ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 196, No. 1, August, pp. 88-92, 1979

Specificity and Site of Action of a Mammary Gland Thioesterase Which Releases Acyl Moieties from Thioester Linkage to the

Fatty Acid Synthetasel

STUART SMITH2 AND LOUIS J. LIBERTIN13

Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center, 51st and Grove Streets, Oakland, California 94609

Received January 29, 1979; revised March 20, 1979

Previous work from this laboratory (L. J. Libertini and S. Smith, 1979, Arch. Biochem. Biophys. 192, 47-60) has shown that thioesterase II, a mammary gland-specific enzyme, modifies the product specificity of the fatty acid synthetase multienzyme by removing acyl chains attached to the multienzyme through thioester linkage. In this study, two experi- mental approaches were used to identify the site of action of thioesterase II. (i) The two thioesterase I domains were removed from the fatty acid synthetase with trypsin. The modified multienzyme was incubated with [2-%]malonyl-CoA, acetyl-CoA, and NADPH to form [14C]acyl-multienzyme thioesters, which were shown to be susceptible to hydrolysis by thioesterase II. Following peptic digestion and [‘%]acyl-peptide purification, the site of attachment of the acyl moiety to the modified multienzyme was identified as a pantetheine thiol. (ii) The ability of thioesterase II to hydrolyze a number of cysteine- and cysteamine- containing thioesters was compared. Cysteamine thioesters were good substrates for thio- esterase II, which showed the following preference: S-decanoyl-pantetheine > S-decanoyl- CoA > S-decanoyl N-acetylcysteamine. None of the cysteine thioesters were effective sub- strates for thioesterase II. These two lines of evidence clearly indicate that the site of action of thioesterase II is the thioester bond linking the acyl moiety to the 4’-phosphopantetheine component of the fatty acid synthetase.

In mammary gland, biosynthesis of me- dium-chain fatty acids by the fatty acid syn- thetase requires participation of an additional enzyme which is not part of the multienzyme complex (1, 2). We have shown that acyl moieties assembled on the fatty acid syn- thetase can be released from their thioester linkage to the multienzyme complex by this enzyme (3); the enzyme has been designated thioesterase II (2) to distinguish it from the

’ Supported in part by Grants AM16073 and RR05467 from the National Institutes of Health, DHEW, and Grant BMS 7412723 from the National Science Foundation.

2 Part of this work was carried out while the author was the recipient of an Established Investigatorship of the American Heart Association.

3 Supported by a National Institutes of Health Fellowship, F32 CA05752, during part of this study. Present address: Department of Biochemistry and Bio- physics, Oregon State University, Corvallis, Oreg. 97331.

two thioesterase I domains (4, 5) covalently associated with the fatty acid synthetase. The identity of the acyl-multienzyme thiol attacked by thioesterase II has not yet been established. Porter’s group (6) has proposed that, during fatty acid synthesis, the grow- ing acyl moieties are transferred from a 4’- phosphopantetheine thiol to a peripheral cysteine thiol between each round of the elongation sequence catalyzed by the multi- enzyme. This shift allows for reloading of the 4’-phosphopantetheine thiol with a malonyl group, in preparation for the sub- sequent condensation step. We have used two independent approaches to determine whether thioesterase II releases the acyl moiety from a 4’-phosphopantetheine or cysteine thiol: (i) Long-chain acyl-multien- zyme thioesters were synthesized enzymi- tally and shown to be susceptible to hydroly- sis by thioesterase II. The acyl-multienzyme

0003-9861/‘79/090088-05$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

88

SPECIFICITY OF MAMMARY GLAND THIOESTERASE 89

was digested with pepsin and the acyl-pep- tide characterized. (ii) The ability of thio- esterase II to hydrolyze a number of model thioesters containing cysteamine and cyste- ine moieties was compared.

EXPERIMENTAL PROCEDURES

Preparation of enzymes. Thioesterase II and fatty acid synthetase were isolated from lactating rat mam- mary gland (2, 7). The two thioesterase I domains were stripped from the fatty acid synthetase by limited trypsinization (5). The residual core of the multienzyme, which retains all partial activities except the thioester- ase, is referred to as trypsinized fatty acid synthetase.

