Studies on the Mechanism of Fatty Acid Synthesisferric chloride to identifv the acvl-CoA’s. The...

THE JOURNAL OF RIOLOGIC.~ CHEMISTRY Vol. 237, No. 5, May 1962

Printed in i7.S.A

Studies on the Mechanism of Fatty Acid Synthesis

XI. THE PRODUCT OF THE REACTION AND THE ROLE OF SULFHYDRYL GROUPS IN THE SYNTHESIS OF FATTY ACIDS*

RUBIN BRESSLER AND SALIH J. WAKIL

From the Department of Medicine and the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina

(Received for publication, November 20, 1961)

Recent communications from this laboratory (1, 2) have described the preparation and properties of a highly purified enzyme fraction from avian liver which catalyzes the synthesis of long chain fatty acids from acetyl coenzyme A (CoA) and malony-Coh in the presence of reduced triphosphopyridine nucleotide (TPNH). The major component of the synthesized fatty acids formed from acetyl-Coa and malonyl-CoA has been shown to be palmitic acid (1, 3). Equation 1 shows the stoichiometry of the synthetic reactions involved in the formation of palmitic acid (I, 4).

CH&OSCoA + 7HOOCCH2COSCoA + 14TPNH + 14H+ ---f

CH3(CHs)&OOH + 14TPN+ + 7CO2 + 8CoASH + 6Hz0 (1)

Similar stoichiometry has been reported on in preparations from yeast (5)) brain (6), and adipose tissue (7). Malonyl-CoA appears to provide the “Cz units” for condensation, and therefore carbon atoms 1 to 14 are derived from malonyl-CoA. Acetyl- Coil functions as the “primer” and is incorporated into the “tail” end of the fatty acid (carbon atoms 15 and 16) (1,4, 8, 9). The exact mechanism of this polymerization and the nature of the reductive steps have not yet been elucidated.

The dependence of fatty acid synthesis on intact sulfhydryl groups on the enzyme(s) was first demonstrated by Wakil et al. in preparations from avian liver (10, 11). A similar requirement for sulfhydryl groups was later shown in preparations from yeast (5), brain (6), and adipose tissue (7). Lynen and Tada (5) have reported that malonyl groups were covalently bound to the enzymes of fatty acid synthesis prepared from yeast through sulfhydryl groups of these enzymes. Lynen’s scheme of synthesis (Equations 2 through 5) is initiated by a reaction between malonyl-CoA and the sulfhydryl group of the enzyme(s) to form a malonyl-S-enzyme. The malonyl-S-enzyme then con- denses with an acyl-Coa (acetyl-CoA, butyryl-CoA, hexanoyl- CoA, etc.) to form a P-ketoacyl-S-enzyme and simultaneously releases CO*. The B-ketoacyl-S-enzyme undergoes sequential reduction, dehydration, and reduction again to form a saturated acyl-S-enzyme. The complex then reacts with CoiZSH to form a free acyl-CoA and regenerates the sulfhydryl form of the enzyme. The saturated acyl-CoA produced is 2 carbon atoms longer than the original condensing acyl-CoA.

*Aided in part by grants from the National Institutes of Health, United States Public Health Service, Nos. RG-6242 (C,) and OG-5, and from the Cent.er for the Study of Aging, Duke University, Nos. H-3582 and M-2109.

COOH I

CHzCOSCoA + HS-enzyme ti

COOH

&HtCOS-enzyme + CoASH (2)

COOH I

CH3(CHzCH2),COSCoA + CHZCOS-enzyme 4

CH3(CH2CH2)nCCH2COS-enzyme + CoASH + CO2

A

(3)

CH3(CH2),CCHzCOS-enzyme + STPNH + 2H+ + II

0 CHI(CH2CH&,+lCOS-enzyme + 2TPN+ + H20 (4)

CHs(CH&H&+lCOS-enzyme + CoASH +

CH~(CH~CHZ)~+~COSCOA + HS-enzyme (5)

Recent reports from this laboratory (I, 2, 12) do not lend support to this proposed mechanism in fatty acid-synthesizing prep. arations from liver. Our data showed that propionyl-Cod could partially substitute for acety-CoA, but butyryl-CoA could only do so to a much lesser extent. Experiments, includ- ing trapping and dilution techniques, designed to isolate short chain acyl-CoA’s (butyryl-CoA, hexanoyl-CoA) from the reaction mixture during the synthesis of long chain fatty acids from C14. labeled malonyl-CoA were not successful (1). Thus far, the isolation of short chain acyl-CoA’s from other systems in which these compounds (butyryl-CoA, etc.) are assumed to be inter-. mediates has not been reported (5, 6, 9, 13, 14). Our results, obtained by using a highly purified preparation from avian liver, make it unlikely that free acyl-CoA derivatives are intermediates, or in equilibrium with intermediates, in the synthesis of long chain fatty acids (I, 2). Clarification of any differences in the mechanism of fatty acid synthesis between yeast and liver, especially concerning the incorporation of short chain acyl-CoA’s must await further experimentation.

