ENZYMES OF FATTY ACID METABOLISM

IV. PREPARATION AND PROPERTIES OF COENZYME A TRANSFERASE*

BY JOSEPH R. STERN,t MINOR J. COON,1 ALICE DEL CAMPILLO,$ AND MORTON C. SCHNEIDER

(From the Department of Pharmacology, New York University College of Medicine, New York, New York)

(Received for publication, October 28, 1955)

Evidence for the reversible enzymatic transfer of CoAl from succinyl-s- CoA to acetoacetate according to Reaction 1 has been presented in Paper III (1). The enzyme catalyzing Reaction 1 has been named succinyl-/3-

(1) Succinyl-S-CoA- + acetoacetate- $ acetoacetyl-S-CoA- + succinate- + H+

ketoacyl-S-CoA transferase (2) and will be referred to simply as CoA transferase or transferase.2 The first enzyme of this type was described by Stadtman (3) in Clostridium kluyveri extracts and named CoA trans- phorase. It catalyzes the reversible transfer of CoA from acetyl-S-CoA to Ca to Cg monocarboxylic saturated fatty acids, vinylacetic and lactic acids, but not to @-keto acids. This paper describes the preparation and prop- erties of highly purified CoA transferase, including substrate specificity and affinity, pH dependence, and equilibrium constants. The distribu- tion of the enzyme and its significance in cardiac metabolism are discussed. A preliminary account of this work has appeared (4).

* Aided by grants from the United States Public Health Service, the American Cancer Society (recommended by the Committee on Growth, National Research Council), and by a contract (NBonr279, T. 0.6) between the Office of Naval Research and New York University College of Medicine.

t Present address, Department of Pharmacology, School of Medicine, Western Reserve University, Cleveland 6, Ohio.

$ Special Fellow of the National Institutes of Health, United States Public Health Service. Present address, Department of Biochemistry, School of Medicine, Uni- versity of Michigan, Ann Arbor, Michigan.

0 Present address, Department of Biochemistry, School of Medicine, University of Puerto Rico, San Juan, Puerto Rico.

1 The abbreviations employed in Paper III (1) are adhered to. In addition, op- tical density (log lo/Z) is abbreviated as E (extinction). aErlo stands for change in optical density at wave-length 310 mp. EDTA = ethylenediaminetetraacetic acid, FAD = flavin-adenine dinucleotide.

2 A Committee on Nomenclature, 2nd International Conference on Biochemical Problems of Lipides, Ghent, Belgium, July, 1955, has proposed the systematic name acetoacetylsuccinic thiophorase for CoA transferase.

15

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16 ENZYMES OF FATTY ACID METABOLISM. IV

Enzyme Assay and Unit

The direct optical assay is based on the finding of Lynen et al. (5) that, at alkaline pH, acetoacetyl thio esters, e.g. X-acetoacetyl-N-acetylthio- ethanolamine (6) and acetoacetyl-S-CoA (1, 6), exhibit a characteristic absorption band in the range 280 to 320 rnp, with a maximum at 303 rnp. This absorption is attributed to formation of an enolate ion and is in- creased by a decrease in Hf ion concentration. Stern et al. (4) observed that this enolate absorption of acetoacetyl CoA is markedly increased by WP ions, probably through formation of a chelate compound. The presence of Mg++ ions, which are not required for CoA transferase activity, increases the sensitivity of the optical assay. Succinyl CoA has no sig- nificant absorption in the enolate ion region. It is important to note that the enolate ion absorption is abolished by 10Y4 M Hg ions (7) and by 6 X 10h3 M L-cysteine, but not by glutathione3 or thioglycolate.4

The optical assay is performed at wave-length 310 rnp with a Beckman spectrophotometer in 2.0 ml. silica cells of 0.5 cm. light path. To the experimental cells are added 0.10 ml. of 1.0 M Tris-HCl buffer, pH 8.1, 0.01 ml. of 0.8 M MgC12, 0.10 ml. of 1.0 M potassium acetoacetate, 0.20 ml. of 0.0018 M suceinyl CoA, enzyme, and water to a final volume of 1.50 ml. The reference cell contains all the assay components except succinyl CoA. The enzyme is diluted with 0.02 M potassium phosphate buffer, pH 7.4, so that 0.01 ml. contains 3 to 40 y of protein, depending on its activity. The reaction is started by addition of enzyme and the in- crease in optical density at 310 rnp is recorded at 0.5 minute intervals at 25”. The AEOU from 0.5 to 1.0 minute after addition of enzyme is used to calculate its activity.

1 unit of enzyme is defined as the amount which causes an initial rate of increase in optical density (+AEalO) of 0.01 per minute under the above conditions. As illustrated in Fig. 1, the initial rate of +AE~uI is propor- tional to enzyme concentration, provided it does not exceed 0.04 per minute. The initial rate is not maintained beyond 2 to 3 minutes, prob- ably because of the unfavorable equilibrium position when the reaction is measured in this direction. The specific activity is expressed as units per mg. of protein. Protein is determined spectrophotometrically by the method of Warburg and Christian (8). The molecular extinction coeffi- cient (~~0) of acetoacetyl CoA under the assay conditions (pH 8.1, Mg++ present) is 11,900. Therefore, 1 unit corresponds to the formation of 0.0025 pmole of acetoacetyl Co*4. It should be noted that the direct assay can be modified to measure the favored enzymatic conversion of acetoacetyl CoA to succinyl CoA (see below).

