THE .JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 239, No. 2, February 1964

fTintea in U.S.A.

Studies on Specific Enzyme Inhibitors

VI. CHARACTERIZATION AND MECHANISM OF ACTIOX OF THE ENZYME-INHIBITORY ISOMER OF MONOFLUOROCITRATE”

D. W. FAivsHIER,t L. K. GOTTWALD, AND E. KUN$

From the Departments oj” Pharmacology and Biochemistry, School 01 Medicine, and the Department of Pharmaceutical Chemistry, School oj Pharmacy, University of California, San Francisco 22, California

(Received for publication, June 5, 1963)

As an approach to the study of controlling mechanisms of enzyme activity in complex systems, we are in the process of determining the influence of relatively minor chemical altera- tions (e.g. exchange of one or more hydrogen by fluorine atoms) in well known enzyme substrate molecules on their reactivity with purified enzymes. The immediate purpose of these esperi- ments is to obtain specific inhibitors for certain key enzymes, which then are further studied in complex biochemical systems with the aid of these specific inhibitors (l-5). In the course of this work an unexpected observation was made: the enzymatic condensation of acet#yl coenzyme d with P-monofluoro-osaloace- tate yielded an isomer of monofluorocit,rate which did not inhibit aconitase (4). It seemed likely that this enzymatic reaction produced only one of the four isomers of monofluorocitrate. The elucidation of stereochemical and enzyme inhibitory rela- tionships of the remaining three isomers to this enzymatically synthesized fluorocitrate has been the subject of further expari-

ments. It will be shown in this paper that the aconitase- inhibitory effect of synthetic monofluorocitrate is actually due to one isomer, synthesized enzymatically from fluoroacetyl coenzyme A and oxaloacetate. The separation of diastereo- isomers of synthetic monofluorocitric acid and the comparison of chemical and enzyme-inhibitory properties of these with enzymatica,lly formed isomers considerably diminished uncer- tainties regarding the true structure and mode of action of the inhibitory species of monofluorocitric acid.

EXPERIMENTAL PROCEDURES AND RESULTS

Chemical Synthesis of Xonofiuorocitrate-Isolation and stereo- chemical identification as well as analyses of enzyme-inhibitory effects of all four isomers of synthetic monofluorocitric acid are prerequisite to the elucidation of biochemical mechanism of action of this substance. One of the obstacles in the process of

* Supported by grants of the cnited States Public Health Service (C-3211, C-4681), National Science Foundation (G-23739), and the American Heart Association, Inc. For Paper V in this series, see Kun, Gottwald, Fanshier, and Ayling (5) and for earlier papers, References 1 to 4.

t Part of the material reported in this paper has been accepted as a dissertation by the Graduate Division of the University of California (San Francisco Campus) in partial fulfillment of the requirements for Doctor of Philosophy in Comparative Phar- macology and Toxicology for D. W. F. (deceased Oct. 12, 1963).

$ To whom correspondence should be directed.

reaching this goal is the rather low yield of fluorocit.ric acid, when synthetized by the method of Rivett (6). Pure samples of both barium and cyclohexylamine salts were prepared earlier by this method of synthesis in our laboratory (4). A search for a synthetic procedure, which potentially could give better yields, was continued. The condensation of diethyl monofluoro-oxalo- acetate and malonic acid, a synthetic route mentioned by Brown and Saunders (7) but not described in technical detail, was found to produce significantly more and purer fluorocitric acid and therefore is described below.

Diethyl monofluoro-oxaloacetate (15.4 g, 0.075 mole) was added over a period of 30 minutes to a solution of malonic acid (8.0 g, 0.075 mole) in pyridine (20 ml). The reaction mixture was left at room temperature for 3 hours, then heated on a steam bath for 15 to 20 minutes until evolution of CO, ceased, cooled to room temperature, and acidified with slight excess of 10% HaSOk. Tire &ester of monofluorocitric acid was e&acted with

three to four portions of 50 ml of diethyl ether. This organic phase was dried with solid anhydrous Na2S04. After removal of ether under vacuum, 10 g of a brown oil (diethyl monofluoro- citric acid) remained. Analysis of infrared spectra revealed absorpt,ion maxima at 2.9, 3.35, 5.75, 8.25, and 11.6 p.

Triethyl monofluorocitrate was prepared from the diester as follows. Diethyl monofluorocitrate (10 g) was dissolved in 80 ml of absolute ethanol, to which 130 ml of benzene and 0.5 g of p-toluenesulfonic acid were added. This mixture was heated under reflus over a period of 20 to 30 hours. During this time almost 100 ml of distillate, containing ethanol, benzene, and water, were removed by means of a Dean-Stark trap. The sol- vent was further removed under vacuum, leaving 10.6 g of crude triet’hyl fluorocitrate. This material was dissolved in 50 ml of ether, washed with dilute (270) aqueous solution of n’azC03, and finally dried with anhydrous NaeS04. The product was distilled, and yielded purified triethyl monofluorocit’rate (6.3 g, 29%), b.p. 120” at 0.10 mm pressure, Tvhich gave the following ele- mentary analysis.

Calculated: C 49.07,, H 6.5Y,, F 6.5% Found : C 49.@&, H 6.3%, F 6.6%

Refractive index (vi’), 1.4380; infrared spectrum (neat) yielded absorption maxima at 2.9 p (strong) ; 3.37 p (strong); 5.75 p (st,rong); 6.8 p; 6.9 p; 7.28 p; 7.3 p (broad); 7.41 p; 8.4 p (strong, broad); 8.8 p; 9.1 p (strong); and 11.6 p (strong). The

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426 Specific Enzyme Inhibitors. VI

6.0 5.0 4.0 3.0 2.0 1.0 OF

TRAMETHYL

FIG. 1. The nuclear magnetic resonance spectrum was obtained with a Varian 60-megacycle V-43202B spectrophotometer with tetramethylsilane as an internal standard. Deuterochloroform was used as solvent. The position of the hydroxyl (OH) peak

TABLE I

Results of nuclear magnetic resonance analysis of triethyl ester of JIuorocitric acid

GUXlp Chemical shift (s)*

-CH, 1.3 (multiplet) -CH%- 2.95 (singlet) -CHz- (ethyl) 4.20 (multiplet) -OH 3.95 (singlet) -CHF- 4.6, 5.4 (both doublets)

* For nomenclature and interpretation, see Jackman (8).

