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Inhibition of fatty acid synthesis by the antibiotic cerulenin
Biochimica et Biophysics Acta, 326 (1973) 155-166 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA 56348
INHIBITION
OF FATTY
ACID
SYNTHESIS
BY THE
ANTIBIOTIC
CERU-
LENIN SPECIFIC
INACTIVATION
OF
/3-KETOACYL-ACYL
CARRIER
PROTEIN
SYNTHETASE
GIULIANO D’AGNOLO=*, and P. ROY VAGELOS”**
IRA s. ROSENFELD~,
JuICHI
AWAYA~, SATOSHI OMURA~
‘Department of Biological Chemistry, Washington University School of Medicine, St. Louis, MO. 63110 (U.S.A.) and bThe Kitasato Institute and School ofPharmaceutical Science, Kitasato University, Tokyo, (Japan)
(Received July z3rd, 1973)
SUMMARY
Cerulenin, (2s) (3R)2,3-epoxy-+oxo-7,Io-dodecadienoylamide, an antibiotic isolated from the culture filtrate of Cephalosporium caerukns, inhibits the fatty acid synthetase of Escherichia coli by specifically inhibiting P-ketoacyl-acyl carrier protein synthetase, the enzyme which catalyzes the condensation reaction of fatty acid biosynthesis. Although cerulenin and tetrahydrocerulenin inhibited both P-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase, neither dihydrocerulenin nor hexahydrocerulenin, analogs which lack the epoxide ring, inhibited these enzyme systems. Both P-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase were protected to the same extent against inhibition by cerulenin by prior incubation with acetyl-acyl carrier protein. The acyl group of this thioester is bound at the fatty acyl site of /3-ketoacyl-acyl carrier protein synthetase. Detailed studies with homogeneous /I-ketoacyl-acyl carrier protein synthetase indicated that cerulenin inhibited model reactions representing both the fatty acyl transacylase and the malonyl-acyl carrier protein decarboxylase components of the P-ketoacyl-acyl carrier protein synthetase reaction. Inhibition of /?-ketoacyl-acyl carrier protein synthetase by cerulenin was irreversible, and it was associated with the binding of approximately I mole of inhibitor per mole of enzyme when inhibition approached IOO %.
INTRODUCTION
Cerulenin Cephalosporium
is an antibiotic isolated from the culture filtrate of the fungus caerulens’ and has the structure (6’) (3R)q-epoxy-4-0x0-7,10-
* Permanent address: Laboratori di Chimica Biologica, Istituto Superiore Italy. ** To whom reprint requests should be addressed.
di Sanita, Rome,
CERULEMIN
H
Fig.
I. Structure
of cerulenin
H
and its analogs.
dodecadienoylamide*S”‘3 (Fig. I>. Addition of this compound to growing cultures of several! different fungi, bacteria, and yeast-type fungi was found to result in the inhibition of growth of these organisms4*5. ~ubse~uent~y~ it was observed that cerulenin decreased the i~corpo~atiou of ~~4~~a~etate into fatty acids and sterols in yea~t~*~, and this observation provided the first indication of a natural product antibiotic that inhibited lipid synthesis. The inhibition yeast growth could be overcome by addition of lauric or oleic acid to the medium, suggesting that cerulenin acted at least in part at the level of fatty acid synthesis in this organism. Further studies of the fatty acid synthetases curdled from a variety of organisms i~~d~~ated that iow ~o~~e~~rat~o~s of cerulenin inhibited both rn~~t~enz~e complex and eon-associated fatty acid synthetases’. The non-associated fatty acid synthetase of Eschericlzia cnli and the multrenzyme complex of yeast were inhibited to the same degree by comparable levels of the antibiotic. The rat liver fatty acid synthetase multienzyme complex on the other hand was not as sensitive and could be only partially inactivated. Only one of the fatty acid synthetases tested, the acyl carrier protein-dependent ~a~~n~to~~-~o~ dongation system of ~~~0~~~~~~~~1~~phlei, was not inhibited by ~~r~~en~~. In contrast the high molecular weight synthetase of M. phlei was quite sensitive to the antibiotic’. The effect of cerulenin on several of the partial reactions catalyzed by the high molecular weight fatty acid synthetase of M. phlei was investigated with model substratess. Evidence was obtained which indicated that /I-ketoacyl-acyl carrier protein synthetase, the enzyme catalyzing the condensation of fatty acyl-acyl carrier proteins with malo~y~-acy~ carrier protein, was specifically inhibited by cerdenin. The fl-
of
* The positions unpublished).
of the double
bonds
are 7,x0 instead
of
&IO
as previcmsly
reported2*3
(6mura S,?
CERULENIN
EFFECT ON FATTY ACID SYNTHESIS
157
ketoacyl-acyl carrier protein synthetase of this fatty acid synthetase, however, has not been isolated for direct testing with cerulenin. E. coli fi-ketoacyl-acyl carrier protein synthetase has been isolated as a homogeneous protein and studied in detai19-12. The reaction catalyzed by this enzyme proceeds in two partial reactions with an acyl-enzyme intermediate. RCO-S-ACP RCO-S-E+
+ HS-E Z+ RCO-S-E HOOCCH,CO-S-ACP
Sum : RCO-S-ACP ACP-SH
+ ACP-SH
(I)
$ RCOCH,CO-S-ACP
+ HOOCCH,CO-S-ACP
+ CO, + HS-E
Z$ RCOCH,CO-S-ACP
(2)
+ CO, + (3)
where ACP is the acyl carrier protein. Thus in Reaction I the acyl group of an acyl-acyl carrier protein is transferred to a sulfhydryl group of the enzyme to form an enzyme thioester intermediateg. In Reaction z the acyl group of the enzyme thioester is condensed with malonyl-acyl carrier protein to form a P-ketoacyl thioester of acyl carrier protein, CO,, and the free enzyme. The sum reaction, Reaction 3, represents the condensation reaction of fatty acid synthesis. In this report we present the effects of cerulenin and several of its analogs (Fig. I) on the fatty acid synthetase of E. coli and on homogeneous preparations of B-ketoacyl-acyl carrier protein synthetase isolated from this organism. The inhibition of the condensation reaction by cerulenin, the protection against inhibition afforded by acetyl-acyl carrier protein, the inhibition of other catalytic activities of P-ketoacylacyl carrier protein synthetase, as well as the stoichiometry of the reaction between P-ketoacyl-acyl carrier protein synthetase and cerulenin, are reported. MATERIALS
AND METHODS
Substrates and enzymes Sodium [r-14C]hexanoate and sodium [2-3H]acetate were purchased from Amersham/Searle, Arlington Heights, Ill. KH14C0, was obtained from Mallinckrodt, St. Louis, MO. [2-14C]Malonyl-CoA and [r,3-14C]malonyl-CoA were purchased from New England Nuclear, Boston, Mass. Coenzyme A and hexanoyl-CoA were obtained from P-L Biochemicals, Milwaukee, Wise. Malonyl-CoA and /?-hydroxyacyl-CoA dehydrogenase were purchased from Sigma, St. Louis, MO. The preblended liquid scintillation solution, 3a7o, was obtained from Research Products International, Elk Grove Village, Ill. Frozen cells of E coli B (3/4 log) were obtained from Grain Processing Corporation, Muscatine, Iowa. Acyl carrier protein was prepared by the method of Majerus et al.13, and acetylACP was prepared by the method of Alberts et aZ.14.The method of Simon and Sheminr ’ was used to synthesize acetyl-CoA. [I-14C]Hexanoyl-CoA was prepared according to the method described by Goldman and Vagelos16. Malonyl-CoA-acyl carrier protein transacylase was the generous gift of Dr F. E. Ruth, Jr, and was assayed according to the procedure of Ruth and Vagelos17. /I-Ketoacyl-acyl carrier protein reductase was purified and assayed by the method of Vagelos et a1.l’. P-Ketoacyl-acyl carrier protein synthetase was purified to homogeneity and assayed according to published procedures 9,10. A unit of activity of B-ketoacyl-acyl carrier protein synthetase is defined as the amount of enzyme that catalyzes