Evidence will be presented in this report to show that acetyl- CoA serves to protect the sulfhydryl groups of the fatty acid- synthesizing enzyme(s) against inhibition by sulfhydryl-binding agents, whereas malonyl-CoA does not. Propionyl-CoA may partially replace acetyl-CoA in this protective function, but butyryl-CoA only does so to a lesser extent. These observations

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1442 Fatty Acid Synthesis. XI Vol. 237, No. 5

FIG. 1. Scanning of the paper chromatogram for the product of fatty acid synthesis after the condensation of C14-malonyl-CoA and acetyl-CoA. The reaction mixture contained 60 pmoles of potassium phosphate buffer, pH 6.5, 2 Mmoles of mercaptoethanol, 40 pg of Rza, 100 mpmoles of TPNH, 75 mpmoles of malonyl-CoA (160,000 counts), 75 m*moles of acetyl-CoA, 150 mfimoles of palmityl-CoA, and water to a final volume of 0.6 ml. The reaction was run at 37” for 5 minutes and was terminated by the addition of 0.03 ml of 30% perchloric acid, which brought the pH of the reaction mixture to 3. The reaction mixture was chromatographed on Whatman No. 3 paper in isobutyric acid: concentrated NH3:H20 for 20 hours. The chromatogram was scanned for radioactivity and then sprayed with hydroxylamine followed by ferric chloride to identifv the acvl-CoA’s. The snot corresnond- I ing to palmityl hydroxamic acid (Spot C, RF 0.69) shows no radioactivity. Radioactivity is seen in the malonic hydroxamate (Spot A, RF 0.32), acetic hydroxamate (Spot B, Rp 0.46), and palmitic acid (Spot D, RF 0.96) spots.

substantiate our earlier report on the relative affinities of these acyl-CoA’s toward the enzyme of fatty acid synthesis (1). Evi- dence will be presented to show that the inhibition of fatty acid synthesis by sulfhydryl-binding agents is due to a block in the decarboxylation-condensation between acetyl-CoA and malonyl- CoA. The present communication will also present evidence to show that the product of the reaction in fatty acid synthesis is palmitate, rather than palmityl-CoA, as had been reported previously (5, 15).

EXPERIMENTAL PROCEDURE

Preparation of Fatty Acid-synthesizing Enzymes-Highly purified preparations (RZ1) were obtained from pigeon liver extracts by methods previously described (1). Preparations of specific activities of 300 to 1000 mpmoles of TPNH oxidized per minute per mg of protein were used throughout these studies. Fatty acid synthesis was assayed spectrophotomctrically as previously reported (1). Assay of TPNH-acetoacetyl-CoA reductase was done as previously described (2).

Assay of Decarbozylation-Condensation Reaction of HOOCl’- CH&‘OXCoA alad CN&OSCoA-The collection of C1402 in a Warburg flask for the subsequent transfer of the radioactive material for liquid scintillation counting was done by a slight modification of the method described by Snyder and Godfrey (16). The C’~OZ was trapped in Hyamine, which is placed in a removable glass vessrl. The release of COZ from the reaction mixture was effected by the iniection of 0.2 ml of 3 N HzS04 with subsequent shaking in a water bath at 37” for 2 hours. The removable glass vessel contained 0.5 ml of 1 M Hyamine in methanol. Recoveries of Cl*02 ranged over 95 y0 by this method.

Acyl-CoA Derivatives-Acyl-CoA’s were synthesized either chemically or enzymatically by methods previously described

(1,2). p-Hydroxymercuribenzoate and TPNH were obtained from

the Sigma Chemical Company. Coenzyme A was obtained

from Pabst Laboratories. N-Ethylmaleimide was obtained from Chemicals Procurement Laboratories in New York. Hy- amine (p-diisobutylcresoxyethoxyethyl dimethyl benzyl ammo- nium hydroxide in methanol) was obtained from Rohm and Haas Company, Philadelphia.

RESULTS

Product of Synthesis--In their original studies of fatty acid synthesis by pigeon liver supernatant solution, Brady and Gurin showed that long chain fatty acids were formed by the particle- free extracts (17). Porter and Tietz later found that. 60% or more of the fatty acids synthesized by pigeon liver supernatant was palmitic acid, and that smaller amounts of stearic and myris- tic acids were formed (18). Porter and Long reported that the product of synthesis of fatty acids from Cl*-acetic acid, ATP, CoA, and pigeon liver extracts was palmityl-CoA (15). Using a highly purified enzyme system prepared from pigeon liver, we recently reported that palmitate constituted over 80% of the fatty acids synthesized from acetyl-CoA and malonyl-CoA in the presence of TPNH (1). The product of the synthesis was found to be free palmitic acid and not palmityl-CoA. This was shown by direct attempts to isolate the product of the synthesis under conditions in which palmityl-CoA was stable and by trapping and dilution techniques with added palmityl-CoA.