NMR (nuclear magnetic resonance) spectrum of the ester is shown in Fig. 1. Results of NMR analysis are shown in Table I.

Alkaline hydrolysis of the triethyl ester of monofluorocitric acid and crystallization as the cyclohexylamine salt were carried out as described earlier (4). The net yield of this fluorocitrate synthesis was about a-fold of that obtained by the method of Rivett (6), if one accepts the yields reported by this author. In our hands the improvement in yield was closer to 5-fold.

Certain considerations, related to the stereochemical nature of this synthesis, are of importance in connection with identifica- tion of enzymatically formed fluorocitrate isomers. Both Rivett’s (6) method and the one described above would be expected, in accordance with Cram’s rule (9), to yield predomi- nantly a diastereoisomer in which fluorine and hydrosyl are on the same “side” of the molecule.1

1 The assignment of nn-erythro-fluorocitrate as “A” and DL- three-fluorocitrate as “B” has been adopted for simplicity in this paper. It should be emphasized that the exact stereochemical identification of “A” and “B” has not yet been carried out and

was established by adding deuterium oxide to the solution and observing the disappearance of the peak. (See raised portion of the spectrum.) The relative area under each peak was measured with the integral curve shown.

TABLE II

Elenaental composition of cyclohexylamine salts of $uorocitric acid

Calculated for monohydrate: CZ~H,,FN~O,.HZO..

Found : Unresolved mixture of Components

A+B . . Component A*. . Component B*.

I c %

54.85

55.2 9.3 7.7 55.9 9.0 7.1 53.7 8.8 7.3

H N

YO %

9.21 7.99

F

70

3.6

3.3

* The cyclohexylamine salts were prepared from the free acids, isolated and eluted from electrograms, then analyzed without further purification. Neutralization equivalents determined for Components A and B: calculated = 70; found = 70.

Several independent arguments favor this interpretation. Analysis of NMR spectra of the triethyl ester of monofluorocitric acid disclose two -CHF- doublets (Table I). Quantitative analysis of the two peaks in each doublet, by means of integra- tion, shows a 4: 1 ratio of the areas under each peak. These results can be interpreted to indicate the existence of two diaster- eoisomers present in 4 : I proportions in the sample. In addition electrophoretic separation of pure synthetic monofluorocitric acid into a weaker and stronger acid component, in proportions of close to 4 : 1 has also been achieved, as described in detail later. As shown below, infrared spectra of each separated component also exhibit sufficiently marked differences, thus furnishing fur- ther evidence in favor of the existence and success of separation of the two diastereoisomers of fluorocitric acid. There is also a difference in melting points between the two diastereoisomers the assignment of DL-erythro (i.e. “A”) to the major component

of chemically synthesized fluorocitrate is therefore provisional. (Table III).

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February 1964 D. W. Fanshier, L. K. Gottwald, and E. Kun 427

TABLE III

Characteristic of melting points of cyclohexylamine salts of jIuorocitric acid

F-citric sample

Unresolved mixture of Compo- nents A + B

Component A

Component B

Melting point*

148-152” (decomposition)

140-150” (softens at 130-140”, decomposes)

190-192” (decomposes)

* The melting points were found to be dependent upon the rate of heating. The results were obtained with a temperature increase of 1” per minute.

Enzymatic Synthesis and Characterization of MonoJEuorocitric Acid from Fluoroacetyl-CoA and Oxaloacetic Acid--Preparation

of fluoroacetyl-CoA from fluoroacetic anhydride was carried out by a procedure similar to that described by Brady (10) with the

modification that fluoroacetic anhydride was allowed to react with CoA in place of the mixed anhydride of ethyl formate and

fluoroacetic acid as done by Brady (10). Fluoroacetic anhydride was prepared according to Saunders and Stacy (11) from sodium

fluoroacetate and fluoroacetyl chloride (12). Fluoroacetic anhydride was purified by distillation (final product: b.p. lOO-

105” at 25 mm) and stored in sealed 5-ml vials under nitrogen. Preparation of fluoroacety-CoA was done immediately before enzymatic experiments as follows. CoA was dissolved in 0.1 M KHCO, and the solution was chilled to 0” in an ice bath. 9 slight excess of fluoroacetic anhydride in ether was added and the mixture was shaken vigorously. The reaction between the

acid anhydride and acetyl-CoA was instantaneous. The pH

of the reaction mixture containing fluoroacetyl-Coil was quickly adjusted to 6.1 with dilute HCl, excess fluoroacetic anhydride was extracted with ether, and traces of ether were removed from the aqueous phase by a stream of nitrogen, while the solution was kept at 10”. This method yielded fluoroacetyl-CoA free of pyri- dine. This modification may be considered an advantage over

the procedure of Brady (lo), who carried out this reaction in

pyridine. Since this CoA derivative is unstable, it was used directly for enzymatic experiments. Brady (10) has previously obtained spectrophotometric evidence indicating that the en- zymatic condensation of fluoroacetyl-CoA with oxaloacetic acid occurs and yields aconitase-inhibitory fluorocitrate; however,

the isolation and chemical characterization of this acid has not been reported. Our criteria for isolation and enzymatic and chemical characterization of this product were the same as established for the noninhibitory isomer (4).