the synthesis of 1.0 pmole of a~g~~~~~~y~-~~y~ protein carrier per mm. The maionylCo&CO, exchange reaction and the rna~o~y~-~o~-a~~~ carrier protein ~~~arboxy~a~~ reaction, catalyzed by ~-~~toa~y~-a~y~ carrier protein synthetase, were mea§~red as described by Alberts et ai. r’ . The ~r~~a~at~o~ and assay of E CC&fatty acid synthetase ~~di~a~t~vity measurements were determined in a Packard Trihas been reported”. carb liquid scintillation spectrometer, model 3380, equipped wit a *model 544 absolute activity ana!yzer. Assay of the fatty a~y~-~o~-a~y~ carrier protein ~~a~sa~y~a~~ activiPy of ~-~etoa~y~-acy~ carrier protein syntb~ta~e has been previously de~cr~b~d~~. The transacylase assay was modified so that the assay mixture was the same as that utilized in the hexanoyl-CoA binding experiments described below. E. CO&acyl carrier protem (20 nmoles) was included in the assay mixtures. The reactions were dependent upon acyl carrier protein and linear over a wide range (O-O. P unit) of ~-ketoa~y~-a~y~ carrier protein s~r~thetase con~~ntrat~5n§. Binding
of hexanql-Cod In 0.05
to /I-ketoacyl-acyi
carrier protein qxthetase
mixture were contained 2.5 poles of imidazolebuffer, pPI 6.7, 1.0 ymole of a-mercaptoethanol, 16.0 pg of /3-ketoacyl-acyl carrier protein synthetase, and 40.5 nmoles of ~~-14~~hexanoy~~~o~ (5.0 G/mole). After incubation for 30 min at 37 “C, the reactions were term~~~at~d by the addition ofo.4 ml 5 Y/itricb~or~acetic acid. ovine serum a~b~~rn~n (0.5 mg) was added to assure complete recovery of the enzyme upon centrifugation. The mixture was centrifuged for at 3000 x gY and the supernatant solution, containing unreacted hexanoyKo discarded. The sedimented material was suspended in 0.2 ml of 5 % tri&loroacetic acid and centrifuged as before. This washing procedure was continued until no radioin the ~~~e~~atant. The pelleted material finally disactivity could be dete 51. The solution was ~entra~i~~d with I.0 M 1and transsolved in 0.1 ml 1 .o M ferred quantitatively to a liquid scintillation vial. Preblended fInor, 3a709 was adde and the radioactivity of the sample determined. When ~“~etoa~y~-a~~~l carrier protein synthetase was replaced with comparable amounts of bovine serum albumin, no radioactive hexanoyI-@oA was recovered with the protein. The ~~~~~entra~io~ of [a-r4C]hexalloy~~~0A used was demo~~strated to be saturat~~~g in control experi-ments. Binding
ml of reaction
qfcerdenin
to G-ketoacyl-acyE
carrier proteirl synthetase
In 0.05
ml of reaction mixture were contained 5.0 /imoles of potassium phosphate buffer, p]M 7.0, 260 ~lg of ~-~etoacy~-~cy~ carrier protein ~y~tbeta~~, and o.o020.2 pmole of ~3~~cer~~e~in (0.4 ~~~~5~~). The reaction mixtures were ~~~~bated for 5 min at room temperature, and sea~tio~s were terminated by the a~d~~~on of 0.4 ma of 5 0/otrichloroacetic acid. The precipitates were recovered by 6entr~~uga~iQn, washed, and counted as described in the other binding experiments. Control experiments, m which equal amounts of bovine serum albumin were substituted for enzyme, indicated that no non-specific binding of cerulenin occurred under these conditions. The number of moles of cerulenin bound per mole of ~-~etoacyl-a~y~ carrier protein ~y~theta§e was calculated using a rno~~u~ar weight of 66000 for the enzyme”“.
Cerulenin
was isolated
from the culture
filtrate
of C. caerulens”-4.
Cerulenin
CERULENIN
EFFECT
ON FATTY ACID SYNTHESIS
159
labeled with 3H was prepared by growing the organism in the presence of IOO mCi of sodium [2-3H]acetate (approx. 2 Ci/mmole~. The specific activity of cerulenin prepared in this manner was 0.4 Cijmole. Dihydro-, tetrahy~o-, and hexahydrocerulenin were prepared chemically as previously described ‘. Stock solutions of cerulenin and its analogs were prepared in absolute ethanol and stored frozen in liquid nitrogen. Fresh dilutions were prepared in water immediately prior to conducting an experinient. RESULTS
Inhibition qffatty acid synthetase by mulenin and tetruhydrocerzrlenin The results of experiments designed to demonstrate the susceptibility of E. coli fatty acid synthetase to cerulenin and tetrahydrocerulenin are shown in Fig. 2A. It is apparent that the fatty acid synthetase is very sensitive to cerulenin. Tetrahydrocerulenin also inhibited the synthesis of fatty acids, but much higher concentrations of this derivative were required. Thus 50% inhibition was achieved with 30 ,uM cerulenin, whereas 4.8 m&I tetrahydrocerulenin was required to obtain a similar level of inhibition. On the other hand neither dihydrocerulenin nor hexahydrocerulenin, when tested at 5.0mM concentrations, caused any inhibition of this fatty acid synthetase. Examination of the structures of the four derivatives tested (Fig. I) indicates that the two active compounds, cerulenin and tetrahydro~erulenin, contain a &,s epoxide ring; the two inactive derivatives lack the epoxide. Thus the intact epoxide ring appears to be an essential structural feature of this class of inhibitors. The fact that cerulenin caused 50 % inhibition of fatty acid synthesis at 150 times lower concentration than tetrahydrocerulenin indicates the importance of the tram double bonds in cerulenin. Other derivatives of cerulenin, such as those containing a single tram double bond, cis B
0
~
0 0
0
50
100 CERULENIN,
1.25
2.50
150 ,uM 3.75
TETRAHYDRO~~RULENtN,
200
0
25 50 ACETYL-ACP,
75 /d.d
100
5.00 mh!
Fig. 2. (A) Effect of cerulenin and tetrahydrocerulenin on E. co2i fatty acid synthetase. Fatty acid synthetase, 300 pg of protein, was incubated for 5 min at room temperature in 0.1 M potassium phosphate buffer, pH 6.8, in a volume of 0.1 ml, with the concentrations of cerulenin and tetrahydrocerulenin indicated. (B) Acetyl-acyl carrier protein (ACP) protection of fatty acid synthetase from inhibition by cerulenin. Fatty acid synthetase was incubated under the same conditions as (A), for 5 min at room temperature with the indicated amounts of acetyl-ACP. Then cerulenin (150 pM) was added and the incubation continued for another 5 min. After the pr~limina~ incubations for (A) and (B), aliqnots of the reaction mixtures were assayed for fatty acid synthetase activity as described under Materials and Methods. Results are expressed as percentage of reaction rates relative to experiments in which (A) treatment with cerulenin (0-o) and tetrahydrocerulenin (a-0) or (B) treatment with both cerulenin and acetyl-ACP (O--O> were omitted.
double bonds or a tracts epoxide ring are not yet available. They wouid be required to fully elucidate the strn~t~ra~ features ~~~essa~~~for the i~~~~~t~o~ of fatty acid sy~t~es~s~
Previous studies by Bloch and his co-workers8 suggested that the /Sketoacyiacyl carrier protein synfhetase ~~~~o~e~~ of the M. phki fatty aci synthetase m&i-, enzyme cm~plex is the locus of ce~~~e~i~ i~~~b~t~o~ of fatty acid s~~tbes~s. Since the ~-k~toacy~-a~y~ carrier protein sy~t~etase has not been ~iss~~~ated in an active form from the fatty acid synthetese rn~~t~e~~~~le complex, the effect of cerulenin on the isolated component enzyme could not be evaluated in that system. The effect ofcesnlenin and its analogs on homogeneous E. ii /IS-ketoacyl-acyl carrier protein syn was therefore investigated. As shown in ig. 31%certrlenin is a very &Tective in higher c~~~~~t~at~o~sof i~bib~tor were required to achieve s r levels of inhIbitlon. Dihydro- and hexahyd~~~e~u~en~~ were not inhibit ~o~c~~~~at~o~s when tested under similar conditions. It is apparent in 364 that the cancer?trations of cerulenin (or tetrahydr~~er~~~~i~~ required to inhibit the activity of the fatty acid sy~t~etase and ~~ket~a~y~-a~y~ carri rotein synthetase are diEerent. T fatty acid synthetase was 50 % ~~~~b~ted by 30 p whereas the b~moge~eo~s condensing enzyme was 50 @/o ~~~~bited by cerulenin at co~ce~t~at~o~s Bower than 5 p
Since /3-ketoacyl-acyl carrier protein synthetase catalyzes the condensation react&x of fatty acid synthesis, it is apparent that, if this enzyme is rate-limiting for sverd fatty acid synthesis, fatty acid syntbetase should be just as sensitive as ,&ketoacyl-acy!