When Cr4-labeled malonyl-CoA (HOOCCH&?40SCoA) was incubated with Rz, in the presence of acetyl-CoA, TPNH, and carrier palmityl-CoA (prepared enzymatically by the procedure of Kornberg and Pricer (19)) and the reaction mixture was chromatographed in isobutyric-concentrated ammonia-water (66: 1:33) at pH 3.7, radioactivity was found in the palmit,ic acid spot, whereas none was found in the palmityl-CoA spot, as shown in Fig. 1. Under these conditions, palmitic acid had an RF of 0.95; palmityl-CoA, an RF of 0.69; malonyl-CoA, an RF of 0.35; and acetyl-CoA, an RF of 0.45. Palmityl-CoA was identified on the paper chromatogram by its capacity to form a colored hydroxamate when sprayed with hydroxylamine followed by treatment with ferric chloride (20) and by the nitroprusside color test after hydrolysis of the palmityl-CoA by alkali (21). Roth of these tests were positive with Spot C (see Fig. l), which contained no radioactivity and had an RF of 0.69, corresponding to authentic chemically synthesized palmityl-CoA. Spot D, which contained a great deal of radioactivity and had an RF of 0.95, corresponding to pure palmitic acid, showed nega- tive hydroxamate and nitroprusside tests. Furthermore, there was quenching of ultraviolet light when the chromatogram was viewed under ultraviolet light in Spots A, B, and C, only (see Fig. 1). These data show that Spot D is not a CoA derivative. Elution of Spot D from the paper, followed by gas chromatog- raphy, identified it as palmitic acid.

Palmityl-CoA deacylase has been reported in pigeon liver preparations (15,22, 23). The highly purified pigeon liver preparations used in these experiments had traces of palmityl-CoA deacylase present. However, under conditions of the experiment (cf. Fig. I), over 60% of the added palmityl-CoA remained intact. The availability of this large pool of non-radio-labeled palmityl-CoA would strongly mitigate against the possibility that newly synthesized palmityl-CoA is deacylated to palmitic acid soon after its synthesis. I f this were the case, then sub- stantial amounts of the radioactivity of the product of this synthesis would have been trapped in the large pool of non-radio- labeled palmityl-CoA. The results shown in Fig. 1 indicate


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May 1962 R. Bressler and S. J. Wakil 1443

that the product of the synthesis is not palmityl-CoA, nor is it in equilibrium with palmityl-CoA. These observations do not preclude a palmityl-enzyme complex of some sort not in equilibrium with free palmityl-CoA. The observations of Porter and Long (15) on palmityl-CoA as the product of synthesis may be explained by the use of a crude pigeon liver preparation by these workers. The preparation used could have contained the long chain fatty acid-activating enzymes, which, in the presence of the ATP and CoA added by these workers, would convert palmitic acid to palmityl-CoA (19).

Product of Condensation of Acetyl-CoA and Malonyl-CoA- We recently reported on the isolation of a product of condensation of acetyl-CoA and malonyl-CoA in the absence of TPNH (1). The product of condensation was characterized in several chromatographic systems and was found to be distinct from acetic, malonic, acetoacetic, fl-hydroxybutyric, and palmitic acids, and it was ascertained not to be a CoA derivative (1). The compound was also formed by the condensation of acetoacetyl-CoA and malonyl-CoA (2). This compound had an Rp of 0.35 to 0.4 in the butanol-ammonia system of Reid and Lederer (24). Propionyl-CoA and butyryl-CoA could substitute for

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FIG. 2. Formation of the product of condensation of acetyl- CoA and malonyl-CoA with concomitant release of COZ. To 30 mpmoles of malbnyl-CoA in the reference cuvette were added 10 vmoles of KOH and water to a final volume of 0.1 ml. The mixture was allowed to stand at room temperature for 10 minutes. HCl (10 pmoles) was then added. The experimental cuvette contained 30 mpmoles of malonyl-CoA and 10 pmoles of KC1 prepared by mixing 10 pmoles each of KOH and HCI. To both cuvettes were added 26 mpmoles of acetyl-CoA, 60 pmoles of potassium phosphate buffer, pH 6.5,2 Fmoles of mercaptoethanol, and water to a final volume of 1.0 ml. The spectrum was scanned at zero time, with a Cary 14 spectrophotometer, between 220 and 400 mB. Then 100 pg of Rz, were added to both cuvettes and the course of the reaction was followed at 2,10,15,25, and 40 minutes. The reaction was run at 23”. The difference spectra between zero time and 40 minutes is shown separately from the course of the reaction’s spectral changes.

acetyl-CoA to a slight extent to release CO, and form a condensation product. The products of these condensation reactions had Rp values close to, but not identical with, the product of condensation of acetyl-CoA and malonyl-CoA. Their Rp values in the butanol-ammonia system were: propionyl-CoA, 0.41 to 0.42, and butyryl-CoA, 0.42 to 0.43. Spectrophotometric meas- urements of these isolated compounds revealed absorption max- ima at 275 rnE.c. Fig. 2 shows the utilization of acetyl-CoA and malonyl-CoA to form the condensation product. The decrease in absorbancy at 235 rnp represents the disappearance of the thioester linkages of acetyl-CoA and malonyl-CoA which are being consumed in the condensation reaction (25). The increase in absorbancy at 275 rnp represents the appearance of the product of condensation. Acetyl-CoA, in the absence of malonyl-CoA, does not show either a decrease in absorbancy at 235 rnp or an increase in absorbancy at 275 rnp. Similar results were obtained when acetoacetyl-CoA, propionyl-CoA, or butyryl- CoA were substituted for acetyl-CoA. These acyl-CoA’s, however, condensed with malonyl-CoA to a lesser extent than did acetyl-CoA. Propionyl-CoA was approximately 40% as effective as acetyl-CoA, whereas butyryl-CoA was only approximately 5% as effective as acetyl-CoA. The behavior of acetoacetyl- CoA may be due to its condensation with malonyl-CoA as an intact Cc unit or to its cleavage to acetyl-CoA and the subsequent condensation of acetyl-CoA and malonyl-CoA. It has not yet been possible to distinguish between these two mechanisms. The assays for thiolase activity which would effect the cleavage of acetoacetyl-CoA to acetyl-CoA in the Rs enzyme preparation have indicated that thiolase activity is absent from the Rz, preparation (2).