3 F. Lynen, personal communication. 4 J. R. Stern, unpublished observations.

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STERN, COON, DEL CAMPILLO, AND SCHNEIDER 17

Assay of Crude Tissue Extracts-The direct optical method of assay can only be employed with enzyme fractions which are free of acetoacetyl thiolase. Thiolase is removed in Steps 3 and 4 of the purification proce- dure. With cruder fractions and tissue extracts, the transferase can be assayed optically by coupling with thiolase and trapping the acetyl-S-CoA thus generated with oxalacetate, with use of the malic dehydrogenase- condensing enzyme system (9, 10) or with p-nitroaniline and the aromatic

0.06

0 0 2 4 6

TRANSFERASE (pg)

FIG. 1. Optical assay of transferase. The assay conditions are given in the text. Transferase, specific activity 900, was used.

amine-acetylating enzyme of pigeon liver (11, 12). The sequence of re- actions is as follows :

(2) Succinyl-S-CoA + acetoacetate $ acetoactyl-S-CoA + succinate

(3) Acetoacetyl-S-CoA + HS-CoA E& 2 acetyl-S-CoA

(4a) 2 Malate + 2DPN+ + 2 acetyl-S-CoA e

2 citrate + 2HS-CoA + 2DPNH + 2H+

(4b) 2 Acetyl-S-CoA + 2 p-nitroaniline --) 2 p-nitroacetanilide

The reduction of DPNf can be followed at wave-length 340 rnp and the acetylation of p-nitroaniline at 420 rnp (12).

The components of the DPN+ reduction assay (in micromoles) are Tris- HCl buffer, pH 8.1, 100; MgCL, 8; potassium acetoacetate, 100; CoA, 0.2; neutralized GSH, 10; DPN+, 0.3; potassium L-m&late, 10; succinyl CoA, 0.35; purified thiolase, 200 units (specific activity 15,000); purified malic dehydrogenase, 2 y (specific activity 62,000); crystalline condensing enzyme, 16 y (specific activity 330); and CoA transferase fraction, 3 to 250 y. The reaction is started by addition of succinyl CoA. 1 unit of transferase is defined as the amount which causes an initial rate of increase in optical density at 340 rnp of 0.01 per minute at 25”. 1 unit corresponds to the formation of 0.0048 pmole of DPNH or 0.0024 pmole of acetoacetyl CoA.

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18 ENZYMES OF FATTY ACID METABOLISM. IV

If the extract is contaminated with DPNH oxidase (the use of cyanide as inhibitor may be tried (lo)), the p-nitroaniline assay should be used. The components of the latter (in micromoles) are Tris-HCl buffer, pH 8.1, 100; MgC12, 8; potassium acetoacetate, 100; CoA, 0.2; neutralized GSH, 10; succinyl CoA, 0.35; p-nitroaniline, 0.3; purified pigeon liver acetylating enzyme, 0.3 mg. (specific activity 150 to 200) ; and transferase fraction. Purified thiolase (200 units) must also be added after Step 2 if not already supplied in excess by the acetylating enzyme fraction employed. The re- action is started by addition of succinyl CoA. 1 unit of transferase is de- fined as the amount which causes an initial rate of decrease in optical density at 420 rnp of 0.01 per minute at 25”. 1 unit corresponds to the formation of 0.0050 pmole of p-nitroacetanilide or 0.0025 pmole of aceto- acetyl CoA.

PuriJication of Enzyme

Step 1. Preparation of Extract-Pig hearts, removed immediately after death, are packed in ice. All subsequent operations are performed at O-3” unless otherwise indicated. Thirty hearts are trimmed of fat, blood clots, and connective tissue and passed twice through an electric mincer. The minced heart (about 4.5 kilos) is washed five times with 5 volumes of cold tap water in a tall glass cylinder (capacity 37 liters). Cylindrical blocks of ice (volume about 800 ml.) are used to keep the suspension cold. The washed mince is then passed through a large table type Biichner filter and dried as far as possible by suction. 170 gm. portions of the washed mince are placed in a Waring blendor and 1.5 volumes (255 ml.) of 0.05 M potassium phosphate buffer, pH 7.4, containing 0.2 M KC1 are added. The suspension is stirred for 5 minutes at two-thirds the maximal speed; then another 1.5 volumes of buffer mixture are added and the stirring is continued for another 5 minutes at one-third maximal speed. The sus- pension is passed through two layers of cheese-cloth and centrifuged in a Servall angle centrifuge at 13,000 X g for 7 minutes. The deep pink supernatant fluid is passed through ten layers of cheese-cloth to remove floating fat particles. About 10 liters of extract are obtained.

Xtep 2. Fractionation with Ammonium Sulfate-5 liter portions of the extract are brought to 35 per cent saturation with powdered ammonium sulfate (245 gm. per liter). The salt is added slowly over a period of 20 minutes with mechanical stirring. The mixture is stirred for a further 20 minutes and centrifuged at 13,000 X g for 15 minutes. The precipitate is discarded. The supernatant fluid is brought to 65 per cent saturation by adding 210 gm. of ammonium sulfate for each liter of original solution in the manner indicated above. The precipitate is dissolved in 500 ml. of 0.017 M potassium phosphate buffer, pH 6.8, and dialyzed against 20 liters of the same buffer for 16 hours.