Enzymatic synthesis of monofluorocitrate from fluoroacetyl- CoA and osaloacetate was carried out with heart muscle-con- densing enzyme (cf. (4)) ; the malate dehydrogenase-coupled spectrophotometric test of Ochoa, Stern, and Schneider (13) was used to determine the progress of the react.ion. A large

scale experiment is illustrated in Fig. 2 (for details, see legend of Fig. 2), where NADH formation in l-ml samples, withdrawn wit,hin short intervals, marks the rate of reaction. Since fluoro- acetyl-CoA is unstable at pH 7.4 at room temperature, aliquots of this solution were added in succession to the enzymatic reac- tion mixture (as shown in Fig. 2) t’o minimize decomposition.

Quantitative recovery of enzymatically formed fluorocitrate in

this system, its isolation, and characterization are described in

the legend of Fig. 2.

In agreement with Brady (lo), the K, for fluoroacetyl-Cod in the condensing enzyme reaction was found to be 2.5 X 1O-5,

identical with that of acetyl-CoA itself. Fluoroacetyl-CoA is a competitive inhibitor of acetyl-CoA (& = 2.2 x 10W6 for the

condensing enzyme reaction). This value agrees well with that reported by Brady (lo), i.e. 1.3 x lo+.

Separation and Chemical Characterization of Enzymatically Formed and Synthetic Fluorocitric Acids-Fluorocitric acid pre- pared enzymatically from monofluoro-oxaloacetate and acetyl- CoA (designated as fluorocitrate I) (4), from fluoroacetyl-Cob and oxaloacetate, as described above (called fluorocitrate II), and obtained by chemical synthesis were subjected to high voltage paper electrophoresis both on an analytical and a pre-

01 I I I 0 50 100 150

TIME IN MINUTES

FIG. 2. Enzymatic formation of fluorocitrate II from fluoro- acetyl-CoA. L-Malate, 500 pmoles, and NAD+, 200 pmoles, were dissolved in 100 ml of deionized water and the pH was adjusted to 7.0 with solid KHC03. Condensing enzyme (50 mg; specific activity = 2.6 rmoles of citrate per mg of protein per minute) and malic dehydrogenase (10 mg; specific activity = 1.45 pmoles of NADH per mg of protein per minute) in 10 ml of 0.005 M Tris- hydrochloride buffer (pH 7.4) were added and the temperature brought to 25” in an amber Erlenmeyer flask (250.ml volume), equipped with magnetic st,irrer, water jacket, thermometer, and pH electrodes. An aqueous solution of freshly prepared fluoro- acetyl-CoA, containing 100 pmoles in 12.5 ml (pH 6.0), was kept separately in ice bath and aliquots of 0.5 to 2.0 ml were added successively to the reaction mixture as shown. Samples of 1 ml were withdrawn and analyzed spectrophotometrically for NADH. During the course of the-reaction the pH was 8.3, and 65 pmoles of NADH were formed. After 3 hours the reaction mixture was brought to 0” and added directly to a large excess of moist Dowex 50 (H+). The mixture was stirred for 10 minut.es at lo”, and the resin then was removed by filtration, resulting in the removal of protein and a large amount of CoA and pyridine nucleotide (cf. (4)). The clear, acidic filtrate was placed under vacuum for 10 minutes (to remove CO*), then lyophilized to dryness, leaving a white amorphous powder. This was taken up in three 2.ml por- tions of water, and the pH adjusted to 7.0 with solid KHC03. The faintly yellow solution was decolorized by several successive treatments with charcoal and stored at -15”. This solution was free of nucleotides and other ultraviolet light-absorbing material and analyses for fluorocitrate (cf. (4)) yielded a total amount of 60 pmoles, in good agreement with kinetic spectrophotometric data.

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428 Specific Enzyme Inhibitors. VI Vol. 239, No. 2

A

0 0 0 a

0 0 go 000

A ‘I’ ‘Z’

12 3 456 1 23456 7 1 2

B

0

0 00 ()0000 0

FIG. 3. Paper electrophoresis was carried out on a temperature- controlled high voltage apparatus (Pherograph; Brinkman In- struments, Inc.) at 0”. The solvent was 0.1 M ammonium for- mate-formic acid mixture of pH 2.8. Current density was 50 ma with a potential gradient of 33 volts per cm. Time of electro- phoresis was 13 to 2 hours. Part A shows the separation of a mixture of chemically synthesized and enzymatically formed fluorocitrates: 1 = picric acid marker; 2 = synthetic fluorocitrate, separating into Components A and B; 3 = enzymatically formed fluorocitrate from fluoro-oxaloacetate and acetyl-CoA (the second spot is unreacted fluoro-oxaloacetate) called fluorocitrate I; 4 = mixture of chemically synthesized fluorocitrate and enzymatic fluorocitrate I (plus fluoro-oxaloacetate) ; 5 = enzymatically formed Auorocitrate from fluoroacetyl-Coil and oxaloacetate

parative scale. Fluorocitric acid was detected on the filter paper by the pyridine-acetic anhydride color test, which was found to be readily applicable to spot tests on filter paper (4). Results of such an electrophoret)ic separation are shown in Fig. 3 (technical details are described in legend of Fig. 3).

The following results were obtained: (a) pure synthetic fluoro- citric acid yielded two components (called A and B) ; (b) both enzymat’ically formed fluorocit’ric acids (fluorocitrates I and II) gave one single component only corresponding to Component A of the synthetic product; (c) aconitase inhibition, as discussed later in detail, was associated only with Component A of the synthet’ic compound, which corresponds to the enzymatically formed fluorocitrates. Quantitative analysis of Components A and B showed that A is present in the synthetic fluorocitric acid, prepared as described earlier, at 4 times the concentration of B.

In Fig. 3 (A, B, and C) for comparison, synthetic and en- zymatically formed fluorocitric acid samples, containing un- reacted monofluoro-oxaloacetic acid, were separated electro- phoretically, clearly demonstrating t’he resolving power of this

(called fluoroci tri Ite II); and 6 = mixture of chemically synthe-

C

c==J

,i A )

sized fiuorocitrate plus fiuorocitrate II. Part B. 1, 2, and 3, same as in A; 4 = enzymatically formed

fluorocitrat,e I after treatment with 2,4-dinitrophenylhydrazine + charcoal (cf. (4)) ; 5 = enzymatic fluorocitrate I after isolation by paper chromatography (in I-butanol-pyridine-water, 3:2:15, v/v); 6 = monofluoro-oxaloacetic acid; and 7 = mixture of fluoro-oxalo- acetic acid and pure enzymatically formed fluorocitrate I.