Role of Sulfhydryl Groups in Fatty Acid SynthesisThe im- portant role of sulfhydryl groups in the synthesis of long chain fatty acids was studied by Wakil et al. in avian liver (10, 11) and was confirmed by others in yeast, brain, and adipose tissue (5-7, 9). Table I shows the sulfhydryl nature of the enzymes of fatty acid synthesis. The synthesis of fatty acids is stimulated by sulfhydryl reagents such as glutathione, cysteine, and mercaptoethanol and is inhibited by sulfhydryl-binding agents such as cadmium, p-hydroxymercuribenzoate, and N-ethylmaleimide. The stimulation of fatty acid synthesis by EDTA was probably due to reversal of heavy metal inhibition of the Rza sulfhydryl groups. This EDTA stimulation of activity also points to the fact that metal cofactors are probably not involved in the conversion of malonyl-CoA to fatty acids. The role of metal ions in acetyl-CoA carboxylase, a biotin-containing enzyme which catalyzes the conversion of acetyl-CoA to malonyl-CoA, has been shown (26). Fig. 3 shows the inhibition of fatty acid synthesis by HMBI and its reversal by mercaptoethanol. Inhibition of fatty acid synthesis by HMB is approximately 85% at an HMB concentration of 2.5 x 10m5 M. The TPNH-linked acetoacetyl-CoA reductase (2), which catalyzes the conversion of acetoacetyl-CoA to D( -)-&hydroxybutyryl-CoA, is not inhibited by sulfhydryl-binding agents, as shown in Table I. This observation indicates that the reductase is not a sulfhydryl enzyme and supports our earlier data on the distinctiveness of the reductase and the fatty acid-synthesizing enzymes (2). The inhibition of fatty acid synthesis by the various sulfhydryl- binding agents is evidenced by a decrease in TPNH oxidation (Tables I and II), and by a decrease in the incorporation of Cl4-labeled acetyl-CoA into long chain fatty acids (Table III).

* The abbreviation used is: HMB, p-hydroxymercuribenzoate.


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TABLE I

E$ect of thiol compounds, EDTA and sulfhydryl-binding agents on synthesis of fatty acids and acetoacetyl-Coil reductase

The reaction mixtures contained 30 pmoles of potassium phosphate buffer, pH 6.5, 50 mfimoles of TPNH, 20 fig of Rza, the indicated concentrations of the various sulfhydryl agents, EDTA, or sulfhydryl inhibitors, and water to a final volume of 0.38 ml. The reaction mixtures were incubated at 37” for 3 minutes, and the reaction was then started by the addition of 20 mpmoles of acetyl- CoA and 30.mpmoles of malonyl-CoA in the case of fatty acid synthesis, and by the addition of 60 mpmoles of acetoacetyl-CoA in the case of acetoacetyl-CoA reductase. The concentrations of the additions listed were those giving maximal stimulation or inhibition.

Addition Concentration

None Glutathione Cysteine Mercaptoethanol EDTA p-Hydroxymercuribenzoat

N-Ethylmaleimide

Cadmium chloride

M

2.5 X 10-4 2.5 X 1O-4

5 x 10-S 5 x 10-c 5 x 10-S

7.5 x 10-b 2.5 X lo- 1.2 x 10-S 2.5 X 1O-6 2.5 X 10-h 7.5 x 10-b

- TPNH oxidized

Fatty acid synthesis

Acet;r;tyl-

reductase

4.1 6.2 7.3

12.7 5.8 1.5 0.2 0.0 1.8 0.3 1.3 0.5

1.0 1.1

1.0

1.2 1.1 1.0 1.1 1.2

PHMB Mercapto- I Ethanol

B/( 2 4 6 8 IO 12 14 16

Minutes

FIG. 3. Reversal of HMB inhibition of fatty acid synthesis by mercaptoethanol. The reaction mixture contained 60 pmoles of potassium phosphate buffer, pH 6.5, 150 mpmoles of TPNH, 75 mrmoles of acetyl-CoA, 130 mpmoles of malonyl-CoA, 30 pg of Rz,, and water to a final volume of 0.5 ml. The reaction was run at 37” and followed spectrophotometrically at 340 rnp. When the reaction had run for 5 minutes, 30 mpmoles of HMB were added. After an additional 5 minutes, 2 pmoles of mercaptoethanol were added.