I!!!,
0 0
25
50 CERULENIN.
L-.-“--.
0
,]I i,___
,
I
75 phi I
60
100
i50
T~TRA~YDROCE~~L~~IN,
too
0
25 50 ACETYL-ACP,
75 /.&I
100
--
ZOO jtM
Fig. 3. (A) Effect of cernlenin and t~trahydro~eru~enjn on E. eoli ~-~etoacy~-a~yl carrier protein (ACP) 3.0 pmoles of potassium phosphate buffer, pW 7.0, 5.0 synthetase. The reaction mixtures contained pnoles of z-mercaptoethanol, 0.1 pmole of EDTA, 2.0 ,ug of /?-ketoacyi-ACP synthetase and the indicated amounts of cerulenin or tetrahydrocerulenin in 0.1 ml. Incubations were at room temperature for 5 min. (B) Acetyl-ACP protection of ,&ktoacyl-ACP synihetase from inhibition by cerulenin. P-Ketoacyl-A@P synthetase was incubated under the same conditions as (A) with the indicated concentrations of acetyl-ACP. Then ceruleain (50 ,dvf) was added and the incubation continued for another 5 min. After these ~rel~l~inary incubations, aliquots of the reaction mixtures in (A.1 and (S were assayed for &ketoacyl-ACP synthetase activity as described under Materials and Methois. Results are expressed as percentage of reaction rates relative to experiments in Lvbich(A) treatmeet with cerulenin (O-O) and tetrahydrocerulenin (e-0) or (B) treatment with both cerulenin and acetyl-ACP (0 - 3) were omitted.
CERULENIN
EFFECT ON FATTY ACID SYNTHESIS
161
carrier protein synthetase to inhibition by cerulenin. The fact that this is not the case suggests that j?-ketoacyl-acyl carrier protein synthetase is not rate-limiting in fatty acid synthesis by the crude E. coli fatty acid synthetase. The difference in sensitivity of fatty acid synthetase and P-ketoacyl-acyl carrier protein synthetase to cerulenin might also be due in part to the differences in protein concentrations in the two assays. The fatty acid synthetase preparation contains many proteins and was tested at high protein concentrations, whereas the homogeneous /I-ketoacyl-acyl carrier protein synthetase was tested at much lower protein concentrations. Since it was difficult to demonstrate a parallel effect of cerulenin on fatty acid synthetase and P-ketoacyl-acyl carrier protein synthetase when inhibition was studied directly, an indirect approach was used. It was known from earlier studies of the E. coli /I-ketoacyl-acyl carrier protein synthetaseg-I2 that the enzyme contains a cysteine residue in the active site which is specifically aikylated by iodoacetamide. When the enzyme is incubated with acetyl-acyl carrier protein, the enzyme accepts an acetyl group and is converted to an acetyl-enzyme (Reaction I). The presence of the acetyl group in the active site of the enzyme protects the enzyme against inactivation by alkylating agents. Assuming that the cerulenin might be acting at the active site of $ketoacyl-acyl carrier protein synthetase, the effect of the prior addition of acetylacyl carrier protein was examined. As noted in Fig. 313 the addition of acetyl-acyl carrier protein to the enzyme prevented inhibition by an amount of cerulenin (45 PM) which normally caused over 95 ‘A inhibition. The protection of enzymatic activity afforded by acetyl-acyl carrier protein increased with increasing concentration of the thioester. At 75 pM, acetyl-acyl carrier protein protected the enzyme almost completely. Thus, conversion of P-ketoacyl-acyl carrier protein synthetase to acetylenzyrne prevents the inhibition by cerulenin. Since acetyl-acyl carrier protein reacts directly and specifically with ~-ketoacyl-acyl carrier protein synthetase in the reaction sequence of fatty acid synthesis’, the effect of acetyl-acyl carrier protein pretreatment ‘of the fatty acid synthetase was examined. Following addition of acetyl-acyl carrier protein to the fatty acid synthetase, cerulenin was added at a concentration (150 I_~M) that normally causes about 90 % inhibition. As noted in Fig. 2B, the prior addition of acetyl-acyl carrier protein protected the fatty acid synthetase from inhibition by cerulenin. The effect of acetyl-acyl carrier protein in protecting the fatty acid synthetase (Fig. 2B) is very similar to its effect with fi-ketoacyl-acyl carrier protein synthetase (Fig. 3B). The concentration required to decrease inhibition to 50 % in both systems was about I I PM. The similarity of the effect of acetyl-acyl carrier protein in protecting j?-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase against inhibition by cerulenin indicates that ~~ketoacyl-a~yl carrier protein synthetase is the site of cerulenin in~bitio~ of fatty acid synthesis. The assay of P-ketoacyl-acyl carrier protein synthetase is carried out with a coupled enzyme system involving two additional enzymes, malonyl-CoA-acyl carrier protein transacylase and P-ketoacyl-acyl carrier protein reductase. The effect of cerulenin on these two enzymes was tested directly. Concentrations of cerulenin (IOO ,uiM) that caused complete inhibition of /3-ketoacyl-acyl carrier protein synthetase had no effect on either malonyl-~oA-a~yl carrier protein transacylase or P-ketoacylacyl carrier protein reductase. The inability of cerulenin to inhibit these two enzymes, which are components of the fatty acid synthetase, further illustrates the specificity of the inhibitor for /3-ketoacyl-acyl carrier protein synthetase.
162
, r 6. D ArjNOk3
ef iii.
The assay of /I-ketoacyl-acyl carrier protein synthetase measures the overa~ji activity ofthe enzyme, shown in Reaction 3, and this activity is inhibited by ceruienin,, ~-~etoacy~-acy~ carrier protein sy~t~et~se also catalyses a ~l~rn~er of model reactions which represent the component reactions and therefore the e&ct of cerulenin on each of these reactions was investigated. The first series of experiments tested the effect of cerule~in on the fatty acyl tran5acy~ase activity of the enzyme ( eaction I). Fatty acyB thioesters of Cob have been shown to be active substrates in Reaction I”: and the transacylase activity can be readily demonstrated by incubating a radioactive thioester, such as [14C]hexanoyl-CoA, with acyl carrier protein and en2yme. The enzyme is initially acylated (reaction I, forward ~rect~ou~ and then the acyl group is tra~s~e~~~d to acyl carrier protein forming ~~4~]~~e~a~oy~-~cy~ carrier protein (~~~~~t~o~ I, reverse direction). Transacylase activity of /3-ketoacyl-acyl carrier rotein synthetase, measured as the rate of [14C]hexanoyl-acyl c in synthesis, was inhibited by cerulenin forward d~~ect~o~~ was also measured (Fig. 4). Acyl transfer to the enzyme ( substrate ~~a~t~t~es of enzyme with directly. This was accomplished by 1 [‘4C]hexanoyl-CoA and then measuring the formation of E‘“C]hexanoyl-euszyane. Cerulenin aiso inhibited this reaction, and it is apparent (Fig. 4) that the inhibition of the transacylase activity of the enzyme, when measured in either of the two assays described, ~ar~~~eled the ~~~~~ib~t~o~ of overall ~~ketoacy~-~c~~ carrier protein synthetase activity. In another model reaction P-ketoacyi-acyl carrier protein syntketsse catalyzes the decarboxy~at~o~ of rn~~~~y~-a~~~ carrier ~rQt~~~ in the abseilce of fatty asyl-acyi component of carrier protein I1 . This activity probably represents the decarboxylation the condensation-decarboxylation reaction shown in Reaction 2. As seen in Fig. 5 malonyl-acyl carrier protein decarboxylase activity was i~b~b~ted by ce both partial reactions catalyzed by ~-ketoacy~-~cy~ carrier protege syn tions I and 2) are inhibited by cerulenin. A final model reaction, the rna~o~y~-~o~~-~~~ exchange reaction which is dependent upon both the fatty acyl transacylase and the condensation-decarboxyla~~on functions of ~-k~toacy~-ache carrier protein synthetase” I, w2s also ~nh~b~t~d by cer~~e~~i~~ as predicted from the results witln the component reactions. inhibition of both malonyl-acyl carrier protein de~arboxylase and malonyl-CoA-CO, exchange activities paralleled the ~n~~bit~o~ of /3-ketoacyl-acy! carrier protein synthetase activity (Fig. 5).