Part C. Large scale separation of Components A (oL-erythro) and B (DL-three), the diastereoisomers from synthetic fluorocitric acid; 1 = picric acid marker. All samples were located by spray- ing with pyridine-acetic anhydride (4). Components A and B were isolated by elution of filter paper and crystallization of fluorocitric acids as the cyclohexylamine salt (4).

procedure. The identity of each electrophoretic constituent was further established by isolation of each component from large scale electrophoretic runs as t’he crystalline cyclohexylamine salt, determination of elementary composition (Table II), melt- ing point (Table III), infrared spectra (Fig. 4), and finally acid dissociation constants of both electrophoretic components (Table IV). These data strongly support the view, already proposed earlier in this paper (see under “Chemical Synthesis of Mono- fluorocitrate”), that separation of two diastereoisomers is readily accomplished by procedures based on a difference between these molecular species. It is of interest to not,e that recrystallization of chemically synthesized fluorocit’rate as the cyclohexylamine salt results in substantial loss of Component B (DL-three), owing to the greater solubility of this diastereoisomer.

Earlier attempts made by Ward and Peters (14) to separate synthetic fluorocitric acid into its maximally expected four components by means of column chromatography on silicic acid resulted in no less than 12 components. These results cannot be explained unless it, is assumed that the synthetic starting material

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February 1964 D. W. Fanshier, L. K. Gottwald, and, E. Kun

WAVENUMBER CM-’ > 3000 2000 1400 I200 1000 900 800 700 I I I , I I I I I I I I I I -

u u I I I I I I I I I 1 1 I I

3 4 5 6 7 8 9 IO II 12 13 14 15 I WAVELENGTH IN MICRONS

FIG. 4. Infrared spectra of citric and fluorocitric acids. The infrared spectra of the cyclohexylamine salts were determined on a Beckman model In-5 spectrophotometer with KBr pellets containing 0.5% sample.

TABLE IV

Apparent acid dissociation constants (pK,) of citric and juorocitric acid (Components A and B)

pKa (temperature, 22”) (carboxyl groups)

I II

III

Citric acid

3.1 4.5 5.9

F-citric acid

component A* Component B

2.7 2.6 3.8 3.2 5.6 4.7

* Prepared either from electrophoretically separated samples or by repeated recrystallization of the synthetic mixture of fluorocitrate. Cyclohexylamine salts of DL-eryfhro and nn-three diastereoisomers are separable by recrystallization since the DL-threo isomer is more soluble in ethanol. The apparent pK, values were read directly from titration curves.

was extremely impure or a decomposition of fluorocitric acid occurred on the column. We have repeated Ward and Peter’s (14) chromatographic method and found it unsuitable for pre- parative or analytical purposes.

Comparison and Kinetics of Aconitase-inhibitory Properties of Electrophoretically separated Fluorocitric Acids-Enzymatic analyses were performed with the coupled aconitase-isocitrate dehydrogenase system of sonically disrupted mitochondria (4) and also with the aconitase reaction itself, by measurement of aconitate formation from both citrate or isocitrate (cf. (4)). Kinetic analyses revealed that the enzyme-inhibitory effect was due to the isomer of fluorocitrate which is formed enzymatically from fluoroacetyl-CoA and oxaloacetate (fluorocitrate II). This isomer corresponds, with respect to electrophoretic behavior, to Component A of synthetic fluorocitrate, which, as predicted, has an inhibitory potency of one-half of pure fluorocitrate II.

Comparison of initial rates of reaction in the coupled aconitase- isocitrate dehydrogenase test (4), in the presence of varying concentrations of fluorocitrate II (the inhibitory isomer, pro- duced enzymatically from fluoroacetyl-Co-4 and osaloacetate), revealed a competitive type of inhibitory mechanism. Graphic

ATE

FIG. 5. Effect of fluorocitrate II (i.e. enzymatically formed from fluoroacetyl-Coil and oxaloacetate) on the initial enzymatic rates of NADPH formation (cf. (4)) in the presence of citrate as substrate (upper three lines) and with cis-aconitate (lower line). Each cuvette contained 0.1 ml of NADP+ (200 pg), 0.05 ml of MnSOl (5 pmoles), 0.67 ml of 0.1 M Tris buffer (pH 7.4), substrate, and inhibitor to make up a final volume of 1 ml. The rate of absorbance changes at 340 rnp was determined in the first 2 min- utes at 15-second intervals. Fluorocitrate did not inhibit the reaction when cis-aconitate was the substrate (lowest line), up to 8 X 10-s M inhibitor concentration.

representation of this effect is shown in Fig. 5. Kinetic ab- normalities of the “substrate inhibition” type are noticeable with respect to citrate. The inhibitory constant (KJ for fluoro- citrate was estimated to be 2.5 x 1OP. Essentially identical results were obtained when the aconitase reaction itself was measured. It is of interest to note that fluorocitrate does not inhibit NADPH formation when the substrate is cis-aconitate instead of citrate in the coupled enzyme system (4) (Fig. 5) even at high inhibitor concentrations.