Protection against Inhibition of Fatty Acid Synthesis by HMB- Protection against the inhibition of fatty acid synthesis by sulfhydryl-binding agents such as HMB was afforded by various acyl-CoA’s (acetyl-CoA, propionyl-CoA, and butyryl-CoA). When the fatty acid-synthesizing enzymes were preincubated with these compounds before the exposure of the enzymes to the

TABLE II

Protection against inhibition of fatty acid synthesis by p-hydroxymercuribenzoate

The reaction mixtures contained 30 mpmoles of potassium phosphate buffer, pH 6.5, 100 mmoles of TPNH, 20 pg of Rz,, the indicated acyl-CoA’s in Experiments 3 to 6, and water to a final volume of 0.4 ml. The reactions were incubated at 37” for 3 minutes; then p-hydroxymercuribenzoate (HMB) to a final concentration of 2.5 X 10-E M was added to Experiments 2 to 6, and the mixtures were reincubated at 37” for 3 minutes. The synthetic reaction was then started by the addition of either 30 mpmoles of acetyl-CoA (Experiment 6), 30 mpmoles of malonyl- CoA, as in Experiments 3 to 5, or both, as in Experiments 1 and 2.

1 2 3

Reaction mixture

Regular reaction HMB 15 mpmoles of acetyl-CoA;

then HMB 75 mpmoles of propionyl-CoA;

then HMB 75 mpmoles of butyryl-CoA;

then HMB 25 mpmoles of malonyl-CoA;

then HMB

- I TPNH oxidized

mpmoles/min

3.8 0.6

3.2

2.3

1.4

0.8

sulfhydryl-binding agents, these compounds afforded various degrees of protection against the inhibition of fatty acid synthesis by the sulfhydryl-binding agents. This protection by acyl-CoA’s against the inhibition of fatty acid synthesis by sulfhydryl-binding agents (p-hydroxymercuribenzoate, N-ethylmaleimide) was evidenced by both an increase in TPNH oxidation and an increase in the incorporation of W-labeled substrates into long chain fatty acids (cf. Table III).

The protection of the fatty acid-synthesizing enzymes by the various acyl-CoA compounds was proportional to the amount of the acyl-CoA added during the preincubation period (cf. Fig. 4 and Table III).

Acetyl-CoA is the most effective agent in protecting against the inhibition. Both propionyl-CoA and butyryl-CoA protect, but to lesser extents. Acetyl-CoA (15 mpmoles) afforded 85% protection, whereas maximal protecting amounts of propionyl- CoA and butyryl-CoA (75 mpmoles of each) afforded only 60 and 35% protection, respectively (cf. Table II). The relation- ship between the amount of acyl-CoA added and the protection obtained is shown in Table III and Fig. 4. At higher levels of acyl-CoA there appears to be inhibition of fatty acid synthesis. This phenomenon of inhibition of fatty acid synthesis by higher levels of acyl-CoA’s has been demonstrated in the over-all process and in the absence of sulfhydryl inhibition (1).

Malonyl-CoA did not afford protection even at higher levels, as shown in Fig. 4. The slight protection shown was probably due to the enzymatic conversion of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (27,28). This enzyme has been found in trace amounts in the highly purified fatty acid-synthesizing enzyme preparations (Rz,) (1).

Longer chain acyl-CoA’s such as hexanoyl-Cob afforded little protection. The relative capacities of the various acyl-CoA’s to protect against the inhibition of fatty acid synthesis by sulfhydryl-binding agents reflects the same type of behavior that


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May 1962 R. Bressler and S. J. Wakil

these acyl-CoA’s show in the conversion of malonyl-CoA to long chain fatty acids and in the enzymatic decarboxylation of malonyl-CoA (1). These observations may be related to the over-all synthesis of fatty acids. It has been shown that acetyl-CoA, propionyl-CoA, and, to a lesser extent, butyryl- CoA are involved in the conversion of malonyl-CoA to long chain fatty acids in the presence of Rz, and TPNH (1). These acyl- CoA’s are also involved in the enzymatic decarboxylation of malonyl-CoA by Rza in the absence of TPNH. Acetyl-CoA was shown to be a better substrate for both the synthesis and the decarboxylation-condensation reactions than propionyl-Coil or butyryl-CoA. This was reflected in the Michaelis-Menten con- stants previously reported from this laboratory (1) : acetyl-CoA, 2.3 X lO+ M; propionyl-CoA, 5.7 X 1W5 M; butyryl-CoA, 3.9 X

10-4 M. The ability of these acyl-CoA’s to protect against inhibition of fatty acid synthesis by sulfhydryl-binding agents is proportional to their affinities for the fatty acid-synthesizing enzymes. These data point to a common center on the enzyme concerned with the decarboxylation-condensation reaction, the over-all synthesis of fatty acids, and the sulfhydryl groups of the enzyme. The acyl-CoA, i.e. acetyl-CoA, which promotes the best synthesis of fatty acids gives the best decarboxylation- condensation reaction and is the most efficient in protecting the sulfhydryl groups of the fatty acid-synthesizing enzymes against