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STERN, COON, DEL CAMPILLO, AND SCHNEIDER 19

Step 3. Fractionation with Acetone-The 35-65 ammonium sulfate frac- tion is made 0.067 M with respect to potassium phosphate buffer, pH 6.8. Cold acetone (- 10’) is then added to a concentration of 40.5 per cent by volume with mechanical stirring, the temperature of the mixture being gradually lowered to -5’ during the 30 to 40 minute period required for the addition. Stirring is continued for another 15 minutes at -5’. The mixture is centrifuged at 1100 X g for 30 minutes at a temperature setting low enough to insure that the temperature of the supernatant solution will be -5” to -6”. The precipitate is dissolved in 150 ml. of 0.017 M potas- sium phosphate buffer, pH 7.4, and the solution is dialyzed overnight against 20 liters of the same buffer. This O-40.5 fraction is used for the purification of thiolase. The cloudy supernatant fluid is brought to 57 per cent acetone concentration by volume at - 9” to - 10” and, after cen- trifugation at this temperature, the precipitate is dissolved in 150 ml. of 0.017 M potassium phosphate buffer, pH 7.4, and the solution is dialyzed overnight against 20 liters of the same buffer.

In practice, stocks of the O-40.5 and 40.5-57 acetone fractions are ac- cumulated as starting material for the further purification of thiolase and transferase, respectively, and kept frozen. Transferase is remarkably stable at all stages of purification and can be kept in the deep freeze for many months with little or no loss of activity.

Step 4. Heat and Acid Treatment-The 40.5-57 acetone fraction is diluted with 0.017 M potassium phosphate buffer, pH 7.4, to bring the pro- tein concentration to 10 mg. per ml. 300 ml. portions, in a 1 liter glass beaker, are placed in a bath at 55” and stirred constantly. The tempera- ture of the solution is allowed to rise to 50” during an interval of 9 minutes, the bath temperature falling to about the same reading. The solution is maintained at 50’ for another 6 minutes and then placed in an ice bath. The precipitate is removed by centrifugation at 13,000 X g for 20 minutes and discarded. About 65 ml. of 0.1 N acetic acid are added rapidly with mechanical stirring to the supernatant fluid until the pH falls to 5.8. The precipitate is removed by centrifugation at 13,000 X g for 5 minutes and discarded.

Step 5. Adsorption on Alumina Gels--The solution from Step 4 is quickly adjusted to pH 6.65 by addition of about 4 ml. of 1.0 M potassium bicarbonate. Alumina C-y gel (dry weight, 13 mg. per ml.) is added to make a final concentration of 30 per cent by volume and the mixture is stirred for 10 minutes at 0”. The gel is separated by centrifugation and the supernatant fluid is discarded. The gel is suspended in a volume of cold 0.1 M potassium phosphate buffer, pH 7.4, equal to that of the starting solution and stirred continuously for 10 minutes at 0”. After centrifuga-

6 We are indebted to Dr. S. Kaufman and Dr. C. Gilvarg, who first worked out this step for the purification of P enzyme from pig heart.

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20 ENZYMES OF FATTY ACID METABOLISM. IV

tion, this eluate (718 ml. containing 1750 mg. of protein, specific activity 4) is discarded and the gel is again eluted by suspending it in a similar volume of 0.2 M potassium phosphate buffer, pH 8.0 (20”), and stirring at 20” for 20 minutes. After removal of gel by centrifugation, the eluate at pH 8, containing about 1 mg. of protein per ml., is concentrated by addi- tion of solid ammonium sulfate to 85 per cent saturation, centrifuging, and dissolving the precipitate in a volume of 0.017 M Tris buffer, pH 7.5, equal to 5 per cent of that of the eluate. This fraction is dialyzed overnight against the same buffer to remove residual inorganic phosphate as well as ammonium sulfate.

Step 6. Ethanol FTacfionation in Presence of Zinc-The dialyzed eluate at pH 8 is diluted to a protein concentration of about 5 mg. per ml. with 0.017 M Tris buffer, pH 7.5, and 0.025 volume of 1.0 M potassium suceinate, pH 6.2, is added. 0.25 volume of 0.1 M zinc acetate is added slowly with stirring at 0’. After 5 minutes the mixture is centrifuged and the pre- cipitate (89.4 mg. of protein, specific activity 4) is discarded. Cold abso- lute ethanol is added to the clear supernatant fluid at 0” to a concentration of 15 per cent by volume and, after stirring for 10 minutes, the mixture is centrifuged and the red brown precipitate (262 mg. of protein, specific activity 12) is discarded. The supernatant solution is brought to 35 volumes per cent ethanol concentration, stirred for 10 minutes at O”, and then centrifuged. The yellowish precipitate is dissolved in 10 ml. of 0.1 M potassium phosphate buffer, pH 7.4, containing 0.01 M potassium EDTA and 0.1 per cent GSH, and the solution is dialyzed overnight against 0.017 M potassium phosphate buffer, pH 7.4. Any turbidity which may appear is removed by centrifugation, leaving a clear yellow solution.

Step 7. Fractionation with Ammonium Sulfate-The procedure is that of Step 2. The 15-35 zinc-ethanol fraction is diluted to a protein concen- tration of 5 mg. per ml. with 0.017 M potassium phosphate buffer, pH 7.4, and then brought to 45 per cent saturation with ammonium sulfate. The precipitate (5.6 mg. of protein, specific activity 168) is discarded and the supernatant fluid obtained after centrifugation is brought to 58 per cent saturation with ammonium sulfate. This precipitate is collected and dis- solved in a few ml. of 0.017 M potassium phosphate buffer, pH 7.4, to give a clear yellow solution which is dialyzed overnight against the same buffer. Data on the purification of the enzyme are summarized in Table I.

Properties of Enzyme

Purity-The 45-58 ammonium sulfate fraction is practically the most active transferase preparation which has been obtained. Its specific ac- tivity falls in the range 1000 f 50. This fraction, concentrated solutions of which were visually yellow, contained a fluorescent flavoprotein. Ul-

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STERN, COON, DEL CAMPILLO, AND SCHNEIDER 21

tracentrifugal analysis6 showed that it contained two protein components. The major component, comprising about 80 per cent of the total protein, was the transferase (see below). The minor component, which sedi- mented somewhat less rapidly and represented about 20 per cent of the protein, was the flavoprotein. The sedimentation constant (sZ~,~) of the CoA transferase component was 5.08 X 1FL3 per second for a 1.22 per cent solution in 0.02 M potassium phosphate buffer, pH 7.4. Assuming a partial specific volume of 0.75 and a frictional ratio (f:fo) between 1.0 and 1.8, the molecular weight would be from 46,000 to 112,000.

TABLE I

PuriJicatio,n o.f CoA Transferuse

step x0.

1. Phosphate extract?. ......... 10,500 181,000 151,000 2. (NH,)zSOh (35-65). .......... 1,240 156,000 25,900 3. Acetone ppt. (40.5-57). ...... 474 86,700 8,250 4. Acid supernatant fluid ....... 723 68,300 4,880 5. Alumina eluatefi ............ 46 63,400 576 6. Zinc-ethanol (1535). ........ 15.t 42,700 95.1 7. (NHh)zS04 (45558). .......... 1.z 34,300 34.3

Volume

mz.

Units Protein

mg. 1

Specific activity’

units per ng. protein

1.21 6.05

10.55 14.011

w 449/l

10001/

Yield

per cent

100 86 48 38 35 24 18

* Calculated in units of direct assay. t From 4.5 kilos of heart. .$ p-Nitroaniline assay. 0 DPN+ reduction assay. 11 Direct assay. 7 After concentration.

Identijkation of Components-Paper electrophoresis7 confirmed the pres- ence of two protein components in the 45-58 ammonium sulfate fraction. The migration of the flavoprotein could be readily followed by its fluores- cence in ultraviolet light. In a typical experiment, 0.42 mg. of purified enzyme (specific activity 1000) was placed on a paper strip (Whatman No. 1) and run with a current strength of 1.0 ma., a solvent of 0.05 M potassium phosphate buffer, pH 7.9, and a temperature of 3’. After 20 hours, the position of the components was determined on a separate paper strip con- taining 0.11 mg. of the enzyme and treated identically. On both strips the flavoprotein had migrated 5.4 f 0.2 cm. toward the anode. Staining the

6 We are indebted to Dr. I. B. Wilson, College of Physicians and Surgeons, Colum- bia University, for the ultracentrifuge runs.

7 We are grateful to Dr. C. V. Tondo and Dr. K. G. Stern, Polytechnic Institute of Brooklyn, for use of their facilities.

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22 ENZYMES OF FATTY ACID METABOLISM. IV

control strip with Amido Schwarz 10B revealed, in addition, a second pro- tein component, which had migrated 4.0 & 0.4 cm. toward t)he anode. The corresponding areas of the experimental strip were eluted wit’h 1.0 ml. of 0.1 M potassium ph0sphat.e buffer, pH 7.4. The eluted proteins representing 69 per cent recovery were assayed enzymatically. The slower moving protein gave a positive transferase assay. Its specific activity of 83 indicated considerable inactivation of the enzyme during the electro- phoretic run, The flavoprotein gave a negative assay for transferase and a positive diaphorase test (see below). When subjected to paper electro- phoresis in 0.05 M potassium phosphate buffer, pH 6.0, the flavoprotein migrated toward the anode and the transferase toward the cathode. This suggested that the isoelectric point of the flavoprotein was below, and that of transferase above, pH 6. Densitometer analysis of the stained paper electrograms gave a calculated 77 per cent of the total protein as trans- ferase.

Nature of Flavoprotein-The absorption spectrum of the solution of the 45-58 ammonium sulfate fraction was typical of a flavoprotein and showed three absorption bands with maxima at 278,355, and 455 mp. The pros- thetic group of the flavoprotein could be resolved by heat denaturation of the protein (pH 7.4), but not by acidification to pH 2.3 in 45 per cent ammonium sulfate solution (12). It was identified as flavin-adenine dinu- cleotide by its reactivity with n-amino acid oxidase apoprotein (13). The native flavoprotein in this fraction gave negative tests for diphosphopyri- dine nucleotide- and triphosphopyridine nucleotide-linked cytochrome c reductases, D- and L-amino acid oxidases, and fumarate reductase. It possessed fairly active diaphorase activity and, in an optical test system (d = 0.5 cm.) consisting of 0.067 M Tris-HCl buffer, pH 8.5, 2 X 10d4 M DPNH, and 0.9 X 10e4 M 2,6-dichlorophenolindophenol in a volume of 1.50 ml., caused a - AEc20 of 0.8 per mg. of total protein. Under the same conditions the flavoprotein component isolated by paper electrophoresis causes a - AE620 of 1.0 per mg. of protein, suggesting some inactivation on electrophoresis. While several of its properties, fluorescence, tightly bound FAD, and absorption spectrum, paralleled those of Straub’s heart di- aphorase (14), no further attempt was made to characterize this flavopro- tein enzyme.

Removal of Flavoprotein-The flavoprotein can be largely, but not en- tirely, removed from the 45-58 ammonium sulfate fraction (2.0 mg. of protein per ml.) by adsorption on 0.12 volume of alumina CT gel at pH 6.0. Higher concentrations of gel result in significant adsorption of the transferase. The specific activity of the CoA transferase in the gel super- natant fluid, which shows very slight fluorescence under ultraviolet light, is thereby increased about 20 per cent. The flavoprotein itself is readily eluted from the alumina C-r with 0.2 M potassium phosphate buffer, pH

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STERN, COON, DEL CAMPILLO, AND SCHNEIDER 23

8.0. Assuming that the flavoprotein contains one flavin group per mole- cule and that the molecular extinction coefficient of flavoproteins at wave- length 455 ml* (~455) is 1040 (15), it can be calculated that its minimal molec- ular weight is 75,000.

Turnover Number-The specific activity of the purest transferase, which is obtained after alumina CT t,reatment and represents practically pure enzyme, falls in the range 1200 f 50. Assuming that the pure CoA transferase would have a specific activity of 1300, it would catalyze the transfer of 325 moles of CoA per minute from succinyl-S-CoA to aceto- acetate per 100,000 gm. of enzyme at pH 8.1 and 25”. As shown below, the enzyme catalyzes the transfer of CoA from acetoacetyl-S-CoA to suc- cinate at a rate about 25 times faster than that of the reverse reaction cor-

TABLE II

Substrate Speci$city of PuriJied Transjerase

The specific activity was determined under standard assay conditions with 100 pmoles of P-keto acid. Activity of acetoacetate taken as 100.

P-K&o acid Specific activity Relative activity Relative activity*

Acetoacetate.......... p-Ketovalerate. P-Ketoisocaproate. p-Ketocaproate. P-Ketooctanoate P-Ketoadipate.

702 100 100 491 70 61 403 57 10 228 32 34

0 0 0 0 0 0

* Calculated from measurements (18) of the rate of citrate synthesis by a crude ethanol fraction of pig heart which contained CoA transferase (specific activity -10) and excess thiolase and condensing enzyme.

responding to a turnover number of 8000. It is noteworthy that the turn- over number of CoA transferase is of the same order of magnitude as that of crystalline citric condensing enzyme (turnover number 5000) also pre- pared from pig heart (16).

The purified transferase (specific activity 1140) is free of thiolase, cro- tonase, P-hydroxybutyryl-S-CoA dehydrogenase, condensing enzyme, aco- nitase, the P enzyme system, and succinyl-S-CoA deacylase. It contains traces of fumarase hydrating 0.44 pmole of fumarate per mg. per minute at pH 8.1 and 25’. Since the turnover number of crystalline fumarase is about 100,000 (17), this transferase preparation contained about 0.09 per cent fumarase. On the basis of the maximal specific activity, it can be calculated that 0.09 per cent of the protein in the initial heart extract is CoA transferase.

Spec$city-As shown in Table II, the purified CoA transferase catalyzes the transfer of CoA from succinyl-S-CoA to acetoacetate, ,&ketovalerate,

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24 ENZYMES OF FATTY ACID METABOLISM. IV

,&ketoisocaproate, and /?-ketocaproate in order of decreasing activity. It

is seen that, P-ketoisocaproate excepted, the relative activity of the P-keto acids measured by direct optical assay with the purified enzyme agrees well with that reported earlier (17) for crude heart fractions by using a multienzyme assay based on citrate synthesis. It appears that in crude fractions the cleavage of /?-ketoisocaproyl-S-CoA by thiolase was the rate- limiting step and was considerably slower than the cleavage of /3-keto- caproyl-S-CoA. The purified transferase also transfers CoA from succinyl- S-CoA to cY-methylacetoacetate* but not to /3-ketooctanoate, ,&ketoadipate, benzoylacetate, crotonate, P-methylcrotonate, and saturated fatty acids. It catalyzes CoA transfer from acetoacetyl-S-CoA to succinate, but not to malonate, glutarate, butyrate, ,&hydroxybutyrate, crotonate, or malate. It does not catalyze CoA transfer from crotonyl-S-CoA to succinate, nor from maleyl-S-CoA to acetoacetate. Thus the transferase is strictly speci- fic for the suecinyl moiety in regard to chain length, substitution, and introduction of a double bond. It is less specific for the P-keto acyl moiety which may vary in chain length from Ch to Cg, and which may be substi- tuted in the a-carbon but not the w-carbon atom (viz. a-methyl acetoace- tate and /3-ketoadipate).

Succinyl-S-pantetheine and succinyl-S-glutathione, even in high con- centration, cannot replace succinyl-S-CoA as substrate for the transferase. Acetoacetyl-X-pantetheine is also inactive. Beinert and Stansly (19) have demonstrated that malonate, butyrate, ,&hydroxybutyrate, and valerate, as well as succinate and acetoacetate, increase the formation of labeled acetoacetate from CY4-acetyl-S-CoA in the presence of crude heart frac- tions containing CoA transferase and thiolase. They interpret their re- sults to indicate that these acids can replace succinate as substrate for CoA transferase. Since CoA transferase is strictly specific for the succinyl moiety, this interpretation is untenable, and other factors must have been operative.

Reaction with Acetoacetyl-X-CoA-The purified transferase catalyzes the transfer of CoA from acetoacetyl CoA to succinate (Curve A, Fig. 2) more rapidly than the reverse reaction. When tested under optimal conditions and without Mg++ ions (to avoid competitive chelation effects), the rate was 25 times faster with acetoacetyl CoA than with succinyl CoA as initial reactant (Table III). The reversibility of the reaction is demonstrated in the experiment of Curve B, Fig. 2.

Afinity of Substrates-These determinations were all performed in the absence of Mg++ because of the chelation of Mg++ by high concentrations of succinate and possibly acetoacetate. The Km value, determined by the method of Lineweaver and Burk (20), was 1.6 X 10B5 M for acetoacetyl

8 M. J. Coon, unpublished experiments.

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STERN, COON, DEL CAMPILLO, AND SCHNEIDER 25

CoA and 8.8 X 10e5 M for succinyl CoA. The concentrations required to saturate the transferase were 4.6 X low5 and 3.6 X lob4 M, respectively. The affinity for acetoacetate and for succinate was much less. Accurate K, values were not obtained for technical reasons. However, the rates given in parentheses, expressed as micromoles of CoA transferred per minute per mg. of enzyme, were observed for the concentration of free acid indicated as follows: succinyl CoA to acetoacetate, 1 X lo+ M (O.lS),

0.05 ’ I I I I 1 I 0 5 IO 15 20 25 30

MINUTES

FIG. 2. Optical measurement of the reaction acetoacetyl-S-CoA + succinate ti succinyl-S-CoA + acetoacetate. Silica cells (d = 0.5 cm.); temperature 25”. Curve A, the cells contained lOOpmoles of Tris-WC1 buffer, pH 8.1,0.07pmole of acetoacetyl CoA (containing an additional 0.03 #mole of acetoacetyl-8-glutathione), 100 pmoles of potassium succinate, pH 8.0,0.21 y of CoA transferase (specific activity 572), and water to a final volume of 1.50 ml. The reaction was started at zero time by addition of enzyme. Curve B, the cells contained initially 100 pmoles of Tris-HCl buffer, pH 8.1, 0.20 pmole of acetoacetyl CoA (containing 0.08 wmole of acetoacetyl-S-gluta- thione), 0.75 pmole of potassium succinate, pH 8.0, 42 y of transferase (specific ac- tivity 572), and water to a final volume of 1.50 ml. The reaction was started at zero time by addition of enzyme. At Arrow 1, 100 rmoles of potassium acetoacetate added; at Arrow 2, 10 @moles of potassium succinate added. Thio ester was omitted from the reference cell in both experiments. Readings corrected for dilution after each addition.

5 X 1k3 M (0.36), 2 X 1o-2 M (0.54), 5 X 1(F2 M (1.30), 6.7 X lk2 M

(1.98); acetoacetyl CoA to succinate, 2.5 X 10m3 M (13.4), 2.5 X 10e2 M

(21.5), 5 X 1O-2 M (37.5), 6.7 X lo-’ M (48.6). Effect of pH-As shown in Table IV, the reaction rate for the transfer

of CoA from succinyl CoA to acetoacetate increased continuously over the range pH 7.0 to 9.1 as determined by the optical method. One factor con- tributing to this effect is the dissociation of a proton from acetoacetyl-S- CoA above pH 9.0, for, as shown by Stern (7), the pK’ of the latter is 9.45. At pH 7.5 and above, Mg++ appeared to inhibit the reaction. If,

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26 ENZYMES OF FATTY ACID METABOLISM. IV

as Loewus et al. (21) have demonstrated for oxalacetate and malic dehy- drogenase, the keto form is the active substrate of the transferase, this effect may be more apparent than real and simply inherent in the optical

TABLE III

Relative Rates of Reactions Catalyzed by Transferase

In Experiment 1, the reaction mixture contained (in micromoles) Tris-HCl buffer, pH 8.1, 100; succinyl-S-CoA, 0.6; potassium acetoacetate, 100; and transferase, 4.2 y (specific activity 572, originally 1000). 7.5 pmoles of MgClz added as indicated. Final volume, 1.50 ml.; temperature, 23”. In Experiment 2, the conditions were the same as those for Curve A of Fig. 2.

Experiment No. Substrate CoA transferred per min. per mg. of enzyme

No Mg++ 5 X lo-MM&

pmoles !moles

1 Succinyl-S-CoA 1.98 1.44 2 Acetoacetyl-S-CoA 48.6 40.8 (51.1)*

I ! j

natio Experiment 2 ’ Experiment 1

24.5 28.3 (35.5)

* The values in parentheses have been corrected (approximately) for chelation of added Mg++ by high concentrations of succinate, an effect which decreases the effec- tive concentration of Mg++ and hence the extinction of acetoacetyl CoA.

TABLE IV

Effect of pH on Transferase Activity

Conditions as in Experiment 1, Table III. Tris-HCl buffer used throughout.

PH

7.0 7.5 8.1 9.1

CoA transferred per min. per mg. of enzyme

No Mg++ 5 X lO*MMg+

pmoles pmoles

0.48 0.56 1.00 0.80 1.98 1.44 3.05 1.71

method of assay in which the concentration of the enolate or chelate forms or both is measured (7).

Egdibrium Constants-The equilibrium constant of Reaction 1 is given by the expression

K = [acetoacetyl-S-CoA-][succinate=]IH+l

[succinyl-S-CoA-l[acetoacetate”l

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STERN, COON, DEL CAMPILLO, AND SCHNEIDER 27

We have determined at different pH values, in the presence and absence of added Mg++, the apparent equilibrium constant K’, employing for the cal- culation the total concentration of reactants (ionized and unionized, che- lated and unchelated) at, equilibrium according to the equation

K’=( t ace oacetyl-S-CoA)(succinate)

(succinyl-S-CoA) (acetoacetate)

TABLE V

Equilibrium Constant of Reaction Succinyl-CoA + Acetoacetate G= Acetoaeetyl-S-CoA + Succinate at pH 8.1 in Presence or Absence of ?vfg++

The reaction mixture contained initially Tris-HCl buffer, pH 8.1 (0.10 M), MgClz (5.3 X 1OP M), potassium acetoacetate (66.7 X 1OP M), succinyl-S-CoA (0.54 X 1OP

M in Experiments 1, 3, and 4; 0.41 X 1OP M in Experiment 2), and potassium suc- cinate (2.43 X 1OP M in Experiments 1, 3, and 4; 2.11 X 1OP M in Experiment 2). The reaction was started by addition of 60 y of transferase (specific activity 880). Final volume, 1.5 ml.; temperature, 30”. pH (glass electrode) 8.10. Acetoacetyl-S- CoA formation measured at 310 rnp against a reference cell containing all components except succinyl-S-CoA. Equilibrium was attained in 7 to 10 minutes. A correction was made for 5 per cent loss of succinyl-S-CoA by spontaneous hydrolysis during this period. Silica cells, d = 0.5 cm.

Experiment No.

1 2 3*

4t

Equilibrium concentrations (X 10-a na)

K’ (X 103) Ace$,a;tyl- Succinate Acetoacetate Succinyl-S-&A

0.095 2.55 66.5 0.42 8.67 0.075 2.44 66.6 0.31 8.84 0.053 2.51 66.6 0.46 4.34 0.098 2.55 66.5 0.42 8.96

Average (withMg++)............................................ 8.82

* MgClz omitted. It was not determined whether the other reactants were com- pletely free of Mg++.

t MgCl2 (5.3 X lo+ M) added to cells of Experiment 3 after equilibrium was reached.

At a given pH, K’ was determined by measuring optically at X = 310 rnp the formation of acetoacetyl-S-CoA from succinyl-S-CoA and aceto- acetate with constant concentrations of CoA transferase. Since the initial concentration of all reactants was known, their equilibrium concentration could be calculated from the +AE,, by use of the molecular extinction coefficient (E& of acetoacetyl-S-CoA determined under the same condi- tions. Typical results at pH 8.1 and 30” are shown in Table V. It is seen that the presence of lUg++, which forms a chelate compound w&h acetoacetyl-S-CoA (4,7), doubled the value of K’, and that the same value

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28 ENZYMES OF FATTY ACID METABOLISM. IV

of K’ was obtained whether Mg++ was present initially or was added after the reaction had attained equilibrium in its absence. The average value of K’, in the presence of Mg++, was 8.82 X 10m3.

The effect of hydrogen ion concentration on the apparent equilibrium constant of Reaction 1 is shown in Table VI, both in the presence and ab- sence of added Mg++. From the equation log K’ ‘v log K + pH, one might. expect a plot of log K’ versus pH to give a straight line of slope 1.0. Actu- ally the experimentally determined values of Table VI when so plotted fall on a line of slope 0.24 (with Mg+) and 0.21 (without Mg++), indicating that the active concentrations of the reactants do not approximate the total concentrations within the narrow pH range investigated.

TABLE VI

Apparent Equilibrium Constant K’ As Function of pH in Presence or Absence of Mg++ at 80”

The experimental conditions were the same as those of Table III except that the initial concentrations of succinyl-S-CoA and succinate, respectively, were as fol- lows: pH 7.0 (0.72 and 1.24 X 10e3 M), pH 7.5 (0.54 and 1.10 X 1OF M), pH 9.20 (0.09 and 2.02 X 1OF M). Also, 0.10 M glycine buffer was used at pH 9.20.

K’ (X 108)

PH Without Mg” With Mg++

7.0 7.5 8.1 9.2

2.8 4.9 4.2 5.9 4.3 8.8 8.6 15.0

Distribution-CoA transferase is also present in kidney as demonstrated by our observation (18) that acetoacetate synthesis by pig kidney extract from acetyl-S-CoA (generated from acetyl phosphate and catalytic amounts of CoA-SH with transacetylase) is dependent on the presence of succinate and succinyl CoA deacylase. Moreover, although these kidney extracts form citrate from acetoacetate, ATP, and CoA, in the presence of oxalace- tate, the yield of citrate is considerably augmented by addition of succinate, since the P enzyme system (22, 23) is also present and generates succinyl CoA. Experiments so far have failed to demonstrate CoA transferase in pigeon breast muscle, rabbit skeletal muscle, or pigeon brain. They have established that pigeon brain extract possesses the acetoacetate-activating enzyme (18) which synthesizes acetoacetyl-S-CoA from acetoacetate, CoA, and ATP.

Activators and Inhibitors-No activators of CoA transferase are known. The enzyme is not inhibited by 10e3 M potassium EDTA, confirming that Mg++ is not required for activity. It is not inhibited by 10e2 M iodoacetate.

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STERN, COON, DEL CAMPILLO, AND SCHNEIDER 29

Attempts to apply the direct optical assay to crude extracts by inhibiting thiolase with iodoacetate (6) have given variable results.

DISCUSSION

The free energy (AF”) of Reaction 1 at a given pH can be calculated from the equation AF” = - RT In K’. At pH 7.0 and 25” the free energy change of Reaction 1 is, therefore, AF” = - 1360 log (2.83 X 10e3) = +3470 calories. Within the range pH 7.0 to 9.2 the average decrease in the free energy change of Reaction 1 per unit increase in pH was 300 calories. The discrepancy between this and the theoretical value of 1380 calories is due to changes in the degree of ionization of the reactants. The free energy of hydrolysis of acetoacetyl-S-CoA is obviously greater than that of succinyl-S-CoA. Assuming that the free energy of hydrolysis of succinyl- S-CoA is about equal to that of acetyl-S-CoA,g for which a revised value of -8200 calories (pH 7.0 and 25”) has been calculated by Burton (25) from equilibrium data, the free energy of hydrolysis of acetoacetyl-S-CoA would be - 11,670 calories. It is relevant that, by coupling CoA transferase with the P enzyme system (22, 23), this energy can be transferred to the pyro- phosphate bond of ATP, 1 mole of which may be synthesized for each mole of acetoacetyl-S-CoA generated during fatty acid oxidation.

There can be little doubt that CoA transferase plays an active rale in the utilization of acetoacetate by peripheral tissues (particularly heart and kidney), by converting it to acetoacetyl-S-CoA which may be either re- duced to fatty acids via the fatty acid cycle or oxidized via the citric acid cycle. The latter in turn generates the necessary succinyl-S-CoA by oxi- dation of ac-ketoglutarate. The utilization of acetoacetate (ketone bodies) by the heart has been demonstrated in situ both in man (26) and dog (27, 28). Bing et al. (26) have determined that the rate of blood ketone utiliza- tion by the human heart is normally 0.50 to 6.1 pmoles per 100 gm. of heart per minute at 37”. Assuming (a) that 15 per cent of the heart is protein, (b) that 0.09 per cent of the protein is transferase, and (c) a turnover num- ber of 450 (calculated from data in this paper for pH 7.5 and assuming a 3-fold increase at 37”), then 60 pmoles of acetoacetate could be utilized per minute by 100 gm. of heart if the blood level were lop3 M. By ex- trapolation, this is compatible with the above values determined at blood levels of 3 to 10 X 10m5 M.

Materials

Pabst CoA which assayed 50 to 65 per cent pure and contained up to 20 per cent (molar basis) glutathione was used. Succinyl-S-CoA, maleyl-S-

9 This is indicated by the close agreement between the K’ values for the P enzyme reaction (22) and the aceto-Cob-kinase reaction (24).

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30 ENZYMES OF FATTY ACID METABOLISM. IV

CoA, and crotonyl-S-CoA were prepared by reaction of the anhydride with CoA-SH according to the procedure of Simon and Shemin (29). In the case of succinyl CoA, the actual succinyl-S-CoA content of the solution was about 50 per cent of the total thio ester formation calculated from the disappearance of sulfhydryl. Since succinyl CoA is relatively unstable, the solution was kept frozen at neutral pH and used within 2 weeks. Acetoacetyl-S-CoA was prepared by reacting diketene with CoA-SH at pH 5.0 to 6.0 (cf. (6)). It was stored at -20”. We are indebted to Dr. J. N. Eisendrath, Aldrich Chemical Company, Milwaukee, for a generous sample of purified diketene. Partly purified n-amino acid apooxidase from pig kidney (13) was kindly supplied by Dr. I. Zelitch.

SUMMARY

1. The preparation from pig heart and properties of highly purified CoA transferase, the enzyme which catalyzes reversible CoA transfer be- tween succinate and ,B-keto acids, are described.

2. The enzyme is strictly specific for succinyl-S-CoA and the CA to CS ,&ketoacyl-S-CoA compounds.

3. The equilibrium constant of the CoA transferase reaction, which is markedly in favor of succinyl-S-CoA and acetoacetate, has been deter- mined and the free energy change has been calculated. The free energy of hydrolysis of the thio ester bond of a.cetoacetyl-S-CoA is greater than that of succinyl-S-CoA by some 3500 calories.

4. The distribution of CoA transferase and its role in the utilization of blood acetoacetate by heart are discussed.

The authors wish to thank Professor S. Ochoa for his helpful interest.

Addendum-Since submission of the manuscript, CoA transferase has been shown to be present in extracts of dog skeletal muscle and ox adrenal gland, tissues known to utilize blood ketones. These extracts catalyze the conversion of acetoacetyl-s- CoA to succinyl-S-CoA as measured by the modified direct opt,ical assay. (J. R. Stern, unpublished experiments.)

BIBLIOGRAPHY

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New York, 1, 573 (1955). 3. Stadtman, E. R., J. Biol. Chem., 203, 501 (1953). 4. Stern, J. R., Coon, M. J., and de1 Campillo, A., J. Am. Chem. Sot., 76,1517 (1953). 5. Lynen, F., Wessely, L., Wieland, O., and Rueff, L., Angew. Chem., 64,687 (1952). 6. Lynen, F., Federation Proc., 12, 683 (1953). 7. Stern, J. R., J. Biol. Chem., 221, 33 (1956). 8. Warburg, O., and Christian, W., Biochem. Z., 310, 384 (1941).

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STERN, COON, DEL CAMPILLO, AND SCHNEIDER 31

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Campillo and Morton C. SchneiderJoseph R. Stern, Minor J. Coon, Alice del

TRANSFERASEPROPERTIES OF COENZYME A

ANDMETABOLISM: IV. PREPARATION ENZYMES OF FATTY ACID

1956, 221:15-32.J. Biol. Chem. 

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