It was noticed that when the enzymatic reaction was followed

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430 Specific Enzyme Inhibitors. VI Vol. 239, hT0. 2

beyond the first 3 or 4 minutes, a progressive inhibition by fluorocitrate occurred, even at very low inhibitor concentrations. It became apparent t’hat a secondary type of inhibition appears under these conditions, which cannot be explained by the simpler competitive mechanism observed during initial rate studies. This type of inhibition is illustrated in Fig. 6. When the time required, expressed in minutes, to produce 50% inhibition is plotted against reciprocals of the negative logarithm of fluoro- citrate concentrations, straight lines were obtained. This is shown in Fig. 7, where the potency of the synthetic compound (mixture of both diastereoisomers), electrophoretic Component A (containing m-erythro isomer), and fluorocitrate II (enzy- matically formed from fluoroacetyl-CoA and oxaloacetate) are compared. Inhibit.ory potency clearly follows the predictable order; i.e. the most potent is fluorocitrate II, whereas Component A is less inhibitory, on a weight basis, since it is a mixture of two optical isomers, and least inhibitory is the synthetic unresolved fluorocitrate containing all four isomers in the known unequal proportions (see under “Chemical Synthesis of Monofluorocit- rate”).

The structural specificity of monofluorocitrate as inhibitor is further substantiated by the observation that diethyl mono- fluorocitrate does not inhibit aconitase, and thus all three car- boxy1 groups must be ionizable for this effect. Furthermore a-difluorocit’rate, prepared according to Raasch (15), is also ineffective as an inhibitor or substrate on citrate-condensing enzyme, aconitase, and isocitrate dehydrogenase at a concentra- tion of 1O-3 11.

Specificity and Jleclmnism of Action of Fluorocitrate-It is generally assumed that the mechanism of toxic action of fluoro- citrate is explicable by its specific inhibitory effect on aconitase. This opinion, suggested by Peters (cJ. (4)), does not contain, however, a detailed account of events leading to cessation of physiological cellular functions. The simplest mechanism would suggest an interruption of the citric acid “cycle” at the aconitase level, resulting in the well known accumulation of citrate and an exhaustion of ATP synthesis. In view of recent. advances in the understanding of regulation of mitochondrial enzyme systems achieved notably by Lehninger (16), Chance and Hollunger (17), Klingenberg and Slenczka (18), Klingen- berg (19), Prairie, Conover, and Racker (20)) Danielson and Ernster (al), and many others, it is evident that the operation of mit,ochondrial multienzyme systems involves a series of ATP-

.600 t

0 2 4 6 8 IO 12 14 TIME IN MINUTES

I 16

FIG. 6. Effect of 5 X 1OV M fluorocitrate II added after 1 min- ute on the velocity of the “coupled assay” reaction with 10m3 M citrate as substrate. Conditions of the spectrophotometric test system were the same as described in legend of Fig. 5.

I 7

FIG. 7. Relationship between time required for 507, inhibition and inhibitor concentration with different fluorocitrate samples. Ordinate: negative logarithm of the concentration of fluorocitrate (pl); abscissa: time in minutes, required to achieve 5070 inhibition. Assav conditions are the same as described in the legend of Fig. 5. Fluorocitrate was added (10 ~1) 1 minute after all r&ctants were incubated for 1 minute at 22”. Concentration of citrate was lo+ M. Upper line = chemically synthesized fluorocitrate; second line = electrophoretically separated on-erythro diastereoisomer (Component “A”) of chemically synthesized fluorocitrate; lowest line = enzymatically synthesized fluorocitrate from fluoroacetyl- CoA and oxaloacetate (“ZZ”).

linked reductive and dismutative processes, rendering the func- tional organization of mitochondrial enzymes much more compli- cated than anticipated earlier. Therefore, it is necessary to clarify the detailed consequences and biochemical significance of inhibition of presumably one enzymatic component of this system by fluorocitrate.

WC have reinvestigated the specificity of pure fluorocitrate on certain isolated mitochondrial enzymatic reactions as well as on the respiration of intact mitochondria. Since certain unes- pected observations were made, it appeared to be of interest to incorporate them into this presentation.

Malate dehydrogenase and NADI’+-linked isocitrate dehy- drogenase of sonically disrupted mitochondria of rat tissues (4) were unaffected by the inhibitory isomer of fluorocitrate, even if preincubated in the absence of substrate for 15 minutes with the inhibitor. No inhibition was detectable with concentrations up to 5 x lop3 M. Succinic dehydrogenase activity of the same preparations was, however, inhibited by fluorocitrate (Table V). Kinetic analyses of this effect revealed that fluorocitrate in- hibited succinic dehydrogenase by a purely competitive mecha- nism, with a Ki of 6 x 10e4. Prior incubation of the enzyme preparation with fluorocitrate did not alter the degree or type of inhibition.

The significance of this unexpected inhibitory action of fluoro- citrate was further studied with intact mitochondria. The consumption of O2 in the presence of various substrates was determined both in conventional Warburg type respirometers or

by rapid polarographic analyses of O2 consumption with vibrating platinum electrode (23) (Oxygraph, Gilson Electronics, Madison, Wisconsin). Manometric experiments were considered to

measure O2 consumption of a quasi-steady state reaction condi- tion, when a certain distribution of intermediate metabolites from the added excess substrate already occurred, permitting a

variety of enzymatic reactions to proceed simultaneously. An experiment of this type is illustrated in Table VI. Succinate “respiration” is rapidly inhibited by fluorocitrate in kidney and

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February 1964 D. W. Fanshier, L. K. Gottwald, and E. Km 431

TABLE V

Effect of monofluorocitrate II and oxaloacetate on succinic dehydro- genase activity of sonically disrupted rat

kidney mitochondria*

The assay procedure of Singer and Kearney (22) was reproduced except that catalytic amounts of phenazine methosulfate were also present. The reaction mixture contained (per ml): 20 pmoles of inorganic phosphate, 10 pg of phenazine methosulfate, 60 pg of 2,6-dichlorophenolindophenol, 2 pmoles of KCN, and 50 pg of kidney mitochondrial enzyme protein, with prior incubation for f minute at 22”. The reaction was started by the addition of succinate (or succinate together with inhibitor) and followed spectrophotometrically by measurement of the decrease in ab- sorbance at 600 mp due to the reduction of the dye. Readings were taken every 15 seconds during the first minute; temperature, 22”.