TABLE III Effect of acyl CoA’s on inhibition of fatty acid synthesis by HMB

The reaction mixture contained 30 pmoles of potassium phosphate buffer, pH 6.5, 50 mrmoles of TPNH, and water to a final volume of 0.4 ml. Rzs. (20 rg) was added to experiments with acetyl-CoA, and 30 ,ug of Rzs were added to experiments with propionyl-Coil and butyryl-CoA. The mixture was incubated at 37” for 3 minutes, and the reaction was started by the addition of 30 mpmoles of malonyl-CoA and the indicated amounts of acyl-CoA (acetyl-CoA, 20 mpmoles, 50,000 counts; propionyl-CoA, 20 m/*moles, 30,000 counts; and butyryl-CoA, 40 mpmoles, 250,000 counts). The reaction proceeded for 5 minutes at 37”. The inhibited reaction had 30 mrmoles of HMB incubated with the reaction mixture for 3 minutes before the reaction was started by the addition of malonyl-CoA and the various acyl-CoA’s. The “protected” reactions had the indicated amounts of the various acyl-CoA’s incubated with the enzyme fraction Rz, for 3 minutes before the addition of 30 mrmoles of HMB. The mixture was then incubated for an additional 3 minutes, and the reaction was started by the addition of malonyl-CoA. The formed fatty acids were extracted in pentane and counted in a Packard Tri- Carb scintillation spectrometer.

Reactions

Acyl-CoA incorporated into palmitate

1. Acyl-CoA + malonyl-CoA.. 2. Inhibited reaction. . . 3. Protected with 20 mpmoles of acyl-

CoA . . 4. Protected with 40 mpmoles of acyl-

CoA 5. Protected with 75 mpmoles of acyl-

CoA . 6. Protected with 100 mpmoles of acyl-

CoA . . .

2.1 2.6 0.5 0.4 0.6 0.1

1.3 0.8 0.2

1.9

1.2

1.4

2.1

1.5

0.3

0.2

0

20 40 60 80 100 120 mp moles Prolecting Acyl CoA

FIG. 4. The effect of acyl-CoA’s on the inhibition of fatty acid synthesis by HMB. The reactions were carried out as described in Table III. In the reaction series in which melonyl-CoA was used as the protecting agent the reaction was started by the addition of acetyl-CoA. Fatty acid synthesis was measured by the rate of TPNH oxidation. The rates of TPNH oxidation per 5 minutes with the various acyl-CoA’s and malonyl-CoA were as follows: acetyl-CoA, 140 mpmoles; propionyl-CoA, 95 mpmoles; butyryl-CoA, 65 mbmoles; malonyl-CoA alone, 45 mpmoles.

inhibition by sulfhydryl-binding agents. Experiments 3 and 4 of Table V show that an inhibition of the decarboxylation-condensation reaction between acetyl-CoA and malonyl-CoA is effected by the inhibition by HMB. Here, too, preincubation of the enzyme with acetyl-CoA before the exposure of the enzyme to HMB protects against the HMB inhibition. It seems likely that the inhibition of fatty acid synthesis by sulfhydryl-binding reagents halts the initial condensation-decarboxylation reaction.

Effect of Arsenite on Rza Activity-The observation that arsenite inhibits the synthesis of long chain fatty acids was first reported by Brady, Mamoon, and Stadtman (29). In contrast to the protection against HMB inhibition afforded by mercaptoethanol, sulfhydryl stimulation of the enzyme system markedly augments the inhibition by arsenite (30). The effect of arsenite on the synthesis of fatty acids is shown in Table IV. The arsenite inhibition is only obtained in the presence of mercaptoethanol. The inhibition by arsenite is 85% at a concentration of 2.5 X low6 M in the presence of mercaptoethanol, whereas a concentration of 2.5 X 1c3 M arsenite is not inhibitory in the absence of mercaptoethanol. This is in marked contrast to the inhibition of fatty acid synthesis by HMB, which inhibits 85% at 2.5 x 10e5 M concentrations in the absence of added mercaptoethanol and whose inhibitory action is not manifest at all in the presence of added sulfhydryl compounds. In further contrast to the inhibition of fatty acid synthesis by HMB, the enzymes cannot be protected by preincubation with acyl-CoA’s before exposure to the arsenite (See Tables IV and V).

Our previous work has shown that the activity of the TPNH- linked acetoacetyl-Cob reductase system is not affected by either arsenite or sulfhydryl-binding agents of the HMB type (2). Table V shows that the enzymatic decarboxylation of malonyl- CoA which occurs upon its condensation with acetyl-CoA is inhibited by both arsenite and HMB. Mercaptoethanol pretreat-


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TABLE IV

Effect of mercaptoethanol and acetyl-CoA on inhibition of fatty acid synthesis by arsenite

The reaction mixtures contained 30 rmoles of histidine buffer, pH 6.5,50 mpmoles of TPNH, 20 pg of Rs,,where indicated, 2 pmoles of mercaptoethanol (Reactions 2,4,5,6) or arsenite (Reaction 3), and water to a final volume of 0.4 ml. The reactions were incubated at 37” for 3 minutes, and then additions were made where indicated of arsenite (Reaction 4) or acetyl-CoA (Reactions 5 and 6). The reactions were incubated for an additional 3 minutes, and then arsenite to a final concentration of 2.5 X 10m6 M was added to Reactions 5 and 6. The incubation mixtures were again incubated for 3 minutes at 37”, and then the synthetic reactions were started by the addition of 40 mpmoles of malonyl-CoA (Reactions 5 and 6) or 40 mrmoles of acetyl-CoA and 40 mpmoles of malonyl-CoA (Reactions 1 to 4). The reactions ran for 5 minutes at 37” and were followed spectrophotometrically at 340 w.

