Eur. J. Biochem. 75,465-479 (1977)

The Biosynthesis of Long-Chain Fatty Acids. Stereochemical Differentiation in the Enzymic Incorporation of Chiral Acetates Brian SEDGWICK and John Warcup CORNFORTH Shell Research Limited, Milstead Laboratory of Chemical Enzymology, Sittingbourne Research Centre, Sittingbourne, Kent (Received August 2, 1976)

1. Acetyl-CoA carboxylase and fatty acid synthetase were purified from chicken liver. Fatty acid synthetase was also purified from bakers' yeast. 2. The corresponding CoA thiol esters were prepared enzymically from R-[2H1,3H~]acetate,S[2H1, 3Hl]acetate and [3Hl]acetate, using a linked acetate kinase/phosphotransacetylase system. [2-14C]Acetate was added to the tritiated specimens to provide doubly-labelled products, which were purified by column chromatography on DEAE-cellulose. 3. The three acetyl-CoA specimens thus obtained were incubated separately in a combined acetylCoA carboxylase/fatty acid synthetase system containing the cofactors necessary for synthesis of long-chain fatty acids. 4. The fatty acids formed were extracted into n-pentane, methylated, separated by gas-liquid chromatography and the 3H/14Cisotope ratio determined. 5. In this manner it was shown, using the chicken liver synthetase, that in palmitic acid a higher proportion of tritium was retained from S-[2-I4C, 2H1, 'Hl]acetyl-CoA than from the R-(2-14C, 2 H ,, 3H1)-labelled thiol ester, the non-chiral (2-14C, 3H1)-labelled substrate giving an intermediate result. 6. This result was confirmed using the fatty acid synthetase preparation purified from bakers' yeast in place of the corresponding chicken liver enzyme. 7. In a separate experiment it was shown that the intramolecular tritium isotope effect in the carboxylation of [3Hl]acetyl-CoA is normal, but small. The slightly smaller, normal deuterium isotope effect calculated from this was shown to be in good agreement with the value expected, based on the experiments on tritium incorporation from chiral acetyl-CoA. 8. It is demonstrated that partial exchange of carbon-bound hydrogen must occur on the synthetase during the enzymic synthesis of fatty acids from malonyl thiol esters and that the extent of this exchange differs in synthetases of different origin. 9. A theoretical treatment of the effect of partial hydrogen exchange on the chirality of asymmetrically labelled methylene groups is given. 10. The results indicate the existence of an overall stereospecificity in the reactions involved in the de novo biosynthesis of long-chain fatty acids in different cell types. ~~~

Abbreviations. GLC, gas-liquid chromatography ;NMR, nuclear magnetic resonance. Enzymes. Acetyl-CoA carboxylase or acetyl-CoA:carbon dioxide ligase (ADP forming) (EC 6.4.1.2); acetate kinase or ATP: acetate phosphotransferase (EC 2.7.2.1); phosphotransacetylase or acetyl-CoA : orthophosphate acetyl transferase (EC 2.3.1.8) ; pyruvate kinase or ATP:pyruvate phosphotransferase (EC 2.7.1.40); lactate dehydrogenase o r L-lactate: NAD oxidoreductase (EC 1.1. 1.27); propionyl-CoA carboxylase (EC 6.4.1.3); pyruvate carboxylase (EC 6.4.1.1); malate synthase (EC 4.1.3.2); si-citrate synthase (EC 4.1.3.7); re-citrate synthase (EC 4.1.3.28); 3-hydroxymethyIglutaryl-CoA synthase (EC 4.1.3.5).

The synthesis of long-chain fatty acids de n o w from acetate [ l ] requires prior activation of this C2 fragment to form the c o thiol ~ ester p i , and the subsequent participation Of two enzyme systems, acetyl-CoA carboxylase and fatty acid synthetase 13- 101. The first of these generates malonil-CoA is ;hen utilised by the synthetase to operate, initially with a priming molecule of acetylCOA, in a cyclic series Of condensation-reductiondehydration-reduction reactions (see [ 5 ] , p. 388)

Fatty Acid Biosynthesis from Chiral Acetates

466 CH,COSC~A+ ATP + HCO;

M++

, L

HO,C-CH,-COSCOA Biotin-Enzyme

+ AOP + Pi

S-[2H,,3H,lacetyl-Coa

\

/

A Major

product Major A

o

M a product

product r

3H\

A

A

/

Minor

product o r

n

,COSCoA

c., H0,C' 28-12-'H,

3H,l-

2S-[2-'H,I-

\'H

Z.-[Z-'H,

3H,]-

2R-[2-3H,l-

Z.-12-ZH,,3H,&

2R-[2-3H,l

-

2R-[2--ZH,,3H,l

-

2S-[2-3H,1

-

Scheme 1. Acetyl-CoA carho.uylase reaction, showing tritiated malonyl-CoA species Jbrmedfrom R- and S-acetyl-CoA both with retention and inversion of configuration, assuming a normal intramolecular hydrogen isotope effect (k,/kz, > I )

which in most cells leads to the formation of palmitic acid as the major product. Reduced pyridine nucleotide (usually NADPH) is required in this synthesis which is described by the equation below:

+ + +

CH~COSCOA 7 HOOCCH~COSCOA + 14 NADPH 14 H + -+ CrtjH33COOH + 14 NADP+ 14 HzO .

+ 7 C02

The reaction catalysed by acetyl-CoA carboxylase is illustrated in Scheme 1 in which the triated products from chiral acetates, assuming both retention and inversion of configuration, are shown. The proportions of chiral and non-chiral malonyl-CoA species produced are dependent on the existence and magnitude of the intramolecular kinetic hydrogen isotope effect, which determines the extent to which protium is preferred to deuterium and tritium for abstraction'. Scheme 2 describes the reactions of the fatty acid synthetase but the possible stereochemistry of the reactions has been drawn for the 2S-[2-2H1, 3H1]malonate only, in order to simplify the diagram. For some reactions the stereochemistry has been experimentally proven, such as the 3 R configuration of the 3-hydroxyacyl intermediate [I 1,121 and the stereochemistry, with respect to the dihydronicotinamide moiety, of hydrogen transfer from reduced pyridine nucleotides during the first and second reductions [13], but for the other reactions the stereochemistry is not known. Our objective, therefore, has been to in-

'

In this paper, 'hydrogen' is used to denote all hydrogen isotopes indifferently. Protium designates the 'H species only.

vestigate the stereochemistry of the de novo biosynthesis of fatty acids catalysed by purified enzyme systems with a view to defining the precise stereochemical course of these reactions, and in these studies we have made use of stereospecifically labelled (chiral) molecules as substrates. The thiol ester intermediates in Scheme 2 have been designated by the general formula RCOSX, where the thiol carrier X may be CoA, acyl-carrier protein (ACP) as in many plants [S] and in most bacterial cells [14,15], enzyme-bound thiol groups as in yeast [5] and avian liver [16,17] or model thiols such as panthetheine and N-acetyl-cysteamine which can be used for assays of many synthetase preparations in vitro [6, 11,13, 181.

MATERIALS A ND METHODS Enzymes

Acetyl-CoA carboxylase was purified from chicken liver as described by Goto et al. [19] but using a Sepharose 2 B column instead of gradient centrifugation, the final preparation having a specific activity of 0.6 U/mg protein. Fatty acid synthetase was purified from chicken liver essentially as described by Hsu and Yun [20]. The final preparation had a relatively low specific activity (0.05 U/mg protein), and tended to lose activity rapidly on storage. In several experiments, therefore, the purified fatty acid synthetase was replaced by a dialysed enzyme solution prepared by fractionating the particle-free supernatant (I00000 x g,

B. Sedgwick and J. W. Cornforth

467

/+\

0‘c-s-x ,

’

CH3

RETENTION

ACETYL + MALONYL THIOLESTER THIOLESTER

Ho2c\C/Co-S-X

hH

2S-[2-2H,,3H,I INVERSION

Svnthase Icondensing enzymel

ACETOACETYL THIOLESTER 28- [2--” H , , ~ HI ,

2s- [2-2 H,,3H,1

4

NADPH + H*

[

&Keroacyl Thiolester: NADP Oxidoreducrase] NAOP+

3-HYDROXYBUTYRYL THIOLESTER

12S.3RI-3-hydro~y-[2-~H,

3H,I

12S.3Rl-3-hydr0xy-[2-~H,

3H, 1

[

]

3R-Hydroxyacyl Thiolesrer Dehydrase

trans-CROTONYL THIOLESTER

I [2-iH,l

[Z-,H,I

[Z-,[I

[Z-iH,l

NADPH + Hi

[:

]

trans-2-Enoyl Thgolesrer NADP Oxidoredocrase

1

1

1

NADP‘

ENANTIDMERIC [ z - ~ H , I 0, [ 2 - 3 ~ , 1 BUTYRYL THIOLESTERS

(arwmingsi-attack at carbon 21

Scheme 2. Reactions of the ,fatty acid synthetase, showing the possible strreochemistry of products formed from the 2S-[2-’H1, ’H~]rnalonyl thiol ester during the first cycle qf reactions

90 min) from the initial liver homogenate between 0 and 40% saturation with ammonium sulphate. This enzyme preparation gave results identical to those obtained with the purified enzyme. Fatty acid synthetase from bakers’ yeast was prepared essentially according to the procedure of Lynen [6] up to the stage of the second ultracentrifugation, the final specific activity being in the range 0.6 - 0.7 U/mg protein. The yeast (Distillers Company Ltd) was obtained as fresh as possible from a local bakery in wet-packed 1-lb or 1-kg blocks which had been stored at 4 “C. After suspension in buffer at 0-4 OC, the cells were broken by two passes through a Manton-Gaulin type cell disrupter (model 15 M-SBA, the A.P.V. Co. Ltd, Manor Royal, Crawley, England) operating at a pressure of 570 kg/cm2 (SO00 lb/in2) [21].

Acetate kinase, phosphotransacetylase, pyruvate kinase and lactate dehydrogenase were obtained from Boehringer Corp. (London) Ltd.

Substrates R-[’H1, 3H1]Acetateand S-[’H1, 3Hl]acetate were prepared as described by Lenz et al. [22]. Stock solutions were kept in sterile ampoules containing 1 ml of a solution of 0.1 M potassium acetate having a specific activity of approximately 0.9 Ci/mol. Nonspecifically tritiated acetic acid (500 Ci/mol) and sodium [2-I4C]acetate (55 Ci/mol) were obtained from the Radiochemical Centre, Amersham. Doubly labelled specimens of the tritiated acetates were obtained

468

by adding [2-14C]acetate to give the desired 3H/'4C ratio, the final specific activity being adjusted by the addition of non-radioactive potassium acetate solution. The corresponding CoA thiol esters were prepared enzymically from the individual doubly-labelled acetate samples using a linked acetate kinase/phosphotransacetylase incubation containing the following: 100 mM ethanolamine-HC1 buffer pH 7.4, 2 mM dithiothreitol, 20 mM KCI, 4 mM ATP (dipotassium salt), 8 mM MgC12,0.2 mM CoA, 0.4 mM potassium acetate, 3 pg (3 U) per ml phosphotransacetylase and 10 pg (1.7 U) per ml acetate kinase. The reaction was started by the addition of the appropriate acetate, and under these conditions, about 70% of the CoA was converted to the acetyl thiol ester after 80-min incubation at 37 "C. The formation of acetyl-CoA was followed spectrophotometrically at 232 nm comparing the absorbance of the test cuvette against a control containing all the components except acetate. After 80 min the reaction was terminated by the addition of 10 vol. of 0.01 M lithium acetate/acetic acid buffer pH 4.5, adjusting the final pH to 4.5 as necessary with dilute acetic acid. The solution was loaded on to a column of DEAE-cellulose (DE-52 ion-exchange cellulose, 0.8 cm x 15.0 cm, previously equilibrated in the above buffer) which was then eluted with a linear gradient composed of 100 ml starting buffer plus 100 ml 0.4 M lithium chloride in the same buffer. Fractions of approximately 5 ml were collected at a flow rate of 0.5 ml/min. The ultraviolet absorbance of the eluate was continuously monitored at 253 nm using a Uvicord I (LKB Produkter AB, Stockholm, Sweden) and the radioactivity of the eluate was checked by counting aliquots of each fraction in a liquid scintillation spectrometer. Acetyl-CoA was eluted from the column at 0.12 M lithium chloride and fractions of constant specific activity were pooled and placed in a pressure concentration cell (model 52, Amicon Inc., High Wycombe, England) containing a UM-05 membrane. The cell was pressurised with nitrogen and the volume reduced to 2-3 ml. This was diluted to 20 ml with water and the procedure repeated twice. This process effectively desalts and concentrates the acetyl-CoA at the same time, and the retention characteristics of the UM-05 membrane (theoretical exclusion limit at a molecular weight of 500) are such that only 10% of the product is lost in the filtrate. (This purification procedure was developed in the laboratory of Professor S. J. Wakil at the Department of Biochemistry, Duke University Medical Center, Durham, N. Carolina, U.S.A.) Non-chiral [3H1]acetyl-CoA was prepared essentially according to the procedure of Simon and Shemin [23], by acetylating free CoA (20 mM) in 0.2 M potassium bicarbonate (final pH 8.0) at 0 "C with [3H1]acetic anhydride (100 Ci/mol). The product was

Fatty Acid Biosynthesis from C h i d Acetates

purified by chromatography on DEAE cellulose as described above. Other Materials, Reagents and Equipment The chemicals listed below were obtained from the sources indicated. CoA ('Chromatopure', trilithium salt), acetyl-CoA, malonyl-CoA, NADPH (P-L Biochemicals) from International Enzymes Ltd; ATP (sodium salt), ATP (potassium salt), ADP (sodium salt), DL-isocitric acid, dithiothreitol, NADH, myristic and stearic acids and methyl esters of long-chain fatty acids from Sigma (London) Chemical Co. Ltd; acetyl phosphate (potassium-lithium salt), phosphoenolpyruvate (sodium salt) from Boehringer Corp. (London) Ltd; deuterium oxide (99.7"/,) from Prochem Ltd; [l-'4C]acetyl-CoA (50.6 Ci/mol), [I ,3-'4C]malonylCoA (10.4 Ci/mol) from New England Nuclear Chemicals GmbH ; [3H]aceticacid (500 Ci/mol), [3H]acetic anhydride (100 Ci/mol), sodium [l-14C]acetate (50 Ci/mol), sodium [2-14C]acetate (55 Ci/mol), sodium [U-'4C]acetate (60 Ci/mol), sodium [14C]bicarbonate (44.4 Ci/mol), 14C-labelled fatty acids and their methyl esters, 3H and 14C toluene scintillation standards and tritiated water (5 Ci/ml, 90 Ci/mol) from the Radiochemical Centre, Amersham ; palmitic acid (Fluka AG) from Fluorochem Ltd; bovine plasma albumin (fraction V) from Armour Pharmaceuticals Co. Ltd. Ion-exchange DEAE-cellulose (Whatman DE 52) was obtained from W. and R. Balston Ltd (Maidstone, England) ; Sephadex gel filtration media and Sepharose 2 B from Pharmacia (Uppsala, Sweden). Pre-spread plates of silica gel G plus F254 fluorescent indicator (Merck) for thin-layer chromatography were supplied by Andermann and Co. Ltd. Liquid scintillation counting fluid consisted of a solution in toluene (AR grade) of 2,5-diphenyloxazole (4 g/l) and 1,4-bis-2-(4-methyl-5-phenyloxazolyl) benzene (0.1 g/l), which were obtained from Packard Instrument Co. Inc. Non-aqueous samples were counted in 10 ml of this fluid in glass vials in a Packard Tricarb model 3375 liquid scintillation spectrometer. For counting of aqueous samples 10 ml of a mixture of 2 vol. of the scintillator plus 1 vol. of Triton X-100 was used [24]. Counting efficiences of 45% for tritium and 64% for 14C were obtained using the basic toluene scintillator. The figures for the Triton X-100 scintillator counting 1 ml aqueous sample were 30% for tritium and 35 % for 14C. All other reagents and solvents used were of analytical (AR) or equivalent grade and were obtained from British Drug Houses (B.D.H.) Ltd, Hopkin and Williams or Ralph N. Emanuel Ltd. Spectrophotometric measurements were made in a Zeiss PMQ 11, Cary 14 or Unicam SP-1800 spectrophotometer, as appropriate. Mass spectra were re-

B. Sedgwick and J. W. Cornforth

corded by a model MS-9 machine from A.E.I. Ltd (Manchester, England). Gas-liquid chromatography/ mass spectrometry (GLC-MS) samples were introduced from a Pye model 104 gas chromatograph and routine GLC analysis employed a Varian model 1400 chromatograph. Protein was determined essentially as described by Lowry et al. 1251. Incubation of Acetyl-CoA Specimens with a Combined Acetyl-CoA CarboxylaselFatty Acid Synthetase Enzyme System Preliminary N M R experiments with malonyl-CoA and with model malonyl thiol esters dissolved in deuterium oxide (see following paper) had indicated that under the approximate conditions of enzymic incubation, the time required for 25 % exchange of a protium atom on carbon-2 of the malonate moiety was of the order of 5-6 min. This rate of exchange obviously precluded the isolation and purification of malonyl-CoA formed by the carboxylase prior to incubation with the synthetase, and suggested that even separate sequential incubations using carboxylase followed by synthetase might lead to an exchange of protons which would be unacceptable. Accordingly, it was decided to use a combined enzyme assay in which the acetyl-CoA was incubated simultaneously with carboxylase and synthetase, the components of the system being as follows: 100 mM potassium phosphate buffer pH 7.0, 20 mM potassium isocitrate, 2 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 20 mM potassium bicarbonate, 8 mM magnesium chloride, 2 mM ATP (disodium salt), 0.15 mM NADPH, 0.03 mM acetyl-CoA, 0.1 mg acetyl-CoA carboxylase and a variable amount of fatty acid synthetase. The reaction was started by the addition of the appropriate labelled acetyl-CoA and was usually incubated at 37 "C. Extraction and Separation of Fatty Acids Reactions were terminated by the addition of 1 .O M KOH (pH > l l ) , followed by heating (15 min, 90 "C). After cooling, carrier palmitic and stearic acid (0.5 mg each) were added and the solution acidified (pH < 2) with 1.0 M HCl. The assays were extracted three times with redistilled n-pentane (4 ml/ml aqueous phase) and the combined extracts were taken to dryness under nitrogen, finally drying the residue with acetone. For extraction of fatty acids without addition of carrier (as in experiments involving deuterium labelling), the method of Bligh and Dyer 1261 was employed, but using 2 M KCl in 0.5 M potassium phosphate buffer pH 5 as the aqueous phase used to separate the layers, as suggested by Brindley et al. 1271.

469

The GLC analysis and separation of fatty acid products, after methylation with a dilute ethereal solution of diazomethane, employed a column (3 m x 2 mm) of 10% diethylene glycol succinate on Gas Chrom Q, helium carrier gas (flow rate 30 - 40 ml/min), and a thermal conductivity detector. Individual methyl esters were trapped (75 - 80 % efficiency for C12- C20 acids) in short (4-cm long) glass tubes packed with glass wool and inserted into the exit port of the column. These were placed directly in a vial of scintillation fluid for radioactivity monitoring, or alternatively the individual samples were recovered by elution with n-pentane.

RESULTS Incovpovation of Radioactivity

,pornNon-Chiral Acetyl-CoA Preliminary experiments to examine various parameters of the combined assay system were conducted using non-specifically tritiated acetyl-CoA, also labelled with 14C at carbon-1, and the results of these experiments are presented in Tables 1 and 2. Using the chicken liver enzymes, palmitic acid was the major product (Table 1) and the isotope ratio in palmitic and stearic acids indicated a tritium retention relative to acetyl-CoA of 31 % and 30% respectively. 5-min incubations were used in all subsequent experiments as longer incubations led to significantly lower tritium incorporation (Table 2), presumably due to an increased exchange with the medium of the malonate methylene protons in malonyl-CoA prior to incorporation into fatty acids. If we assume the initial 3H/14Cratio to be 3/1 for acetyl-CoA (in Scheme 3 this is represented by three 3H atoms per methyl but of course the actual proportion is much smaller), the ratio in malonyl-CoA will be 2/1 due to loss of one of the protons on carboxylation. The stoichiometry of biosynthesis requires the incorporation of one intact acetyl unit and seven C2 fragments derived from malonate per molecule of palmitic acid. From the reactions outlined in Scheme 2, it is seen that each malonate unit, in addition to losing carbon dioxide in the condensation, also loses one of the methylene protons in the dehydration reaction. The carbon dioxide lost is that added during carboxylation, hence there is no loss of 14C during incorporation. This leads to a final 3H/'4C ratio of 1 for C2 units incorporated from malonate. Thus the overall 3H/14Cratio for palmitic acid can be represented by (1 x 3/1) (7 x l / l ) = l0/8 = 1.25. As the starting isotope ratio in acetyl-CoA was 3.0, this represents a 3 H incorporation of 1.2y3.0 x 100 = 41.7 relative to I4C. A similar calculation applied to stearic acid gives a figure of (1 x 3/1) + (8 x l / l )

+

Fatty Acid Biosynthesis from Chiral Acetates

410

Table 1, Radiochemical composition ofjatty acids synthesised by a combined acetyl-CoA carboxylaselfatty acid synthetase system purifi:ed,from chicken liver, using [I-14C, 3HI]acetyl-CoA as substrate Substrate 3H/14Cratio = 17.1. Incubations were for 10 min at 37 “C, see text for details. Theoretical 3H retention calculated assuming carboxylase k,,lk3, = 1 Fatty acid

Radioactive product

’H retention

3H/14Cratio

-

~-

cf: theoretical

cf acetyl-CoA

”/,

%total ~~

Short chain < CIO Decanoic CIO Lauric CIZ Myristic C14 Palmitic Clh Stearic ClS Arachidic CZO Longchain > C ~ O

0 0.6 1.7 4.4 74.6 13.9 4.8

0

~

~

-

~~

-

-

-

-

-

-

-

-

5.24 5.26 5.15 5.33

30.6 30.8 30.1 31.2

71.5 73.9 74.0 78.0

-

-

3H 3 H C -14CO-S-CoA 3H

Table 2. EJfect of time ofincubation on the 3 H / i 4 Cratio offatty acids synthesised by a combined acetyl-CoA carhoxylaselfatty acid synthetase system purified,from chicken livw, using [I-I4C, 3Hl]acetylCoA substrate Substrate 3H/14C ratio = 17.1, see text for details of incubations. Theoretical 3H retention calculated assuming carboxylase kH/ k3” = 1

Acetyl-CoA

t

-

-

3H

d HO-CO-C-’4CO-S-CoA

HCO-

3H Malonyl-CoA

3H/’4C = 3/1

3H/’4C = 2/1

~

Time of incubation

3H/14Cratio of fatty acid products

3H retention -~

~

cf

acetyl-CoA

cf theoretical

3H/’4C = 3/1

”/,

min 5 10 15 32.

5.55 5.31 5.10 4.85

32.5 31.0 29.8 28.4

1

I

1/1

j

1/1

,1 [ j

1/1

![

I

1/1

111

1/1

1/1

3H H j 3 H H j 3 H H13H H j 3 H H13H H i 3 H H i 3 H 3HC-14C~C-14C C -14C I c 1 4 C I C - 1 4 C L C - 1 4 C I C - 1 4 C L ~

78.3 74.7 71.8 68.4

3

H

H

H

H

H

H

H

H

H

H

H

H

H

H

- 1 4 ~ 0

H

I s-x

Palmityl Thiolerter 3H/’4C = 10/8 = 1.25 -41.7% cf. Acetyl-CoA

Scheme 3 . Derivution of the theowtical retention i7 palmitic acid (g tritium derived from [3H]ucetyl-CoA, assuming no isotope ejject ik,,/k3,, = 1)

= 1119 = 1.22, equivalent to 40.7 %. This ‘theoretical’ retention ignores all isotope effects.

Table 3. Radiochemical data on c h i d and non-chiral acetyl-CoA samples synthesised enzymically using a linked acetate kinasel phosphotransacetylase sys tem

Incorporation of Radioactivity from Chiral Acetyl-CoA

Compound

The final specific activities and isotope ratios of the acetyl-CoA products from the enzymic syntheses using R, S and non-chiral tritiated acetates are shown in Table 3 . These thiol esters were incubated in the combined carboxylase/synthetase assay described previously, the purified yeast synthetase replacing the chicken liver enzyme in a second series of experiments. The fatty acid products were extracted into n-pentane and separated by GLC as described in Methods, and the isotope ratio in methyl palmitate determined. The results of these experiments are given in Table 4, expressed as mean k standard deviation of five assays per substrate for each synthetase preparation. In all experiments S-[2-14C, 2 H ~ 3Hl]acetyl-CoA , led to the highest retention and R-[2-14C, ’HI, 3Hl]acetyl-

Specific activity of 3H/L4C ratio 3H I4c

~-

R-[2-I4C,’HI, ’H1]Acetyl-CoA S-[2-14C,ZH1, 3H1]Acetyl-CoA [2-I4C, 3Hl]Acetyl-CoA

Ci/mol __ 083 082 062

~~

012 0 12 012

694 690 538

CoA to the lowest retention of tritium in palmitate, the non-chiral substrate giving an intermediate result. Investigation of the Hydrogen Isotope Effect in the Acetyl-CoA Carboxylase Reaction As was mentioned in the introduction, the proportion of chiral species of malonyl-CoA formed

B. Sedgwick and J. W. Cornforth

47 1

Table 4. H/14C ratio and percentage 3 H retention in palmitic acid synthesised from chiral and non-chiral acetyl-CoA substrates using combined acetyl-CoA curboxyluse/fatt,v acid synthetase enzyme systems Incubations were for 5 min at 37 "C, details as described in the text. Results are expressed as mean standard deviation of five assays for each substrate and the percentage 3H retention is calculated from the isotope ratio in acetyl-CoA, normalising 14C incorporation as I O O ~ , Substrate

Palmitic acid product with ___ chicken liver synthetase ~

~

3H/14Cratio

~

yeast synthetase

~~

3H/14Cratio

-

~~

3H retention

3H/14Cratio

3H retention

%

"/,

~~~~~

R-Acetyl-CoA S-Acetyl-CoA [3H]Acetyl-CoA

6.94 6.90 5.38

2.11 k 0.04 2.37 f 0.01 1.74 f 0.04

enzymically from chiral acetyl-CoA samples is dependent on the magnitude of the intramolecular kinetic hydrogen isotope effect. If a large isotope effect is present during this carboxylation, for example k,/kz, = 3, then starting from R-acetyl-CoA one would expect to generate 75 % of the tritiated malonyl-CoA in the 2R-fH1, 3H1] form and 25% in the 2S-[3H~] form (assuming arbitrarily that configuration is retained on replacement of hydrogen). This is illustrated in Scheme 1. The fact that only a small differentiation between R and S acetate enantiomers with respect to tritium retention in fatty acids was observed (Table 4) suggested the possibility of a small isotope effect during carboxylation, which would lead to almost equal populations of R and S tritiated malonates from both enantiomers. An attempt was made to determine the isotope effect experimentally using the purified carboxylase from chicken liver, a non-chiral [3H]acetyl-CoA substrate and a spectrophotometric assay for the accurate determination of carboxylase activity. The carboxylation reaction generates one mole of ADP (from ATP) for each mole of malonyl-CoA formed, and this ADP was assayed using a linked pyruvate kinase/lactate dehydrogenase system (1 ml) containing the following: 100 mM potassium phosphate buffer pH 7.0, 20 mM potassium isocitrate, 2 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 4 mM ATP, 0.5 mM phosphoenolpyruvate, 0.25 mM NADH, 0.09 mM [3Hl]acetyl-CoA (4.01 Ci/mol), 0.01 mg/ml (1.5 U) lactate dehydrogenase, 0.01 mg/ml (3.6 U) pyruvate kinase and 0.1 mg/ml acetyl-CoA carboxylase. The reaction was started by the addition of acetyl-CoA and incubations were for 0-6 min at 37 "C. The formation of malonyl-CoA was monitored by following NADH oxidation spectrophotometrically at 340 nm against a control cuvette containing all components except acetyl-CoA. The reaction was terminated by transferring the contents of the cuvette to a small flask containing 0.01 ml 10 M KOH, which

30.4 k 0.60 34.3 k 0.15 32.3 f 0.70

+

2.54 0.08 2.78 k 0.06 2.01 f 0.07

36.6 k 1.1 39.9 k 0.5 31.5 k 1.2

resulted in a final pH of about 9. At this pH, proton exchange in the malonyl methylene group is at a minimum (as determined by NMR experiments in deuterium oxide), and any free [3H]aceticacid formed during the incubation will be as the potassium salt and will not, therefore, be volatile in the subsequent distillation. Approximately half of the water from the incubation medium was flash distilled under vacuum into a small tube cooled in liquid nitrogen, warming the flask at 37 "C and shaking to prevent freezing of the assay mixture. The tritium content of the distillate was determined by counting an aliquot in 10 ml of the Triton X-100 scintillation fluid, and the total volatile tritium in each assay was calculated from this by multiplying by the appropriate sampling factors, using the approximation that a given volume of distillate corresponded to an equal volume of solution. The data from this experiment are given in Table 5. When the carboxylase was omitted from the assay, there was no net oxidation of NADH or release of tritium into the medium. In experiments containing the carboxylase, acetyl-CoA and all components except ATP (- ATP controls) there was again no net oxidation of NADH, although there was a small, time-dependent release of tritium into the medium. Correction was made for this, as indicated (Table 5, column 4) by subtraction of the tritium release given by the corresponding -ATP control. In addition an approximate correction has been made for the nonenzymic release of tritium from malonyl-CoA formed during the incubation, (column 5 ) based on the experimentally determined half-time for the exchange of protium and an assumed tritium isotope effect of 10 for this reaction (see Discussion). The corrections may seem small, but in fact they affect significantly the calculation of the isotope effect. Accurate determination of the specific radioactivity of the [3Hl]acetyl-CoA is also important in this experiment. The acetyl-CoA was purified by chromatography on DEAE-cellulose (see Methods) immediately

Fatty Acid Biosynthesis from Chiral Acetates

412

Table 5. Release o f 3 H on carboxylation of(3Hl]acetyl-CoA by ucetyl-CoA carboxyluse purified from chicken liver.. Calculation of the intramolecular hydrogen isoiope efject, k11/k3, Specific radioactivity of the [3HHl]acetyl-CoA = 4.01 Ci/mol = 8.90 x lo3 dis. min-' nmol-' Time of incubation

Total volatile 3H in assay

Acetyl-CoA carboxylated

min

nmol

0 1 2 3 3 6 6

0 10 0 23 7 35 7 37 2 64 7 68 3

Correction lor 3H release in -ATP controls

Correction for Net 3H release due to 3H exchange from carboxylation malonyl-CoA

Initia13H content of acetyl-CoA carboxylated

Fraction ( x ) of 3H released

I so tope effect

on

kHIX?,,

8 900 21 090 31 770 33110 57580 60 790

0 285 0 281 0 293 0 301 0 286 0 287

CarbOxyIdtiOn

dis min ~- -

~

900 27 550 63170 98 790 105400 177 870 187700

900 2 060 3250 4430 4430 8 000 8 000

130 600 1340 1380 5 070 5 350

-

~~~~

25 360 59 320 93 020 99 590 164 800 174350

Medn +SD

prior to use, and the radioactivity of the product was carefully determined by liquid scintillation counting using internal standardisation for calculation of the counting efficiency. The acetyl-CoA concentration was determined spectrophotometrically at 232 nm by following the arsenolysis of the thiol ester by phosphotransacetylase in 25 mM potassium arsenate buffer pH 7.0 using for the molar absorption coefficient d&232= 4 . 5 lo3 ~ M-' cm-'. These determinations yielded a value of 4.01 Ci/mol for the specific activity of the acetyl-CoA used in this work, equivalent to 8.90 x lo3 dis. min-' nmol-', and this figure was used in calculating the initial tritium content of the carboxylated acetyl-CoA given in Table 5. Ifthe rate of protium replacement in the carboxylation = kH and the rate of tritium replacement = k3,, then the proportion x , of the total tritium released into the aqueous medium is given by the expression

1 254 1 279

__

1 206 1 161 1248 1 242 -

1232 f 0 042

(derived by Swain et al. [28]) for the calculation of the intramolecular deuterium isotope effect in the carboxylase reaction. A value for k,/k3, = 1.23 corresponds to a value of k,/kzH = 1.15, and these figures will be used throughout the discussion in all calculations. The experimental approach used in this work was necessary because the rate of exchange of the methylene protons in the product made it impracticable to determine the intramolecular hydrogen isotope effect by the use of doubly labelled [14C, 2-3Hl]acetyl-CoA and subsequent purification and analysis of the isotope ratio of the malonyl-CoA formed. Attempts to trap the malonate in a form in which the tritium was stabilised, e.g. by borohydride reduction of the thiol ester to 3-hydroxypropionic acid, gave unsatisfactory results. Determination of Proton Exchange during Fatty Acid Synthesis

remembering that each tritiated methyl group contains two protium atoms and one tritium atom. From this one can derive the expression for the tritium isotope effect, (1 - x) kJk3, = ~~

2x

Using this formula for the calculation of k,/k3, from the experiment data, one arrives at the values given in the last column in Table 5, which give a mean value & S.D. of 1.232 k 0.042. This small, normal tritium isotope effect suggests a slightly smaller, normal deuterium isotope effect, and we have chosen to use the expression kH/k2,

--

=

~~

1.442 1/kH/k3,

From the figures for retention of tritium from nonchiral tritiated acetyl-CoA (37.5 for the yeast enzyme, see Table 4), it was evident that some loss of tritium was occurring in addition to that allowed for in calculating the 41.7 "/, theoretical retention based on the accepted reaction mechanism (see Scheme 3). In order to determine experimentally the extent of this exchange, malonyl-CoA and S-malonyl-N-caprylyl cysteamine [29] were equilibrated several times in deuterium oxide in order to exchange for deuterium all protium on the C-2 of malonate. These labelled compounds were then used as substrates [30] for the synthesis of long-chain fatty acids using the yeast synthetase in a 1-ml incubation containing 100 mM potassium phosphate buffer pH 7.4,2 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 0.15 mM NADPH,

B. Sedgwick and J. W. Cornforth

413

li

I , 269 268

Fig. 1. Purent peak region of GLClmuss spectrum ofthe methyl ~ . \ I w ofpalmitic acid synthesised f r o m deuterated S-malonyl-N-caprylyl cystearnine by the yeas! .fatty acid synthetase

0.04 mM acetyl-CoA, 0.2 mM malonyl-thiol ester and 50- 100 pg synthetase. Incubation was for 10 min at 37 ”C and after saponification and acidification, the fatty acids were extracted by the Bligh and Dyer method [26] without the addition of carrier. The extracts were methylated and analysed for deuterium content by GLC/mass spectrometry. Fig. 1 shows a typical peak profile from the parent peak region of the methyl ester of palmitate synthesised from deuterated Smalonyl-A’-caprylyl cystearnine. Reference to Schemes 2 and 3 shows that palmitic acid synthesised from malonate fragments containing two deuterium atoms at C-2 should retain one of these per malonate condensed, or 7 per molecule of palmitic acid if no exchange reactions occur. Thus the mass spectrum of the methyl ester should show a parent peak at mle = 270 derived from endogenous palmitic acid, a much smaller peak at m/e = 271 due to the natural abundance of 13C, and a major peak at m/e = 277 due to labelled product containing 7 deuterium atoms per molecule. Instead of this, a non-symmetrical distribution of peaks between m/e = 270-277 is found, with a maximum at 275. Thus although not more than one of the methylene hydrogens of malonate ever survives the biosynthesis, some process has occurred leading to partial exchange of the survivor with hydrogen from the medium. This could happen either before or after the elimination reaction leading to loss of one of the methylene protons. If a fraction a of the original deuterium labelling (assumed 1000/0) is exchanged in this way the relative heights of the peaks of mle = 271 - 277 in the mass spectrum (after correction for contributions from I3C peaks) are given by 7a6(1-a), 21a’(1-a)2, 35a4(1-a)3, 35a3(1-a)4, 21a’( 1-a)’, 7 4 1-a)6 and ( 1-a)’ respectively. This binomial derivation assumes that all seven Cz units derived from malonate are equally labelled with deuterium. The experiment with chicken-

liver synthetase, recorded in Table 1, indicates no major variation in labelling of successive CZunits in the series myristic + arachidic acid. Comparing the corrected heights for the three major peaks we find, from the mle ratio 2751276, a = 0.31 and from the mle ratio 2741275, a = 0.304. Thus some 30% of deuterium has been exchanged, and from data (see following paper) on the rate of exchange of deuterium from malonyl thiol esters not more than 4 0/0 exchange could have occurred in the malonate itself during the incubation. It follows that exchange must occur at some stage of fatty acid synthesis on the synthetase, and that this exchange is nonspecific: that is, it must be an incidental and not an essential feature of fatty acid biosynthesis.

DISCUSSION Isotope EJffects on Carboxylation of’ Acetyl-CoA Some previous studies on biotin-dependent carboxylases have included measurements of the isotope effect in hydrogen abstraction. Prescott and Rabinowitz [31] reported kH/k3, = 1 for propionyl-CoA carboxylase as they found the release of tritium from 2R-[2-3H~]propionyl-CoA to proceed at the same rate as overall formation of methylmalonyl-CoA. A kinetic kH/kzH value of 1.13 was also reported for 2 R-[2-’Hz]propionyl-CoA. This carboxylation proceeds without net configurational change at C-2, 2R-[2-3H~]propionyl-CoA yielding tritium-free 2s-methylmalonylCoA. Retey and Lynen [32] and Arigoni et al. [33] had previously reported a similar conclusion and found k H / k z , = 1.16 on the basis of relative rates of carboxylation of unlabelled and [2-zH~]propionylCoA. It must be emphasised that these are intermolecular isotope effects, the enzyme having no choice of

Fatty Acid Biosynthesis from C h i d Acetates

414 hydrogen in a given molecule of propionyl-CoA. This is not necessarily so for carboxylation of a methyl group: assuming that free rotation of the group about its C C bond is possible in the enzyme-substrate complex, there is a choice of three hydrogens for replacement by carboxyl and intramolecular isotope effects can be shown in addition to any intermolecular discrimination between molecules having different isotopic compositions. One biotin-dependent carboxylase of this type has been studied for isotope effect : pyruvate carboxylase. Rose [34] reported retention of configuration in the carboxylation of chiral pyruvate to oxaloacetate on this enzyme, and by studying the release of tritium into the medium when [3-3H1]pyruvate was carboxylated a large tritium isotope effect was observed : if all of it was intramolecular, a k,/ks, of 5.75 can be calculated. It will be shown (following paper) that acetyl-CoA carboxylase also catalyses carboxylation with retention of configuration. However, our experiments show that the intramolecular isotope effect is small, and this is confirmed by the results for fatty acid synthesis from chiral acetates. As will be shown, these results are readily explained on the basis of the experimentally determined small isotope effect but we have found no explanation that could accommodate an isotope effect of the size reported for pyruvate carboxylase. The acetyl-CoA condensations of the type catalysed by malate synthase 135- 371, si-citrate synthase [22, 38,391, re-citrate synthase [38,40,41] and 3-hydroxymethylglutaryl-CoA synthase [42], which also catalyse proton abstraction from the methyl carbon of acetate (as CoA thiol ester) to form a C-C bond, contrast with the carboxylases in that they proceed with inversion of configuration. These enzymes may [40, 421 or may not (see [51], p. 42) catalyse proton exchange in the absence of reaction, and intramolecular hydrogen isotope effects of 1.4-3.5 have been demonstrated (in some cases the true figures may be higher). Retey and Lynen [32] and Mildvan and Scrutton 1431have proposed a mechanism for the transcarboxylation reaction, in propionyl-CoA carboxylase and pyruvate carboxylase respectively, based on the facts that substitution occurs with retention and that hydrogen exchange from the species to be carboxylated is dependent on the presence of the carboxybiotin intermediate. A concerted displacement mechanism of the type illustrated in Scheme 4 is postulated, whereby the proton leaves and the carbon dioxide is inserted from the same side of the molecule, and Lynen subsequently suggested [44] that all biotin-dependent carboxylations have identical mechanisms and proceed with retention of configuration. Acetyl-CoA carboxylase from E. coli has recently been studied in some detail [45,46] and was shown to ~

Scheme 4. Propo.5ed concerted electrophilic displacement mechanom for transcarboxylation in biotin-dependent carhoxylases. (See Retey and Lynen [ 3 2 ] )

consist of three proteins, a biotin-carrier protein, biotin carboxylase and a transcarboxylase component. The sequence of reactions in yeast [5], plants [47] and the cells of higher animals 1481 suggests a similar enzyme structure, though the three individual proteins have not been fully characterised from these tissues. Sumper and Lynen [49] have compared the physicochemical properties of acetyl-CoA carboxylase and pyruvate carboxylase and have found the two enzymes to be quite similar. It is presumed that the substrate specificity of these enzymes is due to significant differences which exist in the binding site of the transcarboxylase component, as was suggested by Vagelos and co-workers [45]. The contrast between the k,/k3, found for the two enzymes could be accounted for in terms of identical reaction mechanisms: for example, if the enzyme-substrate complex from acetyl-CoA does not permit sufficiently free rotation of the methyl group the intramolecular choice of hydrogen would be restricted and a small isotope effect, such as is observed with propionyl-CoA, would operate. Similarly, if the concerted mechanism of Scheme 4 is correct the optimal geometry for transfer of tritium (which has a smaller atomic radius than hydrogen) might be more difficult to attain in one enzyme than in the other, since the active site of the transcarboxylase component must be structurally different in some respects. A possible alternative explanation would be that a similar, and consequently large, intramolecular isotope effect does operate for both enzymes but that in acetyl-CoA carboxylase the carboxylation on the enzyme is repeatedly reversed with intermediate exchange of the displaced hydrogen for hydrogen from the medium, so that each molecule of malonyl-CoA is effectively the product of several carboxylations. This explanation seems to us less likely, since the effect on chiral acetates would be difficult to reconcile with our experimental results. Further, although the carboxylation of acetyl-CoA is reversible [50] the conditions used in the experiment for determination of isotope effect (excess of ATP, continuous removal of ADP) seem to exclude the presence of decarboxylated biotin necessary for this reversal to be significant. Rose has suggested [51] that carboxylation of pyruvate involves the formation of an enzyme-stabilis-

B. Sedgwick and J. W. Cornforth

ed enol intermediate, and this offers a fourth possibility for explaining the marked difference in isotope effects. Enolisation would probably be accompanied by a large isotope effect and if enolisation was the rate-limiting step in the carboxylation of pyruvate, this isotope effect would be demonstrated in the overall reaction, provided the enol was the species carboxylated. Enolisation of acetyl-CoA, on the other hand, seems unlikely and would be difficult to reconcile with the concerted displacement mechanism (Scheme 4) generally proposed [32,43] for the biotin-dependent carboxylases. In discussing the effect of isotopic discrimination in the carboxylation of acetyl-CoA on retention of tritium in fatty acid synthesis we shall assume that the effect is intramolecular. For the closely related propionylCoA carboxylase, where no intramolecular isotope effect is possible, the experimental evidence (see above) is that deuterium substitution at C-2 of propionate has a small effect on the maximal velocity but that normal molecules of substrate are not significantly preferred by the enzyme to 2R tritiated molecules. Since tritium isotope effects are normally larger than deuterium isotope effects it seems that with this enzyme, reaction of the substrate once it is bound is faster than release of unreacted substrate from the complex; or at least that the rate of release of substrate is unaffected by isotopic substitution. Thus when all molecules presented have deuterium substitution, a slightly slower rate of reaction on the enzyme can be seen; but when a small proportion of the molecules have tritium substitution the same proportion will anyway be transformed once they are bound, and if this transformation is slower it will have an insignificant effect on the overall velocity of the reaction and no effect at all on the relative proportions of tritiated and non-tritiated molecules undergoing reaction. If a similar situation obtains with acetyl-CoA carboxylase, the intramolecular isotope effect is the only one observable in our experiments.

Exchange of Hydrogen during Synthesis of Fatty Acids f r o m Acetate

Earlier investigations on tritium retention from acetate incorporated into long-chain fatty acids have yielded results differring according to the preparation used. D’Adamo et al. [52] perfused rat liver with [3H]acetate and after degradation of the labelled fatty acids found 70 % of their tritium in the terminal methyl. This indicates 82% of tritium exchange after formation of malonate, assuming no isotope effect in the carboxylation and assuming that the fatty acids were formed primarily de novo by the malonyl-CoA pathway. Foster and Bloom [53], using rat liver slices incubated in a medium containing doubly-

415

labelled acetate having a 3H/’4C ratio = 3, reported an isotope ratio of 0.6 in palmitic acid, indicating 74% exchange after malonate formation if the additional assumption is made that the acetyl-CoA used for the synthesis had suffered no exchange of tritium. Abraham et al. [54] using rat mammary gland preparations (where the major product of synthesis was decanoic acid) quoted results indicating tritium exchange after malonate formation of 630/;; for tissue slices and 51 % for a high-speed supernatant fraction prepared from a tissue homogenate. With a microsome plus supernatant preparation from rat liver homogenate the tritium retention (in palmitate) indicated a corresponding exchange of 74 ‘%,. Bressler and Wakil [55] using a partially purified enzyme preparation from pigeon liver and a [2-3H]malonyl-CoA substrate (prepared from [3H]acetyl-CoA by use of the carboxylase) reported a maximum tritium exchange of 29% based on overall fatty acid (palmitate) synthesis as measured by NADPH oxidation. Our own investigations, carried out with purified synthetase preparations, confirm that partial exchange of tritium occurs after formation of malonyl-CoA, the extent of this exchange differing with synthetases of different origin. The data are sufficient for an attempt to analyse the factors governing retention of tritium between acetate and palmitate. These factors are (a) the hydrogen isotope effect in carboxylation of acetylCoA, (b) the exchange of C-2 hydrogen in malonylCoA (or some other malonate intermediate, for example an enzyme-bound malonyl thiol ester) with hydrogen from the medium before utilisation in synthesis, (c) the presumably stereospecific elimination of one hydrogen from what was C-2 of malonylCoA, and (d) the partial further exchange of hydrogen from this carbon, occurring either before or after the elimination. Relation between Hydrogen Exchange and Chirality of Methylene Groups

It is useful here to derive a general expression connecting loss of isotope with loss of chirality in the exchange of hydrogen from a methylene group by enolisation or mechanistically similar processes. For a species A - CH,Hb - B, exchanging hydrogen with a large excess of ionisable H, in a solvent, the ratelimiting pseudo-unimolecular step of enolisation produces A- CH,- B and A- CH,- B in the ratio K = kHb/kH,, a kinetic isotope effect. Provided that A and B have no chiral centre or at least do not exert a significant selective effect on the stereochemistry of addition of H,, these two ions will add H, to produce ‘racemic’ (equal numbers of molecules that are R and S at methylene group) or achiral species. Consider a population P of molecules (ACH&- B or A - CH,H, - B) containing an isotope

Fatty Acid Biosynthesis from Chiral Acetates

416

Ha and undergoing hydrogen exchange in a solvent furnishing H, (which may be the same as Hb). The population consists of y molecules that can be grouped in racemic pairs (that is, ‘12 y are R and ‘12 y are S ) and x molecules A-CH,Hb-B that are all R (or all S). For the rate of change in the total P we have, assuming that secondary isotope effects are negligible when HbfHc,

and, for the rate of change of

X,

where kHa and kHb,are rate constants for removal of H, and Hb respectively. Dividing (1) by (2), and substituting K for kHb/kHa,

which integrates to x = CP“ + K ) where C i s a constant. Since P = x + y this is equivalent to (4) If initial values xo, yo and Po are assigned, C is evaluated as

and

X ~

x+y

xo

- ~-

xo+yo

eK

(5)

where e = PIPo. This is an expression for the ‘optical purity’ of the population containing Haafter a fraction (1 - e) of this isotope has been lost. It can be transformed in various ways: for example, starting from an optically pure R (or S) specimen of A-CH3H-B (yo = 0) the proportion of R molecules in the tritiated material remaining after a fraction (1 - e) of the tritium has been exchanged is given by

The same equation applies to a specimen of A - C2H3H- B undergoing exchange, though here K = kZH/k3*instead of k H / k J H and the count of R molecules includes the proportion of R A- CH3HB arising from the exchange. Since, in enolisations, k H / k J is H commonly around 10 and k2H/k3Haround 2 [56], it can readily be seen that even a small exchange of tritium will affect the chirality of A-CH3H-B much more that that of A-C2H3H-B, a difference important to analysis of our results with chiral acetates. Indeed, this difference could in principle be used to elucidate the stereochemistry of all stereospecific conversions of methyl into methylene groups, irrespective

of whether the conversion is associated with an isotope effect.

‘Post-malonate’ Exchange during Conversion of [jHJAcetate into Palmitate

The overall retention of tritium relative to 14C in the conversion of [3H]acetyl-CoA into palmitate is given in Table 4 for the chicken liver and yeast synthetases. The loss of tritium is compounded of the factors (a-d) mentioned above and factor (d) can now be evaluated. Factor (a). In carboxylation of acetyl-CoA the experimentally determined k ~ / k is 3 ~1.23. Consider a population of molecules of acetyl-CoA actually containing tritium : the proportion of tritiated to nontritiated malonyl-CoA molecules produced from this population will be 2 : k+,/kH, i. e. 2 :0.813. Considering for convenience a population of 2813 molecules of tritiated acetyl-CoA, carboxylation will produce 2000 molecules of [3H~]malonyl-CoA (divided equally between R and S species) and 813 molecules of nontritiated (but still 14C-labelled in our experiments) malonyl-Co A. Factor (b). Incubations produzing fatty acids were in general of 5-min duration. The mean age of a malonyl-CoA molecule used for synthesis was therefore not more than 2.5 min, though it could have been less. It has already been indicated that under experimental conditions similar to those used in enzymic incubations (buffer strength, temperature, pH) the time for 25% exchange of available protium on C-2 of malonyl thiol esters is 5 - 6 min. This exchange (as discussed in the following paper) is a base-catalysed enolisation. Isotope effects in such processes have been extensively studied [56] and while their magnitude depends on the nature of the proton-abstracting base, the normal range of kH/kzHis 4- 7 with a corresponding k H / k ~of H 7-12. We shall assume k ~ / k = 2 ~5, k ~ / k= 3 ~10, k 2 ~ / k = 3 ~2 for malonyl thiol ester exchanges. While not precise this is unlikely to be far from the truth and is consistent with the data that we have. Applying this to an estimated 2.5-min exposure time for malonyl-CoA we obtain a mean of 1.25% tritium exchange during this time: that is of the 2813 molecules under consideration the 2000 tritiated molecules have been reduced to 1975 and the 813 nontritiated have increased to 838. Factor (c). The stereospecific loss of hydrogen by elimination, whether it happens before or after ‘postmalonate’ exchange, leads in this case to a loss of half the surviving tritium: the final result is the same whenever this halving is applied. Applying it for convenience before considering factor (d) we have 987.5 tritiated and 1825.5 non-tritiated ‘malonyl units’ on the synthetase.

B. Sedgwick and J. W. Cornforth

471

Factor ( d ) . With the yeast synthetase, the overall retention of tritium, relative to 14C, from [3Hl]acetylCoA in palmitate was 37.5%. Our 2813 malonyl-CoA molecules were joined on the synthetase by 281317 = 402 molecules of tritiated acetyl-CoA to form 402 molecules of palmitate. In this palmitate 37.5% of the original 402 + 2813 = 3215 tritium atoms have survived, i.e. 1206 atoms. Of these, 402 are present in the terminal methyl groups of palmitate, leaving 804 in tritiated C2 ‘malonyl units’. In other words, the 987.5 tritiated units have been reduced by ‘postmalonate’ exchange to 804, a loss of 18.6%. With chicken liver synthetase the overall retention of tritium was 32.3 % and the ‘post-malonate’ loss by exchange, calculated in the same way, was 35.6 %. In comparison, the experiment with yeast synthetase and [2-2H2]malonyl thiol ester showed 31 % deuterium exchange due to a combination of factors (b) and (d). Correcting for factor (b) (maximally 4 %) leaves 27 % for ‘post-malonate’ exchange and suggests that this exchange is subject to a normal isotope effect with k2H/k3H around 2.

Survival of Tritium in Palmitate from Chiral Acetates The effect of factors (a-d) in the experiments with chiral acetates can now be evaluated and compared with the experimental results. We assume for convenience that the carboxylation proceeds with retention of configuration, i. e. that S-acetyl-CoA yields S-malonyl-CoA. Factor ( a ) . For k ~ / k = 3 ~1.23 and kH/kZH = 1.15 in the carboxylation of acetyl-CoA, the values of k3H/kH and kz,/kH are 0.813 and 0.870 respectively. Now the carboxylation of S-CH2H3HCOSCoAwill produce the species S-H02CC2H3HCOSCoA, RH02CCH3HCOSCoA and non-tritiated S-H02CCH2HCOSC~A in the ratio 1 : kz,/kH: k3,/kH respectively, or 1 :0.870:0.813. Starting, again for convenience, with 2683 molecules the numbers of these species will be 1000, 870 and 813. Factor ( b ) . The above population is now subject to exchange of C-2 hydrogen leading to 1.25 % loss of tritium. Considering first the 1000 molecules of S-H02CC2H3HCOSCoA: 987.5 of these will retain tritium and of this total, applying Equation (6) with K = 2, e = 0.9875, ‘12 (1 0.9875’) x 987.5 = 975 will be S and 12.5 R (no distinction is made in these totals between C2H3Hand CH3H species of the same chirality). At the same time, of the 870 molecules of R-H02CCH3HCOSCoA, 859 retain tritium and of these ’I2 (1 + 0.98751°) x 859 = 808 will be R and 51 S. Thus after the exchange, the S-tritiated species totals 1026 and the R species 820.5. Conversely, from 2683 molecules of R-acetyl-CoA the tritiated S-

+

malonyl-CoA will number 820.5 and the R species 1026. Factor (c). Since, in the event, S-acetyl-CoA led to greater retention of tritium in palmitate, it can be assumed that the more abundant species of chiral malonate derived from it retained tritium in the elimination reaction. After this elimination (if it precedes ‘post-malonate’ exchange) 1026 tritiated ‘malonyl units’ will survive from S-acetyl-CoA and 820.5 from R-acetyl-CoA. Factor ( d ) . Application of a loss of 18.6% tritium to the 1026 tritiated units from S-acetyl-CoA leaves 835 tritiated units which are joined by 383 tritiated acetate units from acetyl-CoA to form palmitate. The total retention is then (835 383)/3066 or 39.7 %. By a similar calculation the retention of tritium from Racetyl-CoA is 34.3 % compared with the experimental figures of 39.9% and 36.6% respectively. For the 35.6 % loss calculated for the chicken liver synthetase the calculated values are 34.1 % retention from Sacetyl-CoA and 29.7 % retention from R-acetyl-CoA compared with 34.3 % and 30.4 % found experimentally (Table 4). It would not be difficult to alter slightly and within experimental error the numerical factors used (isotope effects in carboxylation, extent of malonate exchange, isotope effects in malonate exchange, extent of ‘postmalonate’ exchange) so as to get more exact agreement; but we have preferred to use where possible the factors indicated by experiment. The point is that the retentions from R and S acetates are well correlated with the experimental figures and with the retention from [3Hl]acetate, both for the yeast and for the chicken liver synthetases.

+

Nature of ‘Post-malonate’ Exchange In the foregoing calculations it has been assumed that the elimination on the synthetase of one hydrogen, from what was C-2 of malonyl-CoA, preceded ‘postmalonate’ exchange. For a [3Hl]acetyl-CoA precursor this assumption was immaterial since the result would have been the same if elimination had followed exchange. For the chiral acetyl-CoA precursors this is not so, and the point is worth examination. ‘Post-malonate’ exchange is subject to a kinetic isotope effect, as the differences between deuterium and tritium retention on the yeast enzyme show clearly: kz,/k3, is of the order of 2, which indicates that kH/ks, is of the order of 10, a similar effect to that occurring in ‘malonate’ exchange. This fact makes it difficult to construct any mechanism, agreeing with experiment, whereby ‘post-malonate’ exchange occurs before the elimination of one methylene hydrogen. Consider, for example, the hypothesis that the synthetase or some other component of the system

Fatty Acid Biosynthesis from Chiral Acetates

478

catalyses exchange of hydrogen in malonyl-CoA (or malonyl-enzyme) so that the entire exchange occurs at this stage: for the yeast enzyme the total tritium exchange would be 18.6 + 1.25 = 19.85%. Assuming that isotope effects in this exchange are kz,/k3H = 2 and kH/k3H= 10, calculation as given above but with e now 0.8015 leads to 44.1 % retention of tritium from S-acetyl-CoA and 29.8 retention from R-acetylCoA. Similar considerations apply if, in the reaction mechanism pictured in Scheme 2, exchange at the methylene group occurs at the stages RCOCHzCOSX or RCHOHCH2COSX. It is possible to make one rather implausible assumption that removes this discrepancy : that the 'post-malonate' exchange is stereospecific, that it occurs with retention of configuration and that it involves the hydrogen which survives the subsequent elimination. While there is nothing impossible about this assumption is does seem unlikely that an enzyme should catalyse so stereospecific an exchange as a nonessential by-product of its normal activity, especially since the hydrogen normally removed is the epimeric hydrogen and that any specific hydrogen-abstracting groups on the enzyme would tend to be adapted for removal of that hydrogen. On the other hand, if 'post-malonate' exchange occurs after elimination, no such difficulties arise. There seem to be two main possibilities here. First, the condensation reaction between RCOSX and HOzCCH'COSX may not be concerted: i . e . , it does not lead directly to RCOCH2COSX and CO2, but pro/COzH which then ceeds via an intermediate RCOCH

lcosx

undergoes a rate-limiting decarboxylation with addition of hydrogen (H*) from the medium to form RCOCHH*COSX. The intermediate, having a methine hydrogen activated by three adjacent carbonyl groups, might undergo partial exchange of this hydrogen even while bound to the enzyme, and this exchange would be subject to a kinetic isotope effect. This explanation of 'post-malonate' exchange is chemically plausible but it does require the additional proviso that H * in the decarboxylated product is the hydrogen lost in subsequent dehydration of RCHOHCHH*COSX. Moreover, it is contradicted (at least for the condensing enzyme from E. coli) by the work of Arnstadt et ul. [57].These authors reported virtually no loss of hydrogen from C-2 of malonate, and no tritium incorporation from the medium, in 3-hydroxybutyrate synthesized from acetyl-CoA and S-malonyl-N-acetylcysteamine by enzymic condensation and reduction, thus implicating the concerted mechanism of condensation. Second, the 'post-malonate' exchange may occur after the formation of RCH=CHCOSX in the cycle of synthesis. The final stages of the cycle are the formation of RCH2CH2COSX and its transfer to another sulphydryl group in

the enzyme complex [5,16] and it is possible to formulate mechanisms for partial base-catalysed exchange of the methylene hydrogens indicated in boldface type during (or between) these two steps. An interesting (and experimentally testable) consequence would be that the surviving hydrogen from malonate would not necessarily occupy a specific stereochemical position in the product palmitate: for example, the - CH'H-groups in palmitate synthesised from HOZCC~HZCOSCOA would be partially racemised as a result of non-stereospecific hydrogen exchange. This consequence could not arise from the first hypothesis above. Partial exchange of hydrogen at a later stage, at the methylene groups indicated in boldface type in R'CH2CH = CHCOSX or R'CH2COCH2COSX, for example, offers the same possibility for partial racemisation. We thank Mr J. M. Baron and Mrs Caroline Morris for technical assistance, and Dr J. W. Cornforth Jr. for discussions on formulation of the relationship between chirality and isotopic exchange. The chiral acetates used in this work were prepared by D r R. Mallaby.

REFERENCES Rittenberg, D.&Bloch, K . (1945)J.Biol. Chem. 160,417-424. Lynen, F. (1953) Fed. Proc. I2, 683-691. Wakil, S. J. (1961) J . Lipid Res. 2, 1-24. Wakil, S. J . (1962) Annu. Rev. Biochem. 31, 369-406. Lynen, F. (1967) Biochem. J . 102, 381 -400. Lynen, F. (1969) Meihods Eniymol. 14, 17-33. Vagelos, P. R. (1964) Annu. Rev. Biochem. 33, 139-172. Stumpf, P. K. (1969) Annu. Rev. Biochen?. 38, 159-212. Lennarz, W. J., Light, R. J . & Bloch, K. (1962) Proc. NutlAcutl. Sci. U.S.A. 48, 840-846. 10. Porter, J. W., Kumar, S. & Dugan, R. E. (1971) Progr. Biochem. Pharmacol. 6, 1 - 101. 11. Lynen, F. (1961) Fed. Proc. 20, 941-951. 12. Wakil, S. J. & Bressler, R. (1962) J . Bid. C h m . 237, 687-693. 13. Dugan, R . E., Slakey, L. L. & Porter, J. W . (1970) J . Biol. Chem. 245,6312-6316. 14. Wakil, S. J., Pugh, E. L. & Sauer, F. (1964) Proc. Nut1 Ac.ad. Sci. U.S.A. 52, 106-114. 15. Matsumura, S., Brindley, D. N . & Bloch, K . (1970) Biochcm. Biophys. Res. Commun. 38, 369 - 377. 16. Phillips, G . T., Nixon, J. E., Dorsey, J. A,, Butterworth, P. H. W., Chesterton, C . J. & Porter, J . W . (1970) Arch. Biochem. Biophys. 138, 380- 391. 17. Joshi, V. C., Plate, C . A. & Wakil, S. J . (1970) J . Biol. Chcwi. 245, 2857-2867. 18. Kumar, S., Dorsey, J. A , , Muesing, R. A. & Porter, J. W. (1970) J . Biol. Chem. 245, 4732 - 4744. 19. Goto, T., Ringelman, E., Riedel, B. & Numa, S. (1967) Lip Sci. 6, 785 - 790. 20. Hsu, R. Y. & Yun, S. L. (1970) Biochen7istr.y, 9, 239-251. 21. Hetherington, P. J., Follows, M., Dunnill, P. & Lilly, M. D. (1971) Trans. Insr. Chmi. Engrs. 49, 142- 148. 22. Lenz, H., Buckel, W., Wunderwald, P., Biederman, G., Buschmeier, V., Eggerer, H., Cornforth, J. W., Redmond, J . W. & Mallaby, R. (1971) Eur. J . Biochem. 24, 207-215. 23. Simon, E. J. & Shemin, D. (1953) J . Am. ('hem. Sot,. 75, 2520. 24. Patterson, M . S. & Greene, R . C. (1965) A n d . Chem. 37, 854857. 1. 2. 3. 4. 5. 6. 7. 8. 9.

B. Sedgwick and J . W . Cornforth

25. Lowry, 0. H., Rosebrough, N. J . , Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 26. Bligh, E. G. & Dyer, S. W. (1959) Can. J . Biochem. Physiol. 37, 911 -917. 27. Brindley, D. N., Smith, M. E., Sedgwick, B. & Hubscher, G. (1967) Biochim. Biophys. Acta, 144, 285- 295. 28. Swain, C. G., Stivers, E. C., Reuwer, J . F. J r & Schaad, L. J. (1958) J . Am. Chem. Soc. 80, 5885-5893. 29. Eggerer, H. & Lynen, F. (1962) Biochem. Z . 335, 540-547. 30. Sumper, M . & Lynen, F. (1972) FEBS Lett. 28, 142- 144. 31. Prescott, D. J. & Rabinowitz, J. L. (1968) J . Biol. Chem. 243, 1551 - 1557. 32. Retey, J. & Lynen, F. (1965) Biochem. Z . 342, 256-271. 33. Arigoni, D., Lynen, F. & Retey, J. (1966) Helv. Cliim. Acta, 49, 311-316. 34. Rose, I . A. (1970) J . Biol. C/iem. 245, 6052-6056. 35. Cornforth, J . W., Redmond, J. W., Eggerer, H., Buckel, W. & Gutschow, C. (1969) Nature (Lond.) 221, 1212-1213. 36. Retey, J., Luthy, J. & Arigoni, D. (1969) Nature (Lond.) 221, 1213- 1215. 37. Cornforth, J. W., Redmond, J . W., Eggerer, H., Buckel, W. & Gutschow, C. (1970) Eur. J . Biochem. 14, 1 - 13. 38. Eggerer, H., Buckel, W., Lenz, H., Wunderwald, P., Gottschalk, G., Cornforth, J. W., Donninger, C., Mallaby, R. & Redmond, J . W. (1970) Nature, (Lond.) 226, 517-519. 39. Retey, J., Luthy, J. & Arigoni, D. (1970) Nature (Lond.) 226, 51 9 - 521. 40. Gottschalk, G., Ditlbrenner, S., Lenz, H. & Eggerer, H. (1972) Eur. J . Biochem. 26, 455 - 461. 41. Wunderwald, P., Buckel, W., Lenz, H., Buschmeier, V., Eggerer, H., Gottschalk, G., Cornforth, J. W., Redmond, J. W. & Mallaby, R . (1971) Eur. J . Biochem. 24, 216-221.

479 42. Cornforth, J . W., Phillips, G. T., Messner, B. & Eggerer, H. (1974) Eur. J . Biochem. 42, 591 - 604. 43. Mildvan, A. S. & Scrutton, M . C. (1967) Biochemistry, 6, 2978 - 2994. 44. Lynen, F. (1970) Biochem. J . 117, 47P. 45. Alberts, A. W., Nervi, A. M. &Vagelos, P. R. (1969) Proc. Nurl Acad. Sci. U . S . A .63, 1319-1326. 46. Alberts, A. W., Gordon, S. G . & Vagelos, P. R . (1971) Pro(,. Nail Acad. Sci. U.S.A. 68, 1259- 1263. 47. Heinstein, P. F. & Stumpf, P. K. (1969) J . B i d . Chon. 244, 5374-5381. 48. Hashimoto, T. & Numa, S. (1971) Eur. J . Biochem. 18, 319331. 49. Sumper, M. & Lynen, F. (1972) Hoppe-Scyler‘s Z. Physiol. Chem. 353,502. 50. Gregolin, C., Ryder, E. & Lane, M. D. (1968) J . Bid. Chern. 243,4227 -4235. 51. Rose, I. A. ( I 972) Crit. Rev. Biochem. I , 33 - 57. 52. D’Adamo, A. F. Jr, Hoberman, H . D. & Haft, D. (1961) Fed. Proc. 20, 274. 53. Foster, D. W. & Bloom, B. (1962) Biochim. Biophj~s.Actn, 60, 189- 190. 54. Abraham, S., Katz, J., Bartley, J. & Chaikoff, I. L. (1963) Biochim. Biophj,.s. Acta, 70, 690- 693. 55. Bressler,R.&Wakil,S. J.(1961)J. Biol. C/wni.236,1643- 1651. 56. Bell, R. P. (1973) The Proton in Chemistry, 2nd edn, ch. 12, Chapman and Hall, London. 57. Arnstadt, K.-I., Schindlbeck, G. & Lynen, F. (1975) Eur. J . Biochem. 55. 561 - 571.

B. Sedgwick, Milstead Laboratory of Chemical Enzymology, Shell Research Limited, Broad Oak Road, Sittingbourne, Kent, Great Britain, ME9 8AG J . W. Cornforth, School of Molecular Sciences, University of Sussex, Falmer, Brighton, Great Britain, BNI 9QJ