Contents Reaction velocityt

/moles succinate oxidized/?% enzyme protein/milz

Endogenous (no succinate) 1.3 x 10-a Puccinate (1OW 31). _. 6.3 X 1O-3

+ Oxaloacetate (1OW M) 0.1 x 10-z f F-citrate (1.56 X lo-* M). 4.5 x 10-z + F-citrate (7.80 X 10-h M), . 2.3 X 10-s

* Sonically disrupted mitochondria in 0.15 M KC1 were cen- trifuged at 15,000 X g for 30 minutes and the supernatant frac- t,ion containing the enzyme was used directly.

t Calculated from the expression: A~4600 ,,/19.1 = pmoles of succinate oxidized per ml.

at a slower rate in liver mitochondria, whereas glutamate “res- piration” is affected in an opposite fashion in mitochondria of these two tissues. It is striking that the O2 consumption of mitochondria of various tissues exhibited apparent quantitative differences in response to fluorocitrate, reminiscent of the dif- ferences reported for monofluoro-oxaloacetate (4) on mito- chondrial respiration under certain experimental conditions. The reasons for these apparent specific tissue differences are the subject of further studies.

In contrast to the manometric experiments, rapid polaro- graphic recording of O2 consumption (23) of dilute suspensions of mitochondria more closely approached “initial rate” studies since changes in respiration could be measured within 20 to 40 seconds after addition of substrates or inhibitors. These types of esperiments were performed in order to determine the contri- bution of both aconitase and succinic dehydrogenase inhibition by fluorocitrate on mitochondrial respiration. Results of t,hese experiments are summarized in Table VII. In each experiment listed in this table, Cl2 consumption of dilute suspensions of rat kidney mitochondria was recorded in the presence of SDP and inorganic phosphate in 0.15 M KC1 solution (see legend of Table VII). Experiment 1 shows endogenous respiration; Experiment 2, the oxidation of the substrate combination, l)!;ruvate and osaloacetate; and Experiments 3 and 4, the effect of increasing concentrations of fluorocitrate II. As expected from the inhibitory effect of fluorocitrate, primarily on aconitase, the system containing pyruvate and oxaloacetate as substrates was very sensitive to the inhibitor. The rate-limiting role of aconitase on initial O2 consumption is demonstrated in these experiments. The rest of Table VII illustrates the influence of fluorocitrate and osaloacetate on succinate oxidation. As seen

from Experiment 5, 2.5 X 10M3 M succinate produces maximal rates of oxidation, whereas 9.5 X 1OW M succinate diminishes O2 consumption to about one-half. This characteristic dependence of O2 consumption on succinate strongly supports the view that, under these conditions, succinic dehydrogenase is indeed the rate-limiting enzyme in the initial phase of the over-all reaction. Singer, Kearney, and Bernath (24) determined the K, of suc- cinate for the dehydrogenase to be 5.2 X 1O-4 M; thus concentra- tions of succinate in this order of magnitude (Table VII) should diminish O2 consumption to about the extent that was actually observed. Since the lower concentration range of succinate (Experiments 7 to 11) is close to that reported to be present in rat tissues (approximately 1.5 X 10F4 M; cf. (25)), inhibition of succinic dehydrogenase by tissue concentrations of fluorocit- rate, equivalent to LDSO doses (cf. (4)), would certainly be pre- dictable and have direct relevance to the toxicity of this com- pound. Initial rates of succinate oxidation in the presence of high succinate concentration is diminished only by 16 y0 (Experi- ment 6), but, at a lower substrate level, marked inhibition occurs (35%) (Experiment S), in agreement with the competitive type of inhibition of succinic dehydrogenase by fluorocitrate, as mentioned above (Ki = 6 X 10e4). In Experiment 9 the oxida- tion of succinate was allowed to proceed for 150 seconds before the addition of fluorocitrate. Under these conditions a rate- limiting contribution of aconitase to the over-all rate of respira- tion would be expected to occur and the inhibitory effect of 02 uptake by fluorocitrate should become more pronounced. This prediction was substantiated. Comparison of the inhibitory effect of fluorocitrate with a known inhibitor, osaloacetate, is

TABLE VI Effect of 7.8 X 10-d M Jluorocitrate II on respiration

of mitochondria as determined manometrically

Manometric experiments were carried out in conventional Warburg vessels, containing 0.2 ml of potassium phosphate (0.1 M, pH 7.4), 0.1 ml of ATP (0.1 M), 0.05 ml of MgClz (0.1 M), 0.1 ml of substrates (0.1 M), and 1 ml of mitochondrial suspension in 0.15 M KC1 and 0.15 M KC1 to a final volume of 3.0 ml. COz was absorbed by 0.1 ml of 10% KOH, placed together with a strip of filter paper in the center well of the vessels. Rat liver mitochondria, 1 ml, contained 5.2 mg of N; 1 ml of rat kidney mitochondria, 2.6 mg of N. Readings were taken every 5 min- utes and recorded in the table at various time intervals up to 30 minutes. Temperature, 30”; gas phase, air.

Tissue Time

Liver

milt

o-5 1.520 25-30

o-5 15-20 25-30

Kidney o-5 3.9 1.6 Succinate -59

15-20 3.6 1.1 Succinate -70

25-30 3.3 0.8 Succinate -76

o-5 3.5 2.6 Glutamate -25

15-20 3.0 2.8 Glutamate -7

25-30 2.2 2.1 Glutamate -5

- Rate

lontrol / +F-citrate

2.9 2.7 Succinate 3.0 1.7 Succinate 3.2 0.8 Succinate 3.1 3.9 Glutamate 3.1 2.8 Glutamate 3.0 1.8 Glutamate

Substrate

YO -4 -43 -73 $26 -10 -36

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432 Specific Enzyme Inhibitors. VI

TABLE VII

Vol. 239, X0. 2

Polarographic determination of initial rates of 02 consumption of rat kidney mitochondria

The reaction chamber of the recording polarograph contained 2.5 X 10-a M phosphate (pH 7.4), 3 X 10-a M ADP, and enough 0.15 M KC1 to make up a total volume of 2 to 2.3 ml, including mitochondrial suspension (0.17 to 0.2 mg of N per test), substrates, and inhibitors. Calculations of O2 consumption are based on its solubility to be 0.25 rmole PC !r ml. Temperature, 25”.

Experiment

1

2

3

4 5

G

7 8

9

10

11

,

PI

-

32 consump- tion*

nzoles x IOF/ 40 sec/mg N

94

230

139

75 580

500

230 150

108

123

440

-

-

Substrate Inhibitor (final concentration)

M M

Pyruvate + oxaloacetate (9.4 x 10-q

Same F-citrate (7.4 X 10-S)

Same Succinate (2.5 X 1OW)

Same

Succinate (9.5 X 10-l) Same

Same

Same

Same + pyruvate (8.7 X 1OW)

F-citrate (IOW)

F-citrate (7.4 X IOV) -16

3.5 x 10-d -35

Same -53

Oxaloacetate (9 X 1OW) -47

Oxaloacetate (1.8 X 1OW) +s3

‘hange in rate du to inhibitori

YO

-40

-G7

- Conditions

Endogenous

F-citrate added 60 set after substrate

Same Reaction started with suc-

cinate F-citrate added 60 see be-

fore succinate

F-citrate added 60 see be- fore succinate

F-citrate added 150 set after succinate

Oxaloacetate added 120 set after succinate

Second addition of oxalo- acetate + pyruvate$

* Calculated from reaction rates determined in the first 30 seconds after initiation of reaction. t Percentage of change is compared to rate, measured in each experiment in absence of inhibitor. $ Second addition of oxaloacetate plus pyruvate was done 60 seconds after the inhibition by oxaloacetate alone occurred (see Es-

periment 10).

described in Experiments 10 and 11. Oxaloacetate has been found to be a powerful inhibitor of succinic dehydrogenase by Pardee and Potter (26) with a Kc of 1.5 x 10W6, comparable with that of fluorocitrate on aconitase (Ki = 2.5 x 10W6). In agreement with predictions, oxidation of succinate by dilute suspensions of rat kidney mitochondria is inhibited by oxaloace- tate if the latter is present as a single added component of the system (Experiment 10). If, however, pyruvate is also present (Experiment II), inhibition of O2 uptake is not only abolished but is markedly raised above the level observed with succinate alone (compare with Experiment 7), even if the concentration of oxaloacetate is increased to a level which completely inhibits succinic dehydrogenase. Evidently under these conditions other oxidations, notably those linked to pyridine nucleotides, fully compensate for t’he inhibition of O2 uptake, which occurs when succinate is the single substrate.

primarily on the predictable stereochemical specificity (8) of the chemical synthesis of fluorocitrate but also gains support from considerations of spatial configuration of this isomer. By anal- ogy with the known inductive effect of fluorine and chlorine substitution on the acid dissociation constants of malic acid (27) in which it has been found that the erythro diastereoisomers are weaker acids, the larger pK, values determined for Component A of DL-fluorocitrate (Table IV) may reasonably permit the tenative assignment of erythro to this diastereoisomer.

DISCUSSION

The optical rotation of the inhibitory isomer has not yet been determined, since the limited amounts of this enzymatirally synthesized substance has been utilized in biochemical studies. The marked solubility differences between cyclohexylamine salts of erythro and threo diastereoisomers provides a feasible method of separation which eventually may yield sufficient quantities of the individual diastereoisomers for further resolu- tion into pure optical isomers. This work is in progress.

As discussed in a previous paper (4) the enzymatic mechanism The isolation and chemical characterization of two enzy- of citrate-isocitrate conversion will probably gain further clarifi-

matically formed fluorocitrate isomers, one from monofluoro- cation through the complete stereochemical characterization of oxaloacetate and acetyl-CoA (“noninhibitory isomer” (4)) and the enzyme inhibitory isomer of fluorocitrate. It seems reason- one from fluoroacetyl-CoA and oxaloacetate (“inhibitory iso- able to assume that this fluorocitrate isomer should be struc- mer”), and the establishment of their identity with the “weaker turally related to the postulated “enzyme-bound intermediate” acid” diastereoisomer, a major component of pure synthetic between citrate and isocitrate since, in initial rate experiments, fluorocitrate, constitute direct evidence in favor of the postulate, true competitive kinetics has been demonstrated (Fig. 5). At

which has assumed that only one of the four isomers of fluorocit- present it is difficult to propose a mechanism which could explain rate has inhibitory action on aconitase. It is very probable that the fact that the coupled reaction, cis-aconitate --) isocitrate -+ the diastereoisomer containing both enzymatically formed a-ketoglutarate, does not appear to be inhibited by fluorocitrate, fluorocitrates is the DL-erythro isomer. This conclusion is based although substitution of cis-aconitate by citrate renders this

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I;ebruary 1964 D. W. Fanshier, L. K. Gottwald, and E. Km 433

system fluorocitrate-sensitive. It is probably necessary to succinic dehydrogenase by fluorocitrate under nearly physiologi- isolate a stable and pure aconitase preparation before this ques- cal circumstances argues against the “one-enzyme” type of toxic tion can be settled experimentally. This observation, however, mechanism of action of fluorocitrate. Further problems related may be taken as an argument in support of the view of Speyer to the regulation of mitochondrial enzyme systems are also and Dickman (as), who postulate a transition type of inter- challenged by this observation. mediate between citrate and isocitrate, not identical with aconitate itself. STJNMARY

The inhibition of aconitase by fluorocitrate is composed of two distinct kinetic mechanisms. A simple competitive inhibition 1. Monofluorocitric acid was synthesized by condensation of

is apparent when initial rat,es are measured, whereas a progressive diethyl monofluoro-oxaloacetate with malonic acid. Pure

time-dependent inhibition is superimposed when incubation of fluorocitric acid thus obtained was resolved by paper electro-

t,he enzyme with fluorocitrate is carried out. In earlier studies phoresis into a weaker and stronger acid component, present in

by Morrison and Peters (29) aconitase was routinely preincu- the unresolved sample in a ratio of 4 : 1. Identity of both compo-

bated with fluorocitrate and extremely small concentrations of nents was established by elementary analyses, titration of

this inhibitor were found to be effective. These concentrations carboxyl groups, melting point and solubility of the cyclohexyl-

of fluorocitrate were much smaller than would be anticipated amine salts, and infrared and nuclear magnetic resonance spectra.

from comparisons of LDsO and Is,, values based on initial rate On the basis of these data and stereospecificity of chemical

measurements (4). The significance of the secondary time- synthesis the weaker acid component was identified as the DL-

dependent inhibition remains at present unclear. It seems erythro and the stronger acid as the DL-three diastereoisomer.

reasonable that chelation of a metal component of aconitase by 2. Enzymatic synthesis of an isomer of monofluorocitrate was

fluorocitrate may be the basis of this time-dependent inhibition. carried out from osaloacetic acid and synthetic fluoroacetyl

Whether or not this type of enzyme-inhibitor interaction con- coenzyme A. This isomer is t.he aconitase-inhibitory species of

tributes to the enzymatic mechanism of toxic action of fluorocit- the four isomers present in synthetic fluorocitric acid.

rate is at present unsettled. Prior incubation of mitochondria 3. Enzymatically formed fluorocitrates, synthesized either

with fluorocitrate did not yield an unequivocal answer to this from monofluoro-oxaloacetate and acet,yl coenzyme A (non-

question, because of the multiple events occurring in mito- inhibitory) or from fluoroacetyl coenzyme A and oxaloacetate

chondria (e.g. loss of pyridine nucleotides) during incubation in (inhibitory), correspond to the I,L-erythro diastereoisomer of

the absence of oxidizable substrate. On the other hand, direct synthetic fluorocitrate.

evidence of inhibition of succinic dehydrogenase by fluorocitrat’e 4. Two kinetic mechanisms are responsible for the inhibition

invalidates the hitherto accepted vielv that the mode of toxic of aconitase by fluorocitrate: (a) direct competitive inhibition,

action of fluorocitrate is solely due to inhibition of aconitase. detectable by initial rate measurements, and (b) time-dependent

Evidently simultaneous inhibition of both enzymes occurs in progressive inhibition.

mitochondria and an explanation of the detailed mechanisms of 5. In sonic extracts as an enzyme system, fluorocitrate in-

the biochemical consequences of this dual inhibition must take hibits in vitro the conversion of citrate to cis-aconitate, isocitrate

into account this observation. The susceptibility of the two to cis-aconitate, and citrate to isocitrate, but has no effect on

enzymes is markedly different to the same inhibitor: K; for the conversion of ci.s-aconitate to a-ketoglutarate, when the

succinic dehydrogenase is 6 x lo-*; for aconitase, 2.5 X lo+. aconitase reaction is coupled to reduced nitotinamide adenine

It is evident that our earlier comparison of I50 and LDSO values dinucleotide phosphate formation due to isocitrate dehydro-

(in the order of lo+ M) (4), based on the assumption that only genase.

aconitase inhibition is involved in the mechanism of toxicity of 6. Fluorocitrate also inhibits succinic dehydrogenase in a

fluorocitrate, is oversimplified, and the order of numerical agree- competitive fashion. Initial rate studies in vitro, with intact

ment could merely reflect a combined expression of inhibition of mitochondria, under conditions closely reproducing those pre-

at least two enzymes. vailing in vivo when t,oxic amounts of fluorocitrate are present

Our experiments dealing with the complete compensation by (LDSO), show that inhibition of both aconitase and succinic

pyruvate for the inhibitory effect of osaloacetate on a “succinate dehydrogenase occurs. The mechanism of toxic action of

type” of mitochondrial respiration cast doubt on the physiological fluorocitrate is therefore related to the dual enzyme-inhibitory

significance of this inhibition. It seems almost certain that there effect of this substance.

is enough intracellular (or intramitochondrial) pyruvate present 7. Comparison of inhibitory action of fluorocitrate and oxalo-

in physiologically functioning tissues to accomplish an identical acetate on mit,ochondrial respiration was also carried out. De-

effect as shown in Table VII (Experiment 11). Thus inhibition pression of mitochondrial respiration with succinate as substrate,

of mitochondrial respiration by oxaloacetate could become effec- due to oxaloacetate, is compensated by the addition of pyruvate,

tive only under highly artificial circumstances. and results in total restoration of mitochondrial respiration.

Our conclusions concerning the mechanism of toxic action of This demonstrates that under physiological conditions, xv-here

fluorocitrate are in general agreement with those of Gordon (30). there is ample supply of pyruvate, osaloacetate is not likely to

Since fluoroacetyl-Cob, as already stated by Brady (IO), com- inhibit mitochondrial respiration.

petes with acetyl-CoA in a number of enzyme systems, it is very probable that this inhibition must find espression in a variety of AcknowZedgments-We are grateful to Dr. R. M. Silverstein metabolic reactions in which acetyl-CoA participates. These (Stanford Research Institute, Menlo Park, California) for his considerations constitute a basis for the explanation of the ob- suggestions made in the course of interpretation of NMR spectra, served divergence between the effects in viva of fluorocitrate and to Dr. R. KTvok for his help in recording t,hese spectra, and to fluoroacetate, as reported by Gordon (30). The inhibition of Dr. D. Tuck for his permission to use t,his instrument.

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434 Specific Enzyme Inhibitors. VI Vol. 239, No. 2

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D. W. Fanshier, L. K. Gottwald and E. KunMONOFLUOROCITRATE

MECHANISM OF ACTION OF THE ENZYME-INHIBITORY ISOMER OF Studies on Specific Enzyme Inhibitors: VI. CHARACTERIZATION AND

1964, 239:425-434.J. Biol. Chem. 

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