Reaction TPNH oxidized

mpmoles/5 min

1. Acetyl-CoA + malonyl-CoA.. . 2. Acetyl-CoA + malonyl-CoA + mercapto-

8.3

ethanol. _...__.___._....__.._...._.,..... 3. Arsenite _.__..__.............._........... 4. Mercaptoethanol; then arsenite . 5. Mercaptoethanol; then 40 mpmoles of acetyl-

20.8 8.8 3.1

CoA; then arsenite.. . . . . 6. Mercaptoethanol; then 75 mpmoles of acetyl-

3.8

CoA; then arsenite.. . . 3.5

ment of the enzymes reverses the HMB inhibition and potentiates the arsenite inhibition of the reaction. The behavior of mercaptoethanol in the condensation reaction is similar to its effect on HMB and arsenite in the over-all synthesis of fatty acids. Here again, acetyl-CoA protects against HMB inhibition but is ineffective against the inhibition by arsenite.

Brady has suggested that the synthesis of fatty acids requires closely juxtaposed enzyme sulfhydryl groups (6, 30). It has been shown that arsenite forms relatively nondissociable com- plexes with closely situated sulfhydryl groups of lipoate-linked and nonlipoate-linked enzyme reactions (31). The highly purified fatty acid-synthesizing enzyme complex obtained after purification of the pigeon liver supernatant (R& was assayed for lipoic acid by a microbiological assay.2 The assay indicated that there was an insignificant amount of lipoate in the enzyme preparation (1 mole of lipoic acid per 82 X lo6 g of protein). The data presented here lend support to the involvement of sulfhydryl groups in the synthesis of fatty acids. The sensitiv- ity of the condensing reaction to inhibition by arsenite further supports the concept of vicinal sulfhydryl groups (6, 30).

DISCUSSION

The data presented in this communication concern the role of sulfhydryl groups in fatty acid synthesis. The following points are made. (a) Sulfhydryl stimulation of the Rza enzymes markedly increases the synthesis of long chain fatty acids from acetyl- CoA and malonyl-CoA in the presence of TPNH and greatly augments the decarboxylation-condensation reaction of malonyl-CoA and acetyl-CoA in the absence of TPNH. (b) The inhibition of fatty acid synthesis by sulfhydryl-binding agents is

2 We are greatly indebted to Dr. Lester J. Reed of the Uni- versity of Texas at Austin for the lipoate assays.

accompanied by a marked decrease in the decarboxylation-condensation of malonyl-CoA and acetyl-CoA. (c) Preincubation of an acyl-CoA (acetyl-CoA, propionyl-CoA, butyryl-CoA) with the Rza enzymes before the exposure of these enzymes to sulfhydryl-binding agents protects against the inhibition effected by these agents. (d) The protection afforded by these acyl- CoA’s against inhibitions by sulfhydryl-binding agents is proportional to the affinities of these acyl-CoA’s for the enzymes of fatty acid synthesis. Acetyl-CoA was the most effective acyl-CoA in protecting against inhibition by sulfhydryl-binding agents. Propionyl-CoA protected less well, and butyryl-CoA, to an even lesser degree. The degree of protection afforded by these acyl-CoA’s is of the same order as their ability to convert malonyl-CoA to fatty acids in the presence of Rza and TPNH, and as their capacity to effect the enzymatic decarboxylation and condensation of malonyl-CoA in the presence of Rza and the absence of TPNH. The excellent protection afforded by acetyl-CoA against inhibition by sulfhydryl-binding agents and the failure of malonyl-CoA to protect significantly against HMB inhibition indicates that the acyl-CoA is first bound at the locus of synthesis. This would support the concept previously reported that the type of fatty acid synthesized is determined by the acyl-CoA moiety (1, 32). The bound acyl-CoA serves as the anchor point or “tail” at the locus of synthesis.

Recently Lynen and Tada (5) presented a scheme for the synthesis of long chain fatty acids in yeast enzymes which was similar to an earlier mechanism proposed by Lardy for the @ oxidation of fatty acids by the “fatty acid oxidase” (33). They have demonstrated the sulfhydryl nature of the enzyme system and proposed the scheme shown in Equations 2 to 5. The scheme involves the participation of free acyl-CoA’s such as C&oA, C&oA, etc. condensing with a malonyl-S-enzyme complex

TABLE V

Effect of HMB and arsenite on enzymatic decarboxylation of HOOCWH~COSCoA

The reaction mixtures contained 30 pmoles of histidine buffer, pH 6.5,20 rg of Rzp, 2 pmoles of mercaptoethanol where indicated, and water to a final volume of 0.5 ml. The mixtures were incubated at 37’ for 3 minutes, and HMB to a final concentration of 5 X 10e6 M (Reaction 3)) arsenite to a final concentration of 2.5 X 10m3 M (Reaction 5), or 30 mpmoles of acetyl-CoA (Reactions 4 and 6) were added. Mixtures were again incubated for 3 minutes, and HMB was added to Reaction 4 and arsenite was added to Reaction 6. The reaction mixtures were incubated for an additional 3 minutes, and the reactions were started by the addition of 60 mpmoles of malonyl-CoA (40,000 counts) and 30 mpmoles of acetyl-CoA to Reactions 1, 3, and 5 or by the addition of the malonyl-CoA alone to Reactions 2,4, and 6. Reactions were run for 10 minutes.

Reaction

Counts Trapped as C”Oz

1. Acetyl-CoA + malonyl-CoA 2. Malonyl-CoA alone. 3. HMB, . . . . 4. Acetyl-CoA (30 mpmoles); then

HMB 5. Arsenite. 6. Acetyl-CoA (30 mpmoles); then

arsenite . . . . .I

2030 1010 600 270

2070 335

2140 720 970 920

880 730


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May 1962 R. Bressler and S. J. Wakil

as shown. We have been unable to isolate any acyl-CoA’s during the synthesis of long chain fatty acids from acetyl-CoA and malonyl-CoA in the highly purified pigeon liver preparation (1, 2, 8, 12, 14), nor has the isolation of these intermediates ever been reported. We have also been unable to incorporate these longer chain fatty acyl-CoA’s (C&oA, C&oA) into fatty acids with the Rza enzymes, TPNH, and malonyl-CoA (1, 2, 4, 11). We have also shown that the malonyl-CoA decarboxylation reaction is dependent on the presence of acetyl-CoA or propionyl- CoA as a condensing partner, and that the longer acyl-CoA’s (C$,CoA, etc.) do not substitute for acetyl-CoA in this sequence (1). The Lynen scheme fails to account for the product(s) of condensation of acetyl-CoA and malonyl-CoA in the absence of TPNH. This compound, which can be formed from acetyl- CoA, propionyl-CoA, or butyryl-CoA condensing with malonyl- CoA, has an absorption maximum at 275 rnp. Its role in fatty acid synthesis has not yet been resolved (1,2). This unidentified compound absorbing at 275 rnp has never been observed in the fatty acid reaction mixtures containing acetyl-CoA, malonyl- CoA, TPNH, and Rza even when the reaction rate was slowed in the cold. It is most likely from these data that the rate-limiting step in the over-all synthesis is not the reductive one. If the reductive steps were the rate-limiting ones, then some intermediate products of the condensation of acetyl-CoA and malonyl- Co,4 would have been seen in the reaction slowed by the cold. The condensation compound which is characterized by an absorption maximum at 275 rnp may represent an unreduced intermediate in the synthesis. However, the failure to convert this compound to long chain fatty acids (1) makes it likely that if it is an intermediate in the synthesis of fatty acids it has undergone some “inactivating” change, i.e. deacylation of a CoA derivative, ‘(falling off” the enzyme, “cyclization,” or loss of a cofactor. The evidence thus far obtained (1, 2, 4, 11) leads us to propose the following scheme for the synthesis of long chain fatty acids by the soluble pigeon liver system. (a) Both malonyl-CoA and acetyl-CoA are bound to the enzymes of fatty acid synthesis by sulfhydryl groups. (b) The enzyme-bound acyl groups undergo a decarboxylation-condensation reaction, which may be followed either by reduction, dehydration, and reduction again to form an enzyme-bound saturated Cq compound or by repeated condensations of multiple malonyl groups on the acetyl moiety to form an enzyme-bound keto compound. The carbonyl groups of the ketoacyl compound are then successively reduced by TPNH, dehydrated, and reduced again to the saturated acyl derivative, which is then deacylated to the free fatty acid. In the absence of TPNH, the ketoacyl compound is deacylated and set free in the medium as the “275compound.” The latter deacylation process is slower than the palmitate formation. This is illustrated in Scheme 1.

“275-compound” + E

Acetyl-CoA malonyl-CoA or T

slow -k HS--E +

[acetyl-S--El [malonyl--El

f [keto,acyl--El + COZ + CoA

TPNH (malonyl-CoA?)

[palmityl--E]

I Hz0

palmitate + E

SCHEME 1

Compounds such as the one proposed here have been proposed in biological synthetic systems by organic chemists (34) and in a fatty acid synthesis scheme by Knox (35). This scheme would account for the lack of acyl-CoA intermediates and explain the key role of the sulfhydryl groups in the decarboxylation-condensation reaction and in the over-all synthesis. The nature of the condensation product(s) is under investigation. The likeli- hood of two sulfhydryl groups being involved is apparent.

SUMMARY

Fatty acid synthesis by a highly purified preparation from pigeon liver is stimulated by sulfhydryl agents and inhibited by sulfhydryl-binding agents and arsenite. Acetyl coenzyme A (CoA) protects against the inhibition of fatty acid synthesis by sulfhydryl-binding agents, and in this capacity it may be partially substituted for by propionyl-Coil and to a lesser extent, by butyryl-CoA. The final product of fatty acid synthesis has been shown to be free palmitic acid and not palmityl-CoA. The product of the condensation of acetyl-CoA and malonyl-CoA in the absence of reduced triphosphopyridine nucleotide has been discussed. A mechanism of fatty acid synthesis is proposed and discussed.

Acknowledgment-The authors wish to acknowledge the com- petent technical assistance of Miss G. Martin.

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Rubin Bressler and Salih J. WakilSYNTHESIS OF FATTY ACIDS

THE REACTION AND THE ROLE OF SULFHYDRYL GROUPS IN THE Studies on the Mechanism of Fatty Acid Synthesis: XI. THE PRODUCT OF

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