High concentrations of cerulenin inhibited fi-ketoacyl-acyl carrier protein synthetase very rapidly. When low concentrations were used (43 9 ), the time course of inhibition could be observed. As shown in Fig. 6, when acetyl-acyl carrier protein was added at concentrations (100 ,&I) that completeljr protect the enzyme against cerulenin inhibition, the increase in inhibition stopped immediately. Tn addition, it should be noted that during the time course of this experiment, there was no reversal of Jnhibition since enzyme activity was not recovered after the addition of the acetyl-acyl casrkr protein. The inability of acetyl-acyl carrier protein to reverse the inhibition of pketoacyl-acyl carrier protein synthetase by cerulenin indicates that under these conditions the enzyme was irreversibly inactivated. This suggests the ~ossib~~~ty that certiknin might be bound cova~ent~y to t&e enzyme.
CERULENIN
EFFECT
ON FATTY
ACID
SYNTHESIS
8
0 0
25
50 CERULENIN,
75 /_LM
100
0
25
50 CERULENIN,
75
100
PM
Fig. 4. Effect of cerulenin on P-ketoacyl-acyl carrier protein (ACP) synthetase, fatty acyl-CoA-ACP transacylase activity and hexanoyl-Cob binding. P-Ketoacyl-ACP synthetase, 40.0 pg, was incubated in 0.1 M potassium phosphate buffer, pH 7.0, in a volume of 0.03 ml with the indicated amounts of cerulenin. Following incubation at room temperature for 5 min, aliquots were removed and assayed for /?-ketoacyl-ACP synthetase (@-a), fatty acyl-CoA-ACP transacylase (O-O) and [14C]hexanoylCoA binding (n--a) activities as described under Materials and Methods. Results are expressed as percentage of rates relative to experiments in which treatment with cerulenin was omitted. Fig. 5. Effect of cerulenin on malonyl-CoA-CO, exchange and malonyl-acyl carrier protein (ACP) decarboxylase activities of P-ketoacyl-ACP synthetase. The reaction mixtures contained 2.0 pmoles of potassium phosphate buffer, pH 7.0, 5.0 pmoles of z-mercaptoethanol, 0.1 pmole of EDTA, 2.0 fig QI’ /?-ketoscyl-ACP synthetase, and the indicated amounts of cerulenin in 0.1 ml. Incubations were at room temperature for 5 min. Aliquots were removed and assayed for ,%ketoacyl-ACP synthetase (s--e), malonyl-ACP decarboxylase (O--O) and malonyl-CoA-CO, exchange (A--a) activities as described under Materials and Methods. Results are expressed as percentage of reaction rates relative to experiments in which treatment with cerulenin was omitted.
Stoichiometry of the reaction between P-ketoacyl-acyl carrier protein synthetase and cerulenin The possibility that the interaction of P-ketoacyl-acyl carrier protein synthetase and cerulenin leads to the formation of a covalent complex was tested by denaturing the inhibited enzyme with trichloroacetic acid. As shown in Fig. 7, when substrate quantities of enzyme were treated with [3H]cerulenin and subjected to trichloroacetic acid precipitation, the precipitated protein contained bound cerulenin. The amount of cerulenin bound to the enzyme was proportional to the extent of inhibition, and it is apparent that approximately I mole of cerulenin was bound per mole of enzyme when inhibition of the enzyme approached IOO %. DISCUSSION
The results of experiments conducted by Bloch and his coworkers’ with a fatty acid synthetase of M. phlei indicated that cerulenin inhibited this multienzyme complex by inactivating /?-ketoacyl-acyl carrier protein synthetase. The experiments described in this communication support and extend those observations. Cerulenin inhibition of the E. coli fatty acid synthetase was shown to be due to the specific inhibition of the P-ketoacyl-acyl carrier protein synthetase component which could be studied as a homogeneous protein in this system. It was known that E. coli /?-ketoacylacyl carrier protein synthetase contains in its active site a specific cysteine residue’
100
25
0
15
0
30 MINUTES
45
0
I
2 CERULENIN,
3 mhl
4
Fig. 6. Irreversible inactivation of /3-ketoacyl-acyi carrier protein (ACP) synthetase by cerulenin. The reaction mixtures contained 2.0 pmoles of potassium phosphate buffer, pH 7.0, 5.0 pmoles of a-met-captoethanol, 0.1 ymole of EDTA, 4.0 fig of P-ketoacyl-ACP synthetase. 0.4 nmole of cerulenin in 0.1 ml. After 2, 5 and IO min of incubation at room temperature, 10.0 nmoles of acetyi-AC? were added. p-Ketoacyl-ACP synthetase was assayed in the absence (O--O) and in the presence ( of added acetyl-ACP. Results are expressed as percentage of reaction rates relative to experim which treatment with cerulenin was omitted. Fig. 7. Sioichiometry of the reaction between cerulenin and /I-ketoacyl-acyl carrier protein (ACP) synthetase. /?-Ketoacyl-ACP s-ynthetase, 260 pup of protein, was incubated in 0.x M potassium phosphate buffer, pH 7.0, in a volume of 0.05 ml with the indicated amounts of [3HJcerulenin. Foilowing incubation at room temperature for 5 mm, aliquots were removed and assayed for /‘I-ketoacyl-ACP -IB) and ceru!enin binding (O-O) as described under Materials and Methods. Enzymatic activity is expressed as percentage of reaction rates relative to experiments in which treatment with cerulenin was omitted. Cerulenin binding is expressed as moles orC inhibitor bound per mole of enzyme.
which is presumably first partial
involved
reaction
sensitive to alkylation enzyme is protected
in the formation
catalyzed
of an acyl-enzyme
by this enzyme
by ioduacetamide
from iodoacetamide
(Reaction
which inactivates inhibition
intermediate
I.). This
cysteine
the enzyme;
in the is very
however,
the
when an acetyl group is bound
at the active site. The possibihty that cerulenin, a 12 carbon atom fatty acid amide, is bound at the active site of P-ketoacyl-acyl carrier protein synthetase ~2s suggested by the fact that this enzyme can react with fatty acyl thioesters whose chain lengths vary from 2 to 16 carbon The finding
atoms as part of its
that acetyl-acyl
cysteine residue in the active site, protected permitted
the demonstration
the inhibition and
,&ketoacyl-acyl
concentrations
carrier
from
donates
of E. coli fatty acid synthetase is dUe to synthetase.
Fatty
were protected
acid
in parallel
noted previously
carrier protein
synthetase
with iodoacetamide.
by cerulenin
Iodoacetamide
specific cysteine residue and inhibits the first part1‘al reaction catalyzed (Reaction
I). The
fatty acyl transferase component
ed in the fatty acyl-GoA-acyl by iodoacetamide synthetase
of the
carrier protein transacylase
and accounted
by iodoacetamidell.
spnthetase wrions
by
carrier protein.
of /I-ketoacyl-acyl
the inhibition
synthetase
in fatty acid biosynthesisxl,
an acetyl group to the enzyme from inzctivation by ceruienin
carrier protein
protein
of acetyl-acyl
Inhibition
which
the
that inactivation
of ,&ketoacyl-acyl
function
normal
carrier protein,
for the inhibition
enzymatic
dif?ers
alkylates
a
by the enzyme
reaction,
measur-
model reaction, was inhibited
of /3-ketoacyl-acyl
carrier protein
On the other hand, the malonyl-acyl
carrier protein
CERULENIN
EFFECT ON FATTY ACID SYNTHESIS
165
decarboxylase reaction, which represents a component of Reaction 2, was not inhibitboth partial ed by iodoacetamidell. As shown above, model reactions representing Reactions I and 2 (Figs 4 and 5) were equally inhibited by cerulenin. Thus cerulenin differs from iodoacetamide since the former inhibits malonyl-acyl carrier protein decarboxylation whereas the latter does not. /3-Ketoacyl-acyl carrier protein synthetase contains a fatty acyl and a malonyl-acyl carrier protein binding siteg-I’. The relationship between these sites is not known. Alkylation by iodoacetamide of the cysteine in the fatty acyl site apparently does not affect the malonyl-acyl carrier protein site, whereas inactivation by cerulenin affects the activity of both sites. An explanation for this observation might be that both iodoacetamide and cerulenin alkylate the same cysteine residue in the fatty acyl site but that cerulenin inhibits the malonyl site activity because it has a long hydrophobic chain. The hydrophobic chain of cerulenin presumably could prevent the binding of malonyl-acyl carrier protein at the ad_jacent site. The only evidence suggesting that cerulenin reacts with the cysteine at the fatty acyl site is the protection afforded by acetyl-acyl carrier protein. It is conceivable, but not likely, that cerulenin attacks the malonyl-acyl carrier protein site. Experiments attempting to rule out this possibility, by demonstrating that malonyl-acyl carrier protein can protect against inactivation by cerulenin, are not feasible since the enzyme readily decarboxylates malonyl-acyl carrier protein to form acetyl-acyl carrier protein, and the enzyme would be protected by the latter. Thus, specific reaction of cerulenin at the malonyl-acyl carrier protein site cannot be ruled out. It has been established that inhibition of /3-ketoacyl-acyl carrier protein synthetase by iodoacetamide is due to the alkylation of one residue of cysteine per mole of enzymeg. The demonstration that inhibition by cerulenin is irreversible and that it is accompanied by the binding of a single mole of cerulenin per mole of enzyme suggests a highly specific interaction between the enzyme and the inhibitor. It is likely that cerulenin, like iodoacetamide, inhibits /?-ketoacyl-acyl carrier protein synthetase by alkylating the cysteine residue in the active site, but this has not yet been investigated. The cysteine thiol, which is very reactive with other alkylating agentsg-11, could act as a nucleophile1g,20 and initiate an attack upon one of the epoxide ring carbon atoms. The resulting thioether would be expected to be very stable. Such covalent modification of the enzyme could explain the observed inhibition of all the catalytic properties of this enzyme. Although the epoxide group offers an attractive and versatile protein side chain modifying reagent, the broad spectrum of groups that it can potentially modifyZ1 -23 makes it impossible to predict with certainty which amino acid residue of /Sketoacyl-acyl carrier protein synthetase is modified. The structure of cerulenin presents several features of general interest. As the hydrocarbon backbone of the molecule is composed of 12 carbon atoms, cerulenin is a fatty acid of intermediate chain length; however, the chemical features associated with this compound appear to be unique. The presence of two double bonds in a fatty acid is not uncommon, but the fact that both are of tram configuration and that one is 02 is novel. The finding that an internal cis oxirane ring is necessary for biological activity and that a carbonyl function is adjacent to this structure also appears to be unique. Although other epoxy-containing compounds have been used as inhibitors or modifiers of enzymatic processes or proteins21-23, these have always been compounds with terminal oxirane rings, as opposed to those reported here. The epoxy ring is of distinct chemical significance in cerulenin, and it is possible that synthesis of similar
compounds may yield other agents acid and lipid metabolism
specifically
reactive
with 0th
enzymes
uf fa;cp
A(‘KNOWLED
The excellent technical assistance of Mr Tadataka Kcsadfi is gratefully z_cknowiedged. This investigation was supported in part by the National Institutes of Health ( I-Ror-I-IL-10406) and the Nationai Science Foundation (GB-38676X:. 1.S.R. is a Nation4 Institutes of Health Postdoctoral Fellow (5-Fo2AM5236~02). REFERENCES I Sane, Y.. Xomura, S.. Kamio, Y.. &nura, S. and Hata. I‘. (1967) J. ,4rrti!~iot.. Ser. A 20. ?4_+-~@ z &ura. S., Katagiri. &I.. Nakagaxva. A., Sane. Y.. Nomura, S. and llata. T. (1967) j, An~ihi~!. Ser. .ft. 20.349 -354 3 Gmura. S.. Nakagawa. A.. Sekikawa. K.. Otani. \I. and Hata. 1.. (; 969) C’hen~. Ph~~r~~~. h/L 7bkvo i:. 2761-2364 4 Ma&mae.‘A., Nomura. S. and Hata. T. (1963) .I. Anrihior., Scar. A 16. 239-243 5 Matsumae. A., Nomura. S. and Ha:a, T. : 1964) J. Arrrihior., Ser. A I 7. I 7 6 Nomura. S., Horiuchi, T.. Gmara, S. a!ld I-faia, -1‘. (1972.1 J. Bbcit~,u. ‘i’~!~),) 71. 783-796 7 Nomura. S., Iloriuchi, ‘f.. FIata. T. and Gmura, S. (1972) J. Aurihio/. 25. 365 358 8 Vance. l)., Goldberg. I.. Mitsuhashi, 0.. Bloch, K.. Bmura. S. and Nomura. S. (1972) L?kx/~e~r~. .b’iop/q~. RCS. Con~/n/rn. 48, 649 6 j6 9 Greenspan, M. I).. Albcrts. A. W. and Vagelos, I’. R. (196y1 J. Rio/. (I/~/PI. 2~4~ 6f77 6485 10 I’rcscott, D. J. and Vagclo~. I’. R. (1970) J. Biol. C/I~/~I. ~45, 5484-5490 I I Albcrts. A. W., Bell. R. M. and Vagelos. I’. R. (1972) J. Bid. Chem. 247. 3190-3198 II Rosenfcld. I. S., I>‘Agnolo. G. and Vngclos. P. R. (1073) J. f?io/. C‘lww. 248, 2452 2460 r3 Majerus. P. W.. A!berts, A. W. and Vagetos, P. R. i1964) Proc. r\rnrl. .~!cad. SC,;. L;.S.y. iz31 -1338 14 Albcrts. A. W.. Majerus. I’. W. and Vagelos, I’. R. ( I$&)) in Mr/hodv in Enr,:~mo/ogy (l.owcnsre~n. 3. 41.. cd.). Vol. 14. pp. 57 60. Academic Frcss. New York 15 Simon, E. J. and Shemin, D. (1y53) ./. Avr. C‘lwnr. Sot. 75. 2520 16 Goldman, t’. and Vagc!os. I’. R. (1961) J. Bio!. Chem. 236. ~OZO--Z~Z~ 17 Ruth, Jr, F. E. and Vagclos. F. R. (1y73) J. Bid. Chew.. in the press IX Vagelos. I’. R., Albcrts. A. W. and Majcrus. I’. W. (1969)ir: ;~fethou~s in Jlll:,:.~wkq>. (Lowcnstcin. J. M., xl.), Vol. 14, pp. bo--63. Academic Press. New York II) Ross, W. C. J. (1~50) J. (‘hcjm Sot. 2257-2272 20 Farkcr. R. E. and Isaacs, N. S. (IC)~Q) Chew. Rev. ~9. 737-799 21 Thomas, E. W., McKelvy, J. F. and Sl-aron. N. (196~) Mnrure ‘2:: 483. 486 22 Waley. S. G.. Miller. J. C., Rose, I. A. and O’Connel. t. L. (1970) !V’r~!wr 227. r8: 2.3 Chen. K. C. S. and Tang. J. (1972) J. Bioi. (.‘lrr~. 247. 2566 2575
- Printed in The Netherlands
BBA 56348
INHIBITION
OF FATTY
ACID
SYNTHESIS
BY THE
ANTIBIOTIC
CERU-
LENIN SPECIFIC
INACTIVATION
OF
/3-KETOACYL-ACYL
CARRIER
PROTEIN
SYNTHETASE
GIULIANO D’AGNOLO=*, and P. ROY VAGELOS”**
IRA s. ROSENFELD~,
JuICHI
AWAYA~, SATOSHI OMURA~
‘Department of Biological Chemistry, Washington University School of Medicine, St. Louis, MO. 63110 (U.S.A.) and bThe Kitasato Institute and School ofPharmaceutical Science, Kitasato University, Tokyo, (Japan)
(Received July z3rd, 1973)
SUMMARY
Cerulenin, (2s) (3R)2,3-epoxy-+oxo-7,Io-dodecadienoylamide, an antibiotic isolated from the culture filtrate of Cephalosporium caerukns, inhibits the fatty acid synthetase of Escherichia coli by specifically inhibiting P-ketoacyl-acyl carrier protein synthetase, the enzyme which catalyzes the condensation reaction of fatty acid biosynthesis. Although cerulenin and tetrahydrocerulenin inhibited both P-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase, neither dihydrocerulenin nor hexahydrocerulenin, analogs which lack the epoxide ring, inhibited these enzyme systems. Both P-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase were protected to the same extent against inhibition by cerulenin by prior incubation with acetyl-acyl carrier protein. The acyl group of this thioester is bound at the fatty acyl site of /3-ketoacyl-acyl carrier protein synthetase. Detailed studies with homogeneous /I-ketoacyl-acyl carrier protein synthetase indicated that cerulenin inhibited model reactions representing both the fatty acyl transacylase and the malonyl-acyl carrier protein decarboxylase components of the P-ketoacyl-acyl carrier protein synthetase reaction. Inhibition of /?-ketoacyl-acyl carrier protein synthetase by cerulenin was irreversible, and it was associated with the binding of approximately I mole of inhibitor per mole of enzyme when inhibition approached IOO %.
INTRODUCTION
Cerulenin Cephalosporium
is an antibiotic isolated from the culture filtrate of the fungus caerulens’ and has the structure (6’) (3R)q-epoxy-4-0x0-7,10-
* Permanent address: Laboratori di Chimica Biologica, Istituto Superiore Italy. ** To whom reprint requests should be addressed.
di Sanita, Rome,
CERULEMIN
H
Fig.
I. Structure
of cerulenin
H
and its analogs.
dodecadienoylamide*S”‘3 (Fig. I>. Addition of this compound to growing cultures of several! different fungi, bacteria, and yeast-type fungi was found to result in the inhibition of growth of these organisms4*5. ~ubse~uent~y~ it was observed that cerulenin decreased the i~corpo~atiou of ~~4~~a~etate into fatty acids and sterols in yea~t~*~, and this observation provided the first indication of a natural product antibiotic that inhibited lipid synthesis. The inhibition yeast growth could be overcome by addition of lauric or oleic acid to the medium, suggesting that cerulenin acted at least in part at the level of fatty acid synthesis in this organism. Further studies of the fatty acid synthetases curdled from a variety of organisms i~~d~~ated that iow ~o~~e~~rat~o~s of cerulenin inhibited both rn~~t~enz~e complex and eon-associated fatty acid synthetases’. The non-associated fatty acid synthetase of Eschericlzia cnli and the multrenzyme complex of yeast were inhibited to the same degree by comparable levels of the antibiotic. The rat liver fatty acid synthetase multienzyme complex on the other hand was not as sensitive and could be only partially inactivated. Only one of the fatty acid synthetases tested, the acyl carrier protein-dependent ~a~~n~to~~-~o~ dongation system of ~~~0~~~~~~~~1~~phlei, was not inhibited by ~~r~~en~~. In contrast the high molecular weight synthetase of M. phlei was quite sensitive to the antibiotic’. The effect of cerulenin on several of the partial reactions catalyzed by the high molecular weight fatty acid synthetase of M. phlei was investigated with model substratess. Evidence was obtained which indicated that /I-ketoacyl-acyl carrier protein synthetase, the enzyme catalyzing the condensation of fatty acyl-acyl carrier proteins with malo~y~-acy~ carrier protein, was specifically inhibited by cerdenin. The fl-
of
* The positions unpublished).
of the double
bonds
are 7,x0 instead
of
&IO
as previcmsly
reported2*3
(6mura S,?
CERULENIN
EFFECT ON FATTY ACID SYNTHESIS
157
ketoacyl-acyl carrier protein synthetase of this fatty acid synthetase, however, has not been isolated for direct testing with cerulenin. E. coli fi-ketoacyl-acyl carrier protein synthetase has been isolated as a homogeneous protein and studied in detai19-12. The reaction catalyzed by this enzyme proceeds in two partial reactions with an acyl-enzyme intermediate. RCO-S-ACP RCO-S-E+
+ HS-E Z+ RCO-S-E HOOCCH,CO-S-ACP
Sum : RCO-S-ACP ACP-SH
+ ACP-SH
(I)
$ RCOCH,CO-S-ACP
+ HOOCCH,CO-S-ACP
+ CO, + HS-E
Z$ RCOCH,CO-S-ACP
(2)
+ CO, + (3)
where ACP is the acyl carrier protein. Thus in Reaction I the acyl group of an acyl-acyl carrier protein is transferred to a sulfhydryl group of the enzyme to form an enzyme thioester intermediateg. In Reaction z the acyl group of the enzyme thioester is condensed with malonyl-acyl carrier protein to form a P-ketoacyl thioester of acyl carrier protein, CO,, and the free enzyme. The sum reaction, Reaction 3, represents the condensation reaction of fatty acid synthesis. In this report we present the effects of cerulenin and several of its analogs (Fig. I) on the fatty acid synthetase of E. coli and on homogeneous preparations of B-ketoacyl-acyl carrier protein synthetase isolated from this organism. The inhibition of the condensation reaction by cerulenin, the protection against inhibition afforded by acetyl-acyl carrier protein, the inhibition of other catalytic activities of P-ketoacylacyl carrier protein synthetase, as well as the stoichiometry of the reaction between P-ketoacyl-acyl carrier protein synthetase and cerulenin, are reported. MATERIALS
AND METHODS
Substrates and enzymes Sodium [r-14C]hexanoate and sodium [2-3H]acetate were purchased from Amersham/Searle, Arlington Heights, Ill. KH14C0, was obtained from Mallinckrodt, St. Louis, MO. [2-14C]Malonyl-CoA and [r,3-14C]malonyl-CoA were purchased from New England Nuclear, Boston, Mass. Coenzyme A and hexanoyl-CoA were obtained from P-L Biochemicals, Milwaukee, Wise. Malonyl-CoA and /?-hydroxyacyl-CoA dehydrogenase were purchased from Sigma, St. Louis, MO. The preblended liquid scintillation solution, 3a7o, was obtained from Research Products International, Elk Grove Village, Ill. Frozen cells of E coli B (3/4 log) were obtained from Grain Processing Corporation, Muscatine, Iowa. Acyl carrier protein was prepared by the method of Majerus et al.13, and acetylACP was prepared by the method of Alberts et aZ.14.The method of Simon and Sheminr ’ was used to synthesize acetyl-CoA. [I-14C]Hexanoyl-CoA was prepared according to the method described by Goldman and Vagelos16. Malonyl-CoA-acyl carrier protein transacylase was the generous gift of Dr F. E. Ruth, Jr, and was assayed according to the procedure of Ruth and Vagelos17. /I-Ketoacyl-acyl carrier protein reductase was purified and assayed by the method of Vagelos et a1.l’. P-Ketoacyl-acyl carrier protein synthetase was purified to homogeneity and assayed according to published procedures 9,10. A unit of activity of B-ketoacyl-acyl carrier protein synthetase is defined as the amount of enzyme that catalyzes
the synthesis of 1.0 pmole of a~g~~~~~~y~-~~y~ protein carrier per mm. The maionylCo&CO, exchange reaction and the rna~o~y~-~o~-a~~~ carrier protein ~~~arboxy~a~~ reaction, catalyzed by ~-~~toa~y~-a~y~ carrier protein synthetase, were mea§~red as described by Alberts et ai. r’ . The ~r~~a~at~o~ and assay of E CC&fatty acid synthetase ~~di~a~t~vity measurements were determined in a Packard Trihas been reported”. carb liquid scintillation spectrometer, model 3380, equipped wit a *model 544 absolute activity ana!yzer. Assay of the fatty a~y~-~o~-a~y~ carrier protein ~~a~sa~y~a~~ activiPy of ~-~etoa~y~-acy~ carrier protein syntb~ta~e has been previously de~cr~b~d~~. The transacylase assay was modified so that the assay mixture was the same as that utilized in the hexanoyl-CoA binding experiments described below. E. CO&acyl carrier protem (20 nmoles) was included in the assay mixtures. The reactions were dependent upon acyl carrier protein and linear over a wide range (O-O. P unit) of ~-ketoa~y~-a~y~ carrier protein s~r~thetase con~~ntrat~5n§. Binding
of hexanql-Cod In 0.05
to /I-ketoacyl-acyi
carrier protein qxthetase
mixture were contained 2.5 poles of imidazolebuffer, pPI 6.7, 1.0 ymole of a-mercaptoethanol, 16.0 pg of /3-ketoacyl-acyl carrier protein synthetase, and 40.5 nmoles of ~~-14~~hexanoy~~~o~ (5.0 G/mole). After incubation for 30 min at 37 “C, the reactions were term~~~at~d by the addition ofo.4 ml 5 Y/itricb~or~acetic acid. ovine serum a~b~~rn~n (0.5 mg) was added to assure complete recovery of the enzyme upon centrifugation. The mixture was centrifuged for at 3000 x gY and the supernatant solution, containing unreacted hexanoyKo discarded. The sedimented material was suspended in 0.2 ml of 5 % tri&loroacetic acid and centrifuged as before. This washing procedure was continued until no radioin the ~~~e~~atant. The pelleted material finally disactivity could be dete 51. The solution was ~entra~i~~d with I.0 M 1and transsolved in 0.1 ml 1 .o M ferred quantitatively to a liquid scintillation vial. Preblended fInor, 3a709 was adde and the radioactivity of the sample determined. When ~“~etoa~y~-a~~~l carrier protein synthetase was replaced with comparable amounts of bovine serum albumin, no radioactive hexanoyI-@oA was recovered with the protein. The ~~~~~entra~io~ of [a-r4C]hexalloy~~~0A used was demo~~strated to be saturat~~~g in control experi-ments. Binding
ml of reaction
qfcerdenin
to G-ketoacyl-acyE
carrier proteirl synthetase
In 0.05
ml of reaction mixture were contained 5.0 /imoles of potassium phosphate buffer, p]M 7.0, 260 ~lg of ~-~etoacy~-~cy~ carrier protein ~y~tbeta~~, and o.o020.2 pmole of ~3~~cer~~e~in (0.4 ~~~~5~~). The reaction mixtures were ~~~~bated for 5 min at room temperature, and sea~tio~s were terminated by the a~d~~~on of 0.4 ma of 5 0/otrichloroacetic acid. The precipitates were recovered by 6entr~~uga~iQn, washed, and counted as described in the other binding experiments. Control experiments, m which equal amounts of bovine serum albumin were substituted for enzyme, indicated that no non-specific binding of cerulenin occurred under these conditions. The number of moles of cerulenin bound per mole of ~-~etoacyl-a~y~ carrier protein ~y~theta§e was calculated using a rno~~u~ar weight of 66000 for the enzyme”“.
Cerulenin
was isolated
from the culture
filtrate
of C. caerulens”-4.
Cerulenin
CERULENIN
EFFECT
ON FATTY ACID SYNTHESIS
159
labeled with 3H was prepared by growing the organism in the presence of IOO mCi of sodium [2-3H]acetate (approx. 2 Ci/mmole~. The specific activity of cerulenin prepared in this manner was 0.4 Cijmole. Dihydro-, tetrahy~o-, and hexahydrocerulenin were prepared chemically as previously described ‘. Stock solutions of cerulenin and its analogs were prepared in absolute ethanol and stored frozen in liquid nitrogen. Fresh dilutions were prepared in water immediately prior to conducting an experinient. RESULTS
Inhibition qffatty acid synthetase by mulenin and tetruhydrocerzrlenin The results of experiments designed to demonstrate the susceptibility of E. coli fatty acid synthetase to cerulenin and tetrahydrocerulenin are shown in Fig. 2A. It is apparent that the fatty acid synthetase is very sensitive to cerulenin. Tetrahydrocerulenin also inhibited the synthesis of fatty acids, but much higher concentrations of this derivative were required. Thus 50% inhibition was achieved with 30 ,uM cerulenin, whereas 4.8 m&I tetrahydrocerulenin was required to obtain a similar level of inhibition. On the other hand neither dihydrocerulenin nor hexahydrocerulenin, when tested at 5.0mM concentrations, caused any inhibition of this fatty acid synthetase. Examination of the structures of the four derivatives tested (Fig. I) indicates that the two active compounds, cerulenin and tetrahydro~erulenin, contain a &,s epoxide ring; the two inactive derivatives lack the epoxide. Thus the intact epoxide ring appears to be an essential structural feature of this class of inhibitors. The fact that cerulenin caused 50 % inhibition of fatty acid synthesis at 150 times lower concentration than tetrahydrocerulenin indicates the importance of the tram double bonds in cerulenin. Other derivatives of cerulenin, such as those containing a single tram double bond, cis B
0
~
0 0
0
50
100 CERULENIN,
1.25
2.50
150 ,uM 3.75
TETRAHYDRO~~RULENtN,
200
0
25 50 ACETYL-ACP,
75 /d.d
100
5.00 mh!
Fig. 2. (A) Effect of cerulenin and tetrahydrocerulenin on E. co2i fatty acid synthetase. Fatty acid synthetase, 300 pg of protein, was incubated for 5 min at room temperature in 0.1 M potassium phosphate buffer, pH 6.8, in a volume of 0.1 ml, with the concentrations of cerulenin and tetrahydrocerulenin indicated. (B) Acetyl-acyl carrier protein (ACP) protection of fatty acid synthetase from inhibition by cerulenin. Fatty acid synthetase was incubated under the same conditions as (A), for 5 min at room temperature with the indicated amounts of acetyl-ACP. Then cerulenin (150 pM) was added and the incubation continued for another 5 min. After the pr~limina~ incubations for (A) and (B), aliqnots of the reaction mixtures were assayed for fatty acid synthetase activity as described under Materials and Methods. Results are expressed as percentage of reaction rates relative to experiments in which (A) treatment with cerulenin (0-o) and tetrahydrocerulenin (a-0) or (B) treatment with both cerulenin and acetyl-ACP (O--O> were omitted.
double bonds or a tracts epoxide ring are not yet available. They wouid be required to fully elucidate the strn~t~ra~ features ~~~essa~~~for the i~~~~~t~o~ of fatty acid sy~t~es~s~
Previous studies by Bloch and his co-workers8 suggested that the /Sketoacyiacyl carrier protein synfhetase ~~~~o~e~~ of the M. phki fatty aci synthetase m&i-, enzyme cm~plex is the locus of ce~~~e~i~ i~~~b~t~o~ of fatty acid s~~tbes~s. Since the ~-k~toacy~-a~y~ carrier protein sy~t~etase has not been ~iss~~~ated in an active form from the fatty acid synthetese rn~~t~e~~~~le complex, the effect of cerulenin on the isolated component enzyme could not be evaluated in that system. The effect ofcesnlenin and its analogs on homogeneous E. ii /IS-ketoacyl-acyl carrier protein syn was therefore investigated. As shown in ig. 31%certrlenin is a very &Tective in higher c~~~~~t~at~o~sof i~bib~tor were required to achieve s r levels of inhIbitlon. Dihydro- and hexahyd~~~e~u~en~~ were not inhibit ~o~c~~~~at~o~s when tested under similar conditions. It is apparent in 364 that the cancer?trations of cerulenin (or tetrahydr~~er~~~~i~~ required to inhibit the activity of the fatty acid sy~t~etase and ~~ket~a~y~-a~y~ carri rotein synthetase are diEerent. T fatty acid synthetase was 50 % ~~~~b~ted by 30 p whereas the b~moge~eo~s condensing enzyme was 50 @/o ~~~~bited by cerulenin at co~ce~t~at~o~s Bower than 5 p
Since /3-ketoacyl-acyl carrier protein synthetase catalyzes the condensation react&x of fatty acid synthesis, it is apparent that, if this enzyme is rate-limiting for sverd fatty acid synthesis, fatty acid syntbetase should be just as sensitive as ,&ketoacyl-acy!
I!!!,
0 0
25
50 CERULENIN.
L-.-“--.
0
,]I i,___
,
I
75 phi I
60
100
i50
T~TRA~YDROCE~~L~~IN,
too
0
25 50 ACETYL-ACP,
75 /.&I
100
--
ZOO jtM
Fig. 3. (A) Effect of cernlenin and t~trahydro~eru~enjn on E. eoli ~-~etoacy~-a~yl carrier protein (ACP) 3.0 pmoles of potassium phosphate buffer, pW 7.0, 5.0 synthetase. The reaction mixtures contained pnoles of z-mercaptoethanol, 0.1 pmole of EDTA, 2.0 ,ug of /?-ketoacyi-ACP synthetase and the indicated amounts of cerulenin or tetrahydrocerulenin in 0.1 ml. Incubations were at room temperature for 5 min. (B) Acetyl-ACP protection of ,&ktoacyl-ACP synihetase from inhibition by cerulenin. P-Ketoacyl-A@P synthetase was incubated under the same conditions as (A) with the indicated concentrations of acetyl-ACP. Then ceruleain (50 ,dvf) was added and the incubation continued for another 5 min. After these ~rel~l~inary incubations, aliquots of the reaction mixtures in (A.1 and (S were assayed for &ketoacyl-ACP synthetase activity as described under Materials and Methois. Results are expressed as percentage of reaction rates relative to experiments in Lvbich(A) treatmeet with cerulenin (O-O) and tetrahydrocerulenin (e-0) or (B) treatment with both cerulenin and acetyl-ACP (0 - 3) were omitted.
CERULENIN
EFFECT ON FATTY ACID SYNTHESIS
161
carrier protein synthetase to inhibition by cerulenin. The fact that this is not the case suggests that j?-ketoacyl-acyl carrier protein synthetase is not rate-limiting in fatty acid synthesis by the crude E. coli fatty acid synthetase. The difference in sensitivity of fatty acid synthetase and P-ketoacyl-acyl carrier protein synthetase to cerulenin might also be due in part to the differences in protein concentrations in the two assays. The fatty acid synthetase preparation contains many proteins and was tested at high protein concentrations, whereas the homogeneous /I-ketoacyl-acyl carrier protein synthetase was tested at much lower protein concentrations. Since it was difficult to demonstrate a parallel effect of cerulenin on fatty acid synthetase and P-ketoacyl-acyl carrier protein synthetase when inhibition was studied directly, an indirect approach was used. It was known from earlier studies of the E. coli /I-ketoacyl-acyl carrier protein synthetaseg-I2 that the enzyme contains a cysteine residue in the active site which is specifically aikylated by iodoacetamide. When the enzyme is incubated with acetyl-acyl carrier protein, the enzyme accepts an acetyl group and is converted to an acetyl-enzyme (Reaction I). The presence of the acetyl group in the active site of the enzyme protects the enzyme against inactivation by alkylating agents. Assuming that the cerulenin might be acting at the active site of $ketoacyl-acyl carrier protein synthetase, the effect of the prior addition of acetylacyl carrier protein was examined. As noted in Fig. 313 the addition of acetyl-acyl carrier protein to the enzyme prevented inhibition by an amount of cerulenin (45 PM) which normally caused over 95 ‘A inhibition. The protection of enzymatic activity afforded by acetyl-acyl carrier protein increased with increasing concentration of the thioester. At 75 pM, acetyl-acyl carrier protein protected the enzyme almost completely. Thus, conversion of P-ketoacyl-acyl carrier protein synthetase to acetylenzyrne prevents the inhibition by cerulenin. Since acetyl-acyl carrier protein reacts directly and specifically with ~-ketoacyl-acyl carrier protein synthetase in the reaction sequence of fatty acid synthesis’, the effect of acetyl-acyl carrier protein pretreatment ‘of the fatty acid synthetase was examined. Following addition of acetyl-acyl carrier protein to the fatty acid synthetase, cerulenin was added at a concentration (150 I_~M) that normally causes about 90 % inhibition. As noted in Fig. 2B, the prior addition of acetyl-acyl carrier protein protected the fatty acid synthetase from inhibition by cerulenin. The effect of acetyl-acyl carrier protein in protecting the fatty acid synthetase (Fig. 2B) is very similar to its effect with fi-ketoacyl-acyl carrier protein synthetase (Fig. 3B). The concentration required to decrease inhibition to 50 % in both systems was about I I PM. The similarity of the effect of acetyl-acyl carrier protein in protecting j?-ketoacyl-acyl carrier protein synthetase and fatty acid synthetase against inhibition by cerulenin indicates that ~~ketoacyl-a~yl carrier protein synthetase is the site of cerulenin in~bitio~ of fatty acid synthesis. The assay of P-ketoacyl-acyl carrier protein synthetase is carried out with a coupled enzyme system involving two additional enzymes, malonyl-CoA-acyl carrier protein transacylase and P-ketoacyl-acyl carrier protein reductase. The effect of cerulenin on these two enzymes was tested directly. Concentrations of cerulenin (IOO ,uiM) that caused complete inhibition of /3-ketoacyl-acyl carrier protein synthetase had no effect on either malonyl-~oA-a~yl carrier protein transacylase or P-ketoacylacyl carrier protein reductase. The inability of cerulenin to inhibit these two enzymes, which are components of the fatty acid synthetase, further illustrates the specificity of the inhibitor for /3-ketoacyl-acyl carrier protein synthetase.
162
, r 6. D ArjNOk3
ef iii.
The assay of /I-ketoacyl-acyl carrier protein synthetase measures the overa~ji activity ofthe enzyme, shown in Reaction 3, and this activity is inhibited by ceruienin,, ~-~etoacy~-acy~ carrier protein sy~t~et~se also catalyses a ~l~rn~er of model reactions which represent the component reactions and therefore the e&ct of cerulenin on each of these reactions was investigated. The first series of experiments tested the effect of cerule~in on the fatty acyl tran5acy~ase activity of the enzyme ( eaction I). Fatty acyB thioesters of Cob have been shown to be active substrates in Reaction I”: and the transacylase activity can be readily demonstrated by incubating a radioactive thioester, such as [14C]hexanoyl-CoA, with acyl carrier protein and en2yme. The enzyme is initially acylated (reaction I, forward ~rect~ou~ and then the acyl group is tra~s~e~~~d to acyl carrier protein forming ~~4~]~~e~a~oy~-~cy~ carrier protein (~~~~~t~o~ I, reverse direction). Transacylase activity of /3-ketoacyl-acyl carrier rotein synthetase, measured as the rate of [14C]hexanoyl-acyl c in synthesis, was inhibited by cerulenin forward d~~ect~o~~ was also measured (Fig. 4). Acyl transfer to the enzyme ( substrate ~~a~t~t~es of enzyme with directly. This was accomplished by 1 [‘4C]hexanoyl-CoA and then measuring the formation of E‘“C]hexanoyl-euszyane. Cerulenin aiso inhibited this reaction, and it is apparent (Fig. 4) that the inhibition of the transacylase activity of the enzyme, when measured in either of the two assays described, ~ar~~~eled the ~~~~~ib~t~o~ of overall ~~ketoacy~-~c~~ carrier protein synthetase activity. In another model reaction P-ketoacyi-acyl carrier protein syntketsse catalyzes the decarboxy~at~o~ of rn~~~~y~-a~~~ carrier ~rQt~~~ in the abseilce of fatty asyl-acyi component of carrier protein I1 . This activity probably represents the decarboxylation the condensation-decarboxylation reaction shown in Reaction 2. As seen in Fig. 5 malonyl-acyl carrier protein decarboxylase activity was i~b~b~ted by ce both partial reactions catalyzed by ~-ketoacy~-~cy~ carrier protege syn tions I and 2) are inhibited by cerulenin. A final model reaction, the rna~o~y~-~o~~-~~~ exchange reaction which is dependent upon both the fatty acyl transacylase and the condensation-decarboxyla~~on functions of ~-k~toacy~-ache carrier protein synthetase” I, w2s also ~nh~b~t~d by cer~~e~~i~~ as predicted from the results witln the component reactions. inhibition of both malonyl-acyl carrier protein de~arboxylase and malonyl-CoA-CO, exchange activities paralleled the ~n~~bit~o~ of /3-ketoacyl-acy! carrier protein synthetase activity (Fig. 5).
High concentrations of cerulenin inhibited fi-ketoacyl-acyl carrier protein synthetase very rapidly. When low concentrations were used (43 9 ), the time course of inhibition could be observed. As shown in Fig. 6, when acetyl-acyl carrier protein was added at concentrations (100 ,&I) that completeljr protect the enzyme against cerulenin inhibition, the increase in inhibition stopped immediately. Tn addition, it should be noted that during the time course of this experiment, there was no reversal of Jnhibition since enzyme activity was not recovered after the addition of the acetyl-acyl casrkr protein. The inability of acetyl-acyl carrier protein to reverse the inhibition of pketoacyl-acyl carrier protein synthetase by cerulenin indicates that under these conditions the enzyme was irreversibly inactivated. This suggests the ~ossib~~~ty that certiknin might be bound cova~ent~y to t&e enzyme.
CERULENIN
EFFECT
ON FATTY
ACID
SYNTHESIS
8
0 0
25
50 CERULENIN,
75 /_LM
100
0
25
50 CERULENIN,
75
100
PM
Fig. 4. Effect of cerulenin on P-ketoacyl-acyl carrier protein (ACP) synthetase, fatty acyl-CoA-ACP transacylase activity and hexanoyl-Cob binding. P-Ketoacyl-ACP synthetase, 40.0 pg, was incubated in 0.1 M potassium phosphate buffer, pH 7.0, in a volume of 0.03 ml with the indicated amounts of cerulenin. Following incubation at room temperature for 5 min, aliquots were removed and assayed for /?-ketoacyl-ACP synthetase (@-a), fatty acyl-CoA-ACP transacylase (O-O) and [14C]hexanoylCoA binding (n--a) activities as described under Materials and Methods. Results are expressed as percentage of rates relative to experiments in which treatment with cerulenin was omitted. