100 ml of 0.05 M Tris- HCl (pH 8.0) buffer and exposed to sonic oscillation with a Biosonik III in two 50-ml fractions for a total of 5 min with intermittent cooling in order to keep the temperature below 15”. Unbroken cells and cell debris were removed by centrifuga- tion at 48,200 x g at 4” for 30 min in a Sorvall centrifuge. Sub- sequent steps were carried out at 4”, unless otherwise specified.

Heat Treatment-The enzyme is relatively heat stable, and can be heated to 50” for 5 min with very little loss in activity. In the presence of AMP, a deinhibitor of the enzyme, it can be heated to 60” with no loss in activity. The Sorvall supernatant fluid was made 0.01 M with respect to AMP, transferred to a 500-ml Erlenmeyer flask, and heated to 56” in a 60” water bath (12 liters). After reaching 56” (about 5 min), the solution was kept at this temperature for 5 min. The solution was quickly cooled to 5” and centrifuged at 48,200 x g for 20 min. The heated supernatant fluid contained 94% of the original activity. The enzyme was kept frozen overnight with no loss in activity.

Protumine Sulfate Fractionation-A volume of 0.10 ml of a 1 To protamine sulfate solution was added per ml of heated super- natant fluid. After mixing and centrifuging, the protamine sulfate supernatant fluid was obtained. It contained 86% of the original activity.

Ammonium Xuljate Fractionation-The protamine sulfate supernatant fluid was brought to 40y0 saturation with solid ammonium sulfate and then centrifuged for 10 min at 48,200 X g. The resulting supernatant fluid was brought to 60% satura- tion with solid ammonium sulfate. After centrifugation, the precipitate obtained was resuspended in a minimal volume of 0.05 M Tris-HCl (pH 8.0) buffer and dialyzed overnight in 1000 ml of the same buffer. The dialyzed enzyme contained 68% of the original activity.

DEAE-Cellulose Chromatography-A column, 1 x 30 cm, was packed with DEAE-cellulose (DE 52) to a height of 13 cm. The column was washed with 2 resin bed volumes of 0.05 M

Tris-HCl (pH 8.0) buffer. The dialyzed ammonium sulfate fraction was adsorbed onto the column. The column was washed with 1 resin bed volume of buffer before starting the gradient which contained 250 ml of 0.05 M Tris-HCl (pH 8.0) buffer in the mixing chamber and 250 ml of the same buffer containing 0.5 M KC1 in the reservoir chamber. Protein was monitored by its absorbance at 280 rnp. The peak activity fractions were pooled, concentrated by precipitation with solid ammonium sulfate, and dialyzed overnight in 0.05 M Tris-HCl

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TABLE I TABLE II

Inhibition of deinhibition of cifrate synihase from R. capsulata grown under different growth condirions

Assay conditions were the same as described under “Experi-

mental Procedure,” except that DPNH or DPNH plus AMP were added as indicated in the table. The enzyme extracts were

prepared by suspending 0.6 g of cells, wet weight, in 12 ml of 0.05 M glycylglycine (pH 7.0) and exposing them to sonic oscillation with a Biosonik III probe for 3 min with cooling. The sonic extracts were centrifuged at 104,000 X g for I hour in a Spinco model L ultracentrifuge, and the resulting supernatant fluids

were used as the enzyme source. Activity is in micromoles of HS-CoA formed per mg of protein-min.

Summary of purijication procedure for citrate synthase from R. capsulata

One unit equals 1 pmole of HS-CoA formed per min under the

conditions described under “Experimental Procedure” (i.e. at 0.18 mM acetyl-CoA).

step Protein

w/ml

1. Sorvall superna-

tant fluid.. 35 2. Heated superna-

tant fluid. . 35

3. Protamine sulfate supernatant fluid 36

4. Ammonium sulfate

precipitate.. 3. 5. DEAE-Cellulose

column . 3.

6. DEAE Sephadex column. . 2.

12.6 562 100

8.5 525 93.5

3.G4 485 86.3

18.45 379 67.5

2.71 263 46.8

1.50 85 15.1

Growth conditions Activity + DPNH

Glucose, darka. ........ 0.793

Glucose, light. ......... 0.714

Malate, dark ........... 0.467 Malate, light .......... 0.192

Pyruvate, dark ....... 0.711 Pyruvate, light. ....... 0.454 Acetate + HCOz-, dark 1.80

Acetate, dark .......... 1.67 H2 + COZ, light ........ 0.259

Percentage of activity

0.1 0.19 InY nud

Yo Yo

85a 37 20 31

25 19

21 19 5

27 21 12

+AMP, 0.46 IXIM

114

114

116 99

+DPNH (0.19 InM)

I -

YO

98

103

103

0 This particular extract had a highly actJive DPNH oxidase

activitg.

(pH 8.0) buffer. This fraction contained 47% of the original activity.

DEAE-Xephadex Chromatography-A column, 0.5 X 10 cm, was packed with DEAE-Sephadex A-50 to a height of 8.5 cm. The gradient utilized contained 50 ml of 0.05 M Tris-HCl (pH 8.0) in the mixing chamber and 50 ml of 0.05 M Tris-HCl (pH 7.0) containing 0.5 M KC1 in the reservoir chamber. Protein was monitored by its absorbance at 280 rnp. The peak activity fractions were pooled, concentrated by precipitation with solid ammonium sulfate, and dialyzed overnight in 0.05 M Tris-HCl (pH 8.0) buffer. This fraction contained 15% of the original activity. This last step gave only a small increase in the puri- fication. It reduced considerably the endogenous “deacylase” activity. The deacylase activity was about 7% of the citrate synthase activity. This fraction is very stable when kept frozen and is free from the following activities: malate dehydrogenase, citrate lyase, and aconitase. Table II is a summary of the procedure used for the partial purification of citrate synthase from R. capsulata.

Characterization of Product and Xtoichiomety

of Reaction Catalyzed by Citrate Synthase

A reaction mixture containing acetylJ4C-CoA and nonradio- active oxalacetate was incubated with 21-fold purified enzyme for 5 min, and the reaction was stopped by heating. A reaction mixture containing no oxalacetate was run as a control. The reaction mixtures were spotted on silica gel-coated paper strips and developed in Solvent System A. The strips were monitored for radioactivity and sprayed with bromphenol blue (0.3%)-

Total

units

Specific activity

1.34

1.71

3.65

6.65

24.9

28.3”

Fold

1.3

2.7

5.0

18.6

21.1

G At saturating concentrations of acetyl-CoA (2.2 mM) the

specific activity of the enzyme was 45 pmoles of HS-Cob formed per mg of protein-min.

methyl red (0.1%) (in 95% ethanol) solution to detect organic acids. Unreacted acetyl-CoA remains at the origin, and acetate travels with the solvent front in this system. A new radioactive spot appeared in the strip from the complete reaction mixture and was absent in the strip from the reaction mixture containing no oxalacetate. The relative mobility of this spot was similar to the relative mobility of citrate.

The definite characterization of citrate as the actual product formed in the presence of purified citrate synthase was carried out enzymatically. Two reaction mixtures were prepared, each

containing 100 pmoles of Tris-HCl (pH 8.0), 0.2 pmole of oxal- acetate, and 0.178 pmole of acetyl-CoA in a final volume of 1 ml. To Reaction Mixture A, purified citrate synthase (0.9 pg) was added in such an amount that approximately 5Ooj, of the oxalacetate added would be utilized in about 10 to 12 min. To Reaction Mixture B, no citrate synthase was added. After 11 min at room temperature, both reaction mixtures were placed in a boiling water bath for 90 sec. All succeeding steps were the same for both reaction mixtures. An aliquot, 0.5 ml, was withdrawn and added to a l-ml cuvette containing 44 pmoles of triethanolamine (pH 7.6) buffer, 0.08 mg of ZnCL, and 0.2 pmole of DPNH in a final volume of 1 ml. Malate dehydro- genase (Boehringer, 720 units per mg, 5 mg per ml) (diluted 1 :lOO), 0.010 ml, was added, to assay for the remaining oxal- acetate, and the reaction followed in a Cary model 14 recording spectrophotometer at 340 m,u. Fig. 1 shows the change in absorbance at 340 rnh with time. After all the oxalacetate present was reduced, lactate dehydrogenase (Sigma, Type I, 52 units per mg, 25 mg per ml) (diluted 1:25), 0.010 ml, was added, to assay for pyruvate present in the oxalacetate solution, or formed, or both, during the incubation and heating periods. The reaction was followed until no further oxidation of DPNH was observed. At this point, 0.010 ml of citrate lyase (Boeh- ringer, 9.8 units per ml; free of DPNH oxidase activity and free of isocitrate dehydrogenase activity) was added, to assay for citrate formed, and the reaction followed. Reaction Mixture B

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2940 Regulation of Citrate Xynthase Vol. 245, No. 11

A = COMPLETE

B = -CITRATE SYNTHASE

k- + MDH + CITRATE LYASE

ILDH

Tlt.9ELMIN.l

FIG. 1. Characterization of the product. of citrate synthase from R. capsulata. Trace of chart from Cary model 14 recording spec- trophotometer, optical density at 340 rnp versus time. The condi- tions of the experiment are given under “Results.” A is the re- action mixture containing 0.9 pg of citrate synthase (specific activity, 45), B is the reaction mixture containing no citrate synthase. MDH, malate dehydrogenase; LDH, lactate dehydro- genase.

showed no further decrease in absorbance at 340 rnp. The complete Reaction Mixture A showed a further decrease in absorbance at 340 rnp caused by the citrate present being cleaved to oxalacetate (and acetate), and the oxalacetate in turn being reduced by DPNH to malate. From the decrease in absorbance, it was calculated that 0.087 pmole of citrate had been formed per original (1 ml) Reaction Mixture A.

Another aliquot, 0.3 ml, of the heated reaction mixtures was withdrawn and added to a l-ml cuvette containing 75 bmoles of Tris-HCl (pH 8.0) and 0.16 pmole of DTNB in a total volume of 1 ml to assay for the I-IS-CoA formed. It was calculated that in A, 0.081 pmole of HS-CoA had been formed per original (1 ml) reaction mixture, while in 13, 0.005 pmole of HS-CoA had been formed nonenzymatically.

It can thus be concluded that 1 pmole of oxalacetate reacts with 1 pmole of acetyl-CoA to yield 1 pmole of citrate and 1 pmole of HS-CoA.

Inhibition and Deinhibition of Citrate Synthasc

DPNH inhibits citrate synthase at relatively low concentra- tions and this inhibition is completely relieved by 5’-AMP (Table I). A variety of compounds were tested for their effect on the activity of citrate synthase. At a concentration of 1 rnivr the following compounds neither activate nor inhibit: fruc- tose-6-P, fructose-di-P, 2-keto-3-deoxy-6-phosphogluconate, py- ruvate, TPNH, ADP, and ATP. The following compounds, at concentrations of 0.5 and 5 InM, neither activate nor inhibit and do not relieve the inhibition by DPNH: or-keto-glutarate, L-

glutamate, fumarate, L-aspartate, n-malate, succinate, and cis-aconitate. At a concentration of 1 mM, nn-isocitrate gives a 1.3-fold activation, but, does not relieve the inhibition by DPNH. KC1 at a concentration of 0.1 M gives a 1.3- to IA-fold activation (at 0.18 InM acetyl-CoA and 1 mM oxalacetate), and at this concentration also relieves the inhibition by 0.1 mM

DPNH. Inhibition and Deinhibition of Enzyme under Varying Growth

Conditions-Table I shows the inhibition by DPNH and the

TABLE III Adenylates and pyridine nucleotides as deinhibitors of citrate

synfhase from R. caps&ala

Assay conditions were the same as described under “Experi- mental Procedure,” except that the effector molecules were added as indicated in the table. Citrate synthase (specific activity, 45),

0.06 to 0.15pg was added. The value of 100% activity corresponds to the velocity observed for the reaction mixture containing no effector molecules.

Percentage of activity in presence of 0.1 m&c DPNH

DPNf

- Deinhibitor concentration

% % % % 41 110 105 88

112 101 82 92 69 36

% 84

% 51

??cM

0.92-0.95 0.46-0.48

0.046-0.048 Percentage of activity in presence of 1 IIIM DPNH

I i

0.46-0.48 0.046-0.048

TPN+

deinhibition by 5’-AMP of citrate synthase activity in 104,000 x g supernatant fluids obtained from sonic extracts of cells grown under different growth conditions. Although the specific activ- ity of this enzyme varies depending on the conditions of growth, the activity in all cases is inhibited by DPNH, and 5’-AMP completely relieves this inhibition.

Specificity of Deinhibition-Table III shows the effectiveness of adenylates and of pyridine nucleotides as deinhibitors of DPNH inhibition of the 21-fold purified enzyme. At a con- centration of about 1 mM, both AMP and ADP completely relieve the inhibition by 0.1 mrvr DPNH, while ATP and DPN+ only partially relieve this inhibition; however, at a concentration of about 0.05 mM, AMP completely relieves the inhibition by 0.1 MM DPNH, while ADP only partially relieves this inhibition, and ATP does not relieve it at all. When the concentration of DPNH is increased to 1 InM, 0.5 mM AMP completely relieves this inhibition, 0.5 mM ADP only partially relieves it, and 0.5 mrvr ATP has no effect. At the same concentration of DPNH, 0.05 mM AMP only partially relieves this inhibition and 0.05 mM ADP or 0.05 InM ATP have no effect.

Other nucleoside monophosphates and analogues of 5’-AMP were also tested as deinhibitors. Table IV shows the results obtained. Of those tested only 2’-AMP relieves the inhibition by DPNH. At a concentration of 0.5 mM, both 2’-AMP and 5’-,4MP completely relieve the inhibition by 0.1 InM DPNH; however, at a concentration of 0.05 mM, 5’-AMP completely relieves the inhibition by 0.1 InM DPNH, while 2’-AMP only partially relieves this inhibition. When the concentration of DPNH is increased to 0.5 nnw, and the nucleoside monophos- phate concentration is kept at 0.5 mM, 5’-AMP completely relieves this inhibition while 2’-AMP only partially relieves it.

Kinetic Parameters

All the kinetic studies were carried out within the range of enzyme linearity.

Effect of Acetyl-CoA Concentration-Fig. 2 shows the depend- ence of citrate synthase activity on the concentra.tion of acetyl- CoA in the presence and absence of the enzyme’s effector mol-

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TABLE IV 1st A

Nucleosides monophosphates and other analogues as deinhibitors of

citrate synthase from R. capsulata

Assay conditions were the same as described under “Experi-

mental Procedure,” except that the effector molecules were added as indicated in the table. Citrate synthase (specific activit,y, 45)) 0.06 to 0.15 pg was added. The value of 100% activit,y corresponds

to the velocity observed for the reaction mixture containing no effector molecules

+O.l nni DPNH +O.S ITIM DPNH

Compound

No addition 5’-AMP

Adenine Adenosine 2’-AMP

3’-AMP 5’.CMP

5’-GMP 5’-UMP 5’-TMP Cyclic 3’,5’-AMP

Deinhibitor oncentratior

?nM

0.46

0.046 0.49 0.44 0.48

0.048 0.51 0.51

0.51 0.52 0.45

0.45 i

Percentage of activity

% 27

116

101 27 27

106

41 44

28 32 25 21

33

Deinhibitor Percentage oncentratior of activity

%

0.46 96

0.48 46

FIG. 2. Dependence of citrate synthase activity on the concen- tration of acetyl-CoA in the presence and absence of the enzyme’s effector molecules. The conditions of the assay were those given under “Experimental Procedure,” except that the concentration of acetyl-CoA was varied, and either DPNH or AMP or both were added as indicated in the figure; 0.06 to 0.15 pg of citrate synthase (specific activity, 45) was added. The inset is a plot of log v/ v/Vmitr - I’ against the log of acetyl-CoA (AC-CoA) concentra- tion. Ii,,,,, was taken as 45 rmoles of HS-CoA formed per mg of protein-min for all four cases.

ecules. DPNH, 0.1 mM, increases the concentration of

acetyl-CoA required for one-half maximal velocity (S,& from 0.19 to 0.58 mM. The deinhibitor AMP, 0.46 mM, in the pres- ence of 0.1 mM DPNH, restores the SO.s value for acetyl-CoA to that of the control in which no effector molecules are included. AMP (0.46 mM) alone does not alter this parameter. The inset

shows the Hill plots obtained. DPNH increases the fi value for

v AM.? 646mM

A / +DPNH. O.lmM

0 02 0.6 [~XAL~ACETATE].

1.0 d

B

%.s n ItIM)

o -DPNH,-AMP 00214 ,.09

A +DPNH.O.lmM 0055 102 +IO-‘+AMF:046mM +DPNH,OlmM 00089 082

0 ' -1.0 I.0 2.0

log COAA.~MI

FIG. 3. Dependence of cit,rate synthase activity on the concen- tration of oxaloacetate in the presence and absence of the enzyme’s effector molecules. The conditions of the assay were those given under “Experimental Procedure,” except that the concentration of oxalacetate was varied and DPNH or DPNH plus AMP were added as indicated in the figure; 0.06 to 0.15 rg of citrate synthase (specific activity, 45) was added. The inset shows the correspond- ing S/v versus S plot. B is a plot of log v/V,,, - v against the log of oxalacetate (OAA) concentration. V,,, is in micromoles of HS-CoA per mg of protein-min.

acetyl-CoA from 0.99 to 1.47. AMP plus DPNH, or AMP alone, yield the same fi value for acetyl-CoA as the control in which no effector molecules are included. AMP thus acts strictly as a deinhibitor of the DPNH inhibition, since it reverses the effect of DPNH on the S0.j and fi values for acetyl-CoA to the values observed in the absence of the effector molecules.

The activity at saturating acetyl-CoA concentration, 2.26 rnbr, is 1.6-fold greater than at 0.18 mM, at which concentration all other work was performed.

E$ect of Oxalacetate Concentration-Fig. 3A shows the de- pendence of citrate synthase activity on the concentration of oxalacetate at 0.18 mM acetyl-CoA, in the presence and absence of the enzyme’s effector molecules. The inset shows the X/v versus S plots. Fig. 3B shows the corresponding Hill plots. DPNH at a concentration of 0.1 mM decreases the Vmax by 70%, increases the S0.s for oxalacetate from 0.021 to 0.055 mM, and does not significantly change the ti value for oxalacetate. AMP, 0.46 mM, in the presence of 0.1 mM DPNH, restores V,,,

to the control value obtained in the absence of effector mole-

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2942 Regulation of Citrate Xynthase Vol. 245, No. 11

cules, and decreases the S0.s for oxalacetate from 0.021 to 0.009 ~hl. AMP (0.46 mM) alone (not shown in Fig. 3) yields no change in Vm,,, and decreases the So.s for oxalacetate from 0.021 to 0.005 mkt. Thus, AMP not only acts as a deinhibitor of the

- y0344mM AC-&+ 0.41

5mM AC-CoA+0.46mM AMP ) 4

5mM AMP IO.5 n

QmoleJ/m~mirl (mM)

0026 1.22

0074 I I6

-2 log &NH]

A

20.115mM AC&A r;r

0

I 0 0.5 1.0 1.5 20

[DPNH]. mM

FIG. 4. The effect of DPNH concentration on the activity of citrate synthase in the presence and absence of AMP. The condi- tions of the assay were those given under “Experimental Proce- dure,” except that the concentration of DPNH was varied and the levels of aeetyl-CoA (AC-CoA) and of AMP were as indicated; 0.06 to 0.15 pg of citrate synthase (specific activity, 45) was added. The values for 100% activity (micromoles of HS-CoA formed per mg of protein-min) in the presence of 0.115 mM acetyl-CoA was 18 and in the presence of 0.344 IIIM acetyl-CoA was 29. The inset is a plot of log vi/v, - vi against the log of DPNH concentration. o0 is the observed velocity in the absence of DPNH and 2ri is the observed velocity at a given concentration of DPNH.

a DPN++ DPNH=0.2m

o DPN++ DPNH=I.Oml

q DPN++DPNH= I.Omll + AMF: 20pU

I I 0 50% 100%

% Reduced Pyridine Nucleotide

FIG. 5. The effect of the percentage of DPNH, at fixed levels of total diphosphopyridine nucleotides, in the presence and absence of AMP. The conditions of the assay were those given under “Experimental Procedure,” except that DPN+, DPNH, and AMP were also added at the concentrations indicated in the figure; 0.06 to 0.15 pg of citrate synthase (specific activity, 45) was added. The value of 100% activity corresponds to the velocity observed for the reaction mixture containing all of the diphosphopyridine nucleotide in the DPN+ form (i.e. 0% reduced diphosphopyridine nucleotide).

inhibition by DPNH with respect to oxalacetate but also in- creases the apparent affinity of the enzyme for oxalacetate.

Effect of DPNH Concentration-Fig. 4 shows the effect of DPNH concentration on the citrate synthase activity at two concentrations of acetyl-CoA and in the presence and absence of the deinhibitor AMP. At both 0.115 and at 0.344 mM acetyl- CoA concentrations, in the presence of 0.46 mM AMP, no in- hibition is observed even at DPNH concentrations as high as 2.15 mM. In the absence of ,4MP, at 0.115 rnhf acetyl-CoA the concentration of DPNH required for one-half maximal in- hibition (10.5) is 0.026 nnvr. When the acetyl-Co-4 concentration is increased to 0.344 mM, the lo.5 for DPNH is increased to 0.074 mM. The inset shows the Hill plots obtained; at both acetyl- CoA concentrations the fi value for DPNH is 1.2.

Fig. 5 shows the effect of varying the DPN+:DPNH ratio at two different total diphosphopyridine nucleotide concentrations on the citrate synthase activity. At a total diphosphopyridine nucleotide concentration of 0.2 mM, 50% inhibition occurs when the percentage of reduced diphosphopyridine nucleotide is 28%;

I II I I 50 10011 200 300 400 500

[AMP]. PM

DPNH A,, n ITIM) +MJ

0 0.1 4.27 1.52

q 049 26.4 1.77 A 0.98 50.8 1.91

-ID’ 0

I 1.0 2.0

log [AMP,@]

FIG. 6. The effect of AMP concentration on the activity ok citrate synthase in the presence of varying levels of DPNH. The conditions of the assay were those given under “Experimental Procedure,” except that AMP and DPNH were added at the concentrations indicated in the figure; 0.06 to 0.15 pg of citrate synthase (specific activity, 45) was added. The inset shows the corresponding reciprocal plot. B is a plot of log Av/Vmsx - v against the log of AMP concentration. Au is the increase in veloc- ity caused by the addition of AMP, i.e. the velocity obtained upon addition of a certain amount of AMP to the reaction mixture (with DPNH) minus the velocity of the reaction mixture (with DPNH) containing no AMP. V,,, is in micromoles of HS-CoA formed per mg of protein-min.

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Issue of June 10, 1970 L. Eidels and J. Preiss

TABLE V

Kinetic parameters of citrate synthase from R. capsdata

The specific activity of the enzyme was 45 pmoles of HS-CoA formed per mg of protein-min.

2943

Compound Conditions

Acetyl-CoA -AMP, -DPNH

+AMP, 0.40 mM +DPNH, 0.1 mivr

AMP, 0.46 mM +DPNH, 0.1 mM

Oxalacetatec

DPNH

AMPc

SO..? SO2 IO2 nb

-AMP, -DPNH +AMP, 0.46 mivr

+DPNH, 0.1 mM AMP, 0.46 mM

+DPNH, 0.1 mM

ma

0.020 0.007

0.048 0.010

?x.M

0.19 0.16

0.58 0.14

0.99

0.93

1.47 0.93

0.021 1.09 0.005 0.83 0.055 1.02 0.009 0.82

+O. 115 m&r acetyl-CoA 0.026 1.22

+O. 344 mM acetyl-CoA 0.074 1.16

+DPNH, 0.10 mM 4.27 1.52

+DPNH, 0.49 rnnf 26.4 1.77 +DPNH, 0.98 mM 50.8 1.91

a So.5 values were obtained from S/v versus S plots (Fig. 3A). * Values obtained from Hill plots (Figs. 2, 3B, 4, and 6B). c At 0.18 mM acetyl-CoA.

while at a total diphosphopyridine nucleotide concentration of 1 mM, 50% inhibition occurs when the percentage of reduced diphosphopyridine nucleotide is 17.5%. The presence of low levels of AMP (20 PM) at a total diphosphopyridine nucleotide concentration of 1 mM increases the percentage of reduced di- phosphopyridine nucleotide required to yield 50% inhibition from 17.5 to 54%.

Effect of 5’-AMP Concentration-Fig. 6A shows the effect of 5’-AMP concentration, in the presence of three different levels of DPNH, on the citrate synthase activity. Increasing amounts of DPNH increase the degree of sigmoidicity of the deinhibitor saturation curve. At low 5’-AMP concentrations, the higher the DPNH concentration the greater the degree of inhibition;

TABLE VI

Inhibition ancl deinhibition of citrate synthase from R. rubrum grown

under different growth conditions

Assay conditions were the same as described under “Experi- mental Procedure,” except that DPNH or DPNH plus AMP were

added as indicated in the table. The enzyme extracts were prepared by suspending 0.6 g of cells, wet weight, in 12 ml of 0.05 M glycylglycine (pH 7.0) and exposing them to sonic oscilla-

tion with a Biosonik III probe for 3 min with cooling. The sonic extracts were centrifuged at 104,000 X g for 1 hour in a Spinco model L ultracentrifuge, and the resulting supernatant fluids were used as the enzyme source. Activity is in micromoles of

HS-CoAformed per mg of protein-min.

however, increasing the 5’-AMP concentration completely over- comes the inhibition by DPNH. The l/Au versus l/S plot shown in the inset is nonlinear. Fig. 6B shows the corresponding

Growth conditions

Hill plots. The concentration of 5’-AMP required for one-half maximal activation (deinhibition) is 4.27 pM in the presence of 0.1 mM DPNH, 26.4 j.&M in the presence of 0.49 mM DPNH, and Malate, light 0.153

50.8 ~.LM in the presence of 0.98 rnN DPNH. The A value for Malate, dark. . 0.423

5’-AMP is 1.52 in the presence of 0.1 mu DPNH, 1.77 in the Hz + COz, light. 0.206

presence of 0.49 InM DPNH, and 1.91 in the presence of 0.98 mM DPNH.

Activity

+AMP (0.46 my)

+AMP (0.46 maa),

0.186 0.010 0.186

0.470 0.094 0.470 0.206 0.047 0.205

The kinetic parameters of citrate synthase from R. capsulata DISCUSSION

are summarized in Table V.

Citrate Xynthase from Rhodospirillum rubrum

Table VI shows the specific activity of citrate synthase from R. rubrum grown on malate both in the dark and in the light and on hydrogen-carbon dioxide in the light. The citrate syn- thase from R. rubrum, a photosynthetic bacterium that does not grow on glucose, is also inhibited by DPNH, and AMP com- pletely relieves this inhibition.

The specific activity of citrate synthase was found to be dependent on the nature of the carbon source and was 2-fold greater in cells grown aerobically in the dark than in cells grown semianaerobically in the light, when either malate or pyruvate were the carbon sources (Table I). With R. r&rum grown on malate as carbon source, the specific activity in the dark-grown cells was about 3-fold greater than the specific activity in the light-grown cells (Table VI). Anderson and Fuller (15) obtained similar results with citrate synthase from R. rubrum grown on

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2944 Regulation of Citrate Xynthase Vol. 245, No. 11

malate in a minimal salts medium. These observations would be consistent with a more active tricarboxylic acid cycle in the dark than in the light. Since an important function of the tricarboxylic acid cycle is to produce reducing power, the ac- tivity of this cycle would be expected to be decreased in the light where reducing power could also be obtained from photo- synthesis in a photosynthetic bacterium like R. capsulata. This regulation could be accomplished by controlling the amount of enzyme synthesized, or by regulating the activity of the enzyme synthesized, or both. Although the amount of citrate synthase in the light was lower than in the dark (on malate or pyruvate), it still showed the highest activity of any of the enzymes in- vestigated in this organism.’ In light of this, the allosteric regulation of the activity of this enzyme becomes of great interest.

Citrate synthase in R. capsulata catalyzes a physiologically irreversible reaction and it can be considered as the first enzyme in the tricarboxylic acid cycle. Citrate synthase in this orga- nism was found to be a regulatory enzyme. DPNH was a strong inhibitor of this enzyme and AMP was found to be the best deinhibitor.

Although the levels of DPNH and DPN+ in R. capsulata are not known, the levels of these cofactors have been measured in Escherichia coli I) (16); the concentration of DPNH in glucose- grown cells was 2.05 InM and in succinate-grown cells it was 1.2 mM, and the concentration of DPN+ was 1.08 and 1.44 mM in glucose- and in succinate-grown cells, respectively. Jackson and Crofts (17) have shown that in R. rubrum under dark aerobic conditions, the total diphosphopyridine nucleotides were present as DPN’; while under light anaerobic conditions, 70% of the total diphosphopyridine nucleotide pool was in the form of DPNH and 30% in the form of DPN+.

Under conditions of “low energy charge” (i.e. low ATP: ADP + AMP ratio) (18) and “low reducing power” (i.e. low DPNH:DPN+ ratio) the activity of citrate synthase would be maximal resulting in an active tricarboxylic acid cycle. The increase in “reducing power” in the cell, caused by substrate oxidation in the dark or by photosynthesis in the light, with the concomitant increase in energy charge caused by an “excess of ATP production” over that utilized by the cell, would result in the inhibition of citrate synthase of R. capsulata. This inhibi- tion would decrease the activity of the tricarboxylic acid cycle.

Jangaard et al. (3) found ATP to be an inhibitor of citrate synthase from bovine liver, bovine heart, and E. co&. Weitz- man (4) found that the enzyme from E. coli was very sensitive to DPNH inhibition. oc-Ketoglutarate has been reported to also be an inhibitor of the enzyme from E. coli (19). Neither ATP nor oc-ketoglutarate inhibited the enzyme from R. capsulata under the conditions of the assay used. It should be pointed out that Jangaard et al. (3) showed that ATP inhibition of the E. coli enzyme was dependent on pH. Although a similar study was not made in the case of the enzyme from R. capsulata, the pH of the reaction mixtures in the present study (8.0) was the pH reported where the E. coli enzyme was most sensitive to ATP inhibition.

The finding of DPNH inhibition and AMP deinhibition of citrate synthase in R. capsulata is consistent with the observa- tions of Weitzman and Jones (2). They reported that the enzyme from gram-negative organisms was inhibited by DPNH. In this group, those organisms (strict aerobes) that do not me- tabolize glucose via the Embden-Meyerhof pathway but utilize

an alternate pathway (e.g. Entner-Doudoroff) were very sensitive to deinhibition by AMP, while those organisms (facultative anaerobes) that do metabolize glucose via the Embden-Meyerhof pathway were less sensitive to deinhibition by AMP. Organisms included in the former group like Pseudomonas sp. (20) and Xanthomonas sp. (21) metabolize glucose via the Entner-Doudo- roff pathway (22). R. capsulata, which also metabolizes glucose via the Entner-Doudoroff pathway but is not a strict aerobe,l would now be included in this group. E. coli was included in the latter group. The inclusion of E. coli citrate synthase in the AMP-insensitive group would appear to be inconsistent with Weitzman’s (5) previous report that the inhibition by DPNH (1 mM) in E. co& is “virtually completely overcome” by AMP (0.3 mM) under very similar conditions to those utilized in his more recent investigation (2) (i.e. DPNH, 0.8 mM; AMP, 0.5 mM). This discrepancy reported for E. coli should eventually be resolved when more complete kinetic analysis of the inhibi- tion-deinhibition relationship is undertaken for that enzyme.

Weitzman and Jones (2) concluded that organisms in which AMP (or ADP), acting as a “low energy signal,” activates the key glycolytic enzymes such as phosphofructokinase or pyruvate kinase, would not require a similar low energy signal at the level of their citrate sgnthase. On the other hand, organisms in which the Embden-Meyerhof pathway is absent or in which there is no regulation of its key enzymes would require a low energy signal to control the tricarboxylic acid cycle at the level of the entry to the cycle, namely citrate synthase. The latter appears to be the case with R. capsulata.

Weit,zman and Dunmore (23) found that cu-ketoglutarate (1 mM) only inhibited the cit,rate synthase of the Enterobacteriaceae. Wright, Maeba, and Sanwal (19) have shown that ar-ketogluta- rate (a tricarboxylic acid cycle intermediate which is not only used for energy generation but also for biosynthesis of glutamate) acted as a feedback inhibitor of citrate synthase from E. coli. The citrate synthase from R. capsulata was not inhibited by a-ketoglutarate. Citrate synthase activity is not only necessary for energy production but is also necessary for biosynthesis of the glutamate family of amino acids. In light of this, it is interesting to note that the enzyme from R. capsulata could not be completely inhibited by DPNH, a high energy signal (Fig. 4). The residual (noninhibitable) activity could be considered to always be active for biosynthetic purposes.

Rindt and Ohmann (24) have recently reported that in Rhodopseudomonas spheroides (an organism closely related to R. capsulata), ribulose-5-P kinase, a key enzyme of the reductive pentose-phosphate cycle, is activated by DPNH (the inhibitor of citrate synthase) and is inhibited by AMP (the deinhibitor of citrate synthase). This effector relationship is consistent with the findings reported here for R. capsulata. Under condi- tions of active growth in the light, the DPNH levels would be expected to increase while the AMP levels would be expected to decrease. This would lead to activation of ribulose-5-P kinase and inhibition of citrate synthase resulting in low tri- carboxylic acid cycle activity and high reductive pentose- phosphate cycle activity. The reciprocal relationship between citrate sgnthase and ribulose-5-P kinase in photosynthetic bacteria with respect to their modulation by the energy charge and the “reducing state” of the cell could be responsible for the regulation and interaction between two of the carbon path- ways present in these organisms.

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Leon Eidels and Jack PreissRHODOPSEUDOMONAS CAPSULATA

Citrate Synthase: A REGULATORY ENZYME FROM

1970, 245:2937-2945.J. Biol. Chem. 

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