Preparation of long-chain acyl-S-multienzyme and isolation of acyl-peptide thioester. In a typical experi- ment, trypsinized fatty acid synthetase (614 mg) was incubated with NADPH (0.12 mM), acetyl-CoA (0.05 mM), and [2-14C]malonyl-CoA (0.05 mM, 0.16 Ci/mol) in 330 ml of 80 mM potassium phosphate buffer (pH 7). The reaction mixture was incubated for 1.5 min at 3O”C, then trichloroacetic acid (7% final concentration) was added. The precipitated protein was washed three times with 30 ml of 0.2 N acetic acid and finally sus- pended in 12 ml of 1 mM HCl; the pH was 3. Pepsin (25 mg) was added and the suspension incubated for 20 h at 30°C with shaking. The resulting clear solution was lyophilized. The dry powder was partitioned be- tween butan-l-01 and water (l/l, v/v, 20 ml total). The upper phase was collected and the lower phase was washed twice with 5 ml butan-l-01 saturated with water. The upper phases which contained over 90% of the radioactivity, were pooled. The [‘V]acyl-peptide was purified by partition chromatography on a column (95 x 2.5 cm) of Sephadex G-25, equilibrated and eluted with butan-l-01 saturated with water/l% acetic acid (8). Radioactivity eluted as a single zone. Fractions containing radioactivity were pooled and lyophilized. The residue was dissolved in a minimum volume of methanol/water/acetic acid (5/2/l, v/v/v) and applied to a plate coated with silica gel G (20 x 20 x 0.1 cm). The plate was developed in phenohbutan-l-ollwateriace- tic acid (2/2/1/l, v/v/v/v), dried and examined by radio- autography. A single major radioactive zone was re- vealed (R,0.57). A trace of radioactive component with an R,of 0.40 was found in one experiment. Radioactive material was removed from the silica gel by repeated extraction with methanol/water/acetic acid (5/2/l, v/v/v, 7 x 5 ml). Solvent was evaporated under vacuum, the residue was dissolved in butan-l-01, saturated with water/l% acetic acid and stored under nitrogen at -70°C.

Amino acid analysis. Portions of the acyl-peptide thioester were lyophilized and hydrolyzed, under vacuum at llO”C, with 6 N HCl. Duplicate portions were also oxidized with performic acid (9) prior to hy- drolysis. Components were identified by cochromatog-

raphy with authentic standards; a Beckman amino acid analyzer was used.

Gus chromatography. Fatty acids were methylated with diazomethane (10) and analyzed on a Perkin- Elmer 3920gas chromatograph, equipped with an auto- matic capsule injection system and a radioactivity monitor. A column (6 ft x ‘/s in.) containing 10% di- ethylene glycol succinate on Chromosorb W (So-100 mesh) was used.

Synthesis of decanoyl thioesters. Decanoyl pante- theine, S-decanoyl N-acetylcysteamine, S-decanoyl N-acetylcysteine, S-decanoyl N-acetylglutathione, decanoyl benzenethiol, and S-decanoyl3-thiopropionic acid were prepared by slight modifications of the fol- lowing general procedure.

(i) Protection of free amino groups. Compounds con- taining amino groups were N-acetylated prior to acyla- tion of the thiol group. Cystamine or glutathione disul- fide was dissolved in water and the pH adjusted to 7.5 with KOH 40% (w/v). A twofold excess of acetic anhydride was added dropwise while the pH was main- tained at 7.5. Complete disappearance of amino groups was verifled with fluorescamine (11). The N-acetylcys- tamine was recovered by filtration after saturating the solution with NaCl. Potassium ions were removed from the N,N-diacetylglutathione disulfide solution with Bio-Rad AG 50W x8 (H+ form) and the solution lyophilized.

(ii) Reduction of disulfides. Pantethine, N,N-diace- tylglutathione disulfide, and N-acetylcystamine were reduced with sodium amalgam (12) prior to acylation with decanoyl chloride.

(iii) Reaction of thiol with decanoyl chloride. A two- fold excess of decanoyl chloride was added stepwise to a solution of the thiol compound in tetrahydrofuran/ water (l/l, v/v); the pH was maintained between 7.5 and 8.0 by addition of 40% (w/v) KOH. When no further drop in pH was observed, 12 N HCl was added to adjust the pH to 1.5-2.5; this caused the formation of two phases. NaCl was added to saturation and the upper phase was collected; the lower phase was washed with an equal volume of diethyl ether and the ethereal phase combined with the original upper phase. Solvent was evaporated in a stream of nitrogen. Decanoic acid was removed from the thioesters of N-acetylglutathione, N-acetylcysteine, N-acetylcysteamine, and pante- theine by washing three times with 20 vol petroleum ether (bp 35-60°C). Sublimation of the decanoic acid was used to separate it from S-decanoyl3-thiopropionic acid. The S-decanoyl-benzenethiol was purified by vacuum distillation (bp 150°C at loo-150 wm).

(iv) Methylation of thioesters containing ‘free car- boxy1 groups. The carboxyl groups of the S-decanoyl thioesters of N-acetylglutathione, N-acetylcysteine, and d-thiopropionic acid were methylated with diazo- methane (IO).

(v) Assay of thioester content. Thioesters were dis- solved in ethanol (0.3 mg/ml) and a O.&ml portion mixed

90 SMITH AND LIBERTINI

with 0.2 ml freshly prepared, neutralized hydroxyl- amine (14%, w/v). The liberated thiol was estimated (13) at intervals, until a maximum value was established.

The S-decanoyl benzenethiol was a liquid having 60% of the expected thioester content, by weight. S-Dec- anoyl3-thiopropionic acid was a white, crystalline solid (mp 60-62°C); the thioester content was 95%. S-Dec- anoyl cysteine was obtained as a white crystalline com- pound (mp 53-54°C) and contained 85% thioester. Decanoylpantetheine was obtained as a clear viscous, extremely hygroscopic liquid and was 73% thioester. S-decanoyl N-acetylcysteamine was a white solid (mp 69°C) consisting of 93% thioester. TheS-decanoyl N,N- diacetylglutathione was a slightly yellow solid (mp 113-115°C) consisting of 79% thioester.

Materials. The sources of most of the materials and the methods for preparing other substrates have been described in recent publications (2,4). Pepsin was pur- chased from Pentex Inc., Kankakee, Illinois. N-Ace- tylcysteine, N-acetylcysteamine, and pantethine were purchased from Sigma, St. Louis, Missouri.

RESULTS AND DISCUSSION

Identification of Site of Attachment of the Long-Chain Acyl Moiety to Trypsinixed Fatty Acid Synthetase

Fatty acid synthetase which has been stripped of its two thioesterase I domains by limited trypsinization retains the ability to assemble a single long-chain acyl moiety but is unable to release the product from its thioester linkage to the multienzyme (3). Thioesterase II, however, can release this thioester-bound acyl moiety as a free fatty acid. We, therefore, anticipated that identi- fication of the site of attachment of the acyl moiety to the modified fatty acid synthetase would reveal the probable site of action of thioesterase II.

Trypsinizecl fatty acid synthetase was loaded with a long-chain [14C]acyl moiety by incubation with [2J4C]malonyl-CoA, acetyl- CoA, and NADPH. A small portion of the [ 14C]acyl-multienzyme was used to confirm that the bound [14C]acyl moieties could be released by thioesterase II (i.e., without prior treatment with trichloroacetic acid, see Experimental Procedures). At pH 8, 3O”C, thioesterase II (1 pg/ml) released all of the radioactive acyl groups bound to 2 nmol of multienzyme within 5 min; the ki- netics of this process have been described in detail (3). The remaining portion of the

TABLE I

COMPOSITION OF [W]ACYLPEPTIDE THIOESTER ISOLATED FROM PEPTIC DIGEST OF

[%]AcYL-FATTY ACID SYNTHETASE~

Peptide component mol/mol Acyl peptide*

Aspartate 1.0 Serine’ 0.9 Glycine 1.0 Leucine 2.1 P-Alanine 1.0 Taurine 1.1 Fatty acid 1.0

a [W]Acyl-fatty acid synthetase was synthesized by incubating trypsinized fatty acid synthetase with NADPH, acetyl-CoA, and [2-W]malonyl-CoA. The [14C]acyl-fatty acid synthetase was digested with pep- sin and the labeled acyl-peptide purified and analyzed (see Experimental Procedures for details).

* Values are means of three analyses on two differ- ent preparations of [W]acyl-peptide.

r Corrected for losses during hydrolysis.

[14C]acyl-multienzyme was digested with pepsin and the resulting hydrophobic [14C]- acyl-pepticle purified. Radiopurity of the [14C]acyl-pepticle was established by thin- layer chromatography (See Experimental Procedures). Performic acid treatment of the [14C]acyl-pepticle, prior to chromatog- raphy, resulted in a change in mobility of the radioactive component; all of tlie radio- activity migrated as free fatty acid. The composition of the fatty acid released from the [14C]acyl-peptide by performic acid was 44% l&O, 56% 20:0. Although only a single long-chain acyl moiety is assembled on each fatty acid synthetase climer, the extent of elongation of the acyl group depends on the specific reaction conditions, primarily the availability of malonyl-CoA and the duration of the incubation period (3). It is common, therefore, to find at any given time a spec- trum of acyl-multienzyme species at various stages of elongation; elongation normally ceases at 20 or 22 C atoms (3).

Amino acid analysis of the [14C]acyl-pep- tide revealed five a-amino acid residues (Table I). When the pentapepticle was oxi- dized with performic acid prior to hydrolysis with HCl, single residues of taurine and

SPECIFICITY OF MAMMARY GLAND THIOESTERASE 91

z

e 0 100 200 300

Thmester ivM1

FIG. 1. Hydrolysis of model thioester substrates by thioesterase II. Incubation systems, at 3O”C, contained 0.1 M Tris/HCl (pH 8.21, 0.1 M 5,5’-dithiobis (2-nitrobenzoate), and substrate. Reactions were monitored by recording the change in absorbance at 412 nm. Ethanol (15%, v/v, final concentra- tion) was included in incubations with decanoyl benzenethiol, S-decanoyl methylthiopropionate, S-dec- anoyl N-acetylcysteine, and S-decanoyl N-acetylmethylcysteine to facilitate dissolution of these compounds.

p-alanine, but no cysteic acid, were found. Taurine and p-alanine are the predicted products from pantetheine whereas cysteic acid would have been formed had a cysteine residue been present. The lability of the [14C]acyl-peptide to performic acid and the presence of both a pantetheine residue and a serine residue [the 4’-phosphopantetheine moiety is linked to the fatty acid synthe- tase via a serine residue (14)] demonstrated clearly that the acyl moiety was bound to the serine-O-4’-phosphopantetheine thiol of the trypsinized fatty acid synthetase.

Confirmation that thioesterase II can in- deed hydrolyze the acyl moiety from the polypeptide containing a 4’-phosphopante- theine thiol was obtained as follows: thio- esterase II (10 pg) was incubated, at 3O”C, with [14C]acyl-pentapeptide (0.43 nmol) and albumin (5 pg) in 0.5 ml of 50 lllM Tris/ maleate/NaOH (pH 7); 0.4 nmol of 14C-fatty acid was released within 20 min.

Examination of Specificity of Thioesterase II Using Model Thioester &b&rates

The ability of a number of decanoyl thio- esters to serve as substrates for thioester- ase II was studied (Fig. 1, Table II). The best substrates were two thioesters con- taining the pantetheine moiety, decanoyl- pantetheine, and decanoyl-CoA; S-decanoyl

N-acetylcysteamine, which contains only the cysteamine portion of the pantetheine moiety, was somewhat less effective than the two pantetheine-containing substrates (Fig. 1). Although decanoyl-pantetheine is only sparingly soluble in water, brief sonica- tion of a suspension of this compound in water was sufficient to provide a form of the

TABLE II

KINETIC CONSTANTS~FORHYDROLYSIS OFMODEL THIOESTER SUBSTRATESBYTHIOESTERASE II

Substrate V K,

(nmoVm/g) @Ml

S-Decanoyl pantetheine S-Decanoyl pantetheineb S-Decanoyl CoA S-Decanoyl N-acetylcysteamine S-Decanoyl Benzenethiolb S-Decanoyl thiopropionic acid S-Decanoyl methylthiopropionate* S-Decanoyl N-acetylcysteineb S-Decanoyl N-acetyl methyl-

cysteine* S-Decanoyl N-acetyl glutathione S-Decanoyl N-acetyl methyl-

glutathione”

1260 27 1000 40 1050 61

79 20 160 5 90 400 15 33 11 240

26 125 0 -

0 -

a Most of the values were obtained from Line- weaver-Burk plots of the data shown in Fig. 2.

b The assay systems included 10% ethanol.

92 SMITH AND LIBERTINI

substrate acceptable to the enzyme. Since it was necessary to include ethanol in some incubations, to assist in solubilizing certain of the thioesters, we also tested the effect of ethanol (lo%, v/v) on the ability of thio- esterase II to hydrolyze decanoyl-pante- theine. Ethanol reduced V 17% and increased the K, about 50% (Table II). Several com- pounds containing S-decanoyl cysteine moieties were tested as substrates; none of them were very effective. No activity was observed using S-decanoyl N-acetylgluta- thione or its methyl ester. The activity ob- served with S-decanoyl N-acetylcysteine was about 1% of that observed with dec- anoyl-pantetheine; methylation of the car- boxy1 group made little difference to the effectiveness of this compound as a substrate for thioesterase II. Two thioesters unrelated structurally to the pantetheine or cysteine thioesters were tested and found to have slight activity. Surprisingly thioesterase II appeared to have a high affinity for the dec- anoyl ester of benzenethiol (K, 5 pM) in 10% ethanol. The V values for this compound and that for S-decanoyl3-thiopropionic acid, however, were much lower than that for decanoyl-pantetheine.

Those compounds which were not effec- tively hydrolyzed by thioesterase II were tested for inhibition of the enzymatic hydrol- ysis of S-decanoyl-pantetheine, in the pres- ence of 10% ethanol. None of the compounds showed significant inhibition at 100 j&M.

A cysteine site on the fatty acid synthe- tase has been shown to bind fatty acyl moieties, but not malonyl moieties. The S-acylcysteine moiety appears to be involved in the condensation reaction with the S-mal- onylpantetheine moiety of the fatty acid synthetase (6). The inability of thioesterase II to hydrolyze a number of decanoyl thio- esters of cysteine-containing compounds indicates that it is unlikely that the enzyme removes acyl moieties from a cysteine thiol on the fatty acid synthetase. The ability of thioesterase II to hydrolyze decanoyl thio- esters of several pantetheine-related com-

pounds and the direct demonstration that thioesterase II is able to hydrolyze acyl- panthetheine thioesters attached to a fatty acid synthetase polypeptide is strong evi- dence to indicate that this is the site of action of the mammary gland enzyme. Thio- esterase I, the fatty acid synthetase domain, which, in the absence of thioesterase II, terminates growth of the acyl chain at 16 C atoms, is also believed to hydrolyze acyl moieties from their thioester linkage to the 4’-phosphopantetheine site (4, 6). Thus the spatial arrangement of polypeptide in the vicinity of this site is such that both thio- esterases have access to the acyl-4’-phos- phopantetheine thioester.

1.

2.

3.

4.

5.

6.

7.

8.

9. 10.

11.

12.

13.

14.

REFERENCES

KNUDSEN, J., CLARK, S., AND DILS, R. (1977) Biochem. J. 160, 683-691.

LIBERTINI, L. J., AND SMITH, S. (1978) J. Biol. Chem. 253, 1393-1401.

LIBERTINI, L. J., AND SMITH, S. (1979)Arch. Bio- them. Biophys. 192, 47-60.

LIN, C. Y., AND SMITH, S. (1978) J. Biol. Chem. 253, 1954- 1962.

DILEEPAN, K. N., LIN, C. Y., AND SMITH, S. (1978) Biochem. J. 175, 199-206.

PHILLIPS, G. T., NIXON, J. E., DORSEY, J. A., BUTTERWORTH, P. H. W., CHESTERTON, C. J., AND PORTER, J. W. (1970) Arch. Biochem. Biophys. 138, 380-391.

SMITH, S., AND ABRAHAM, S. (1975) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 35, pp. 65-74, Academic Press, New York.

AYLING, J., PIRSON, R., AND LYNEN, F. (1972) Biochemistry 11, 526-532.

HIRS, C. H. (1956) J. Biol. Chem. 219, 611-621. ARNDT, F. (1943) Organic Syntheses, p. 165,

Wiley, New York. UDENFRIEND, S., STEIN, S., BOHLEN, P., DAIR-

MAN, W., LEIMGRUBER, W., AND WEIGELE, M. (1972) Science 178, 871-872.

FIESER, L. F. (1941) Experiments in Organic Chemistry, p. 419, Heath, New York.

ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77.

RONCARI, D. A. K., BRADSHAW, R. A., AND VAGELOS, P. R. (1972) J. Biol. Chem. 247, 6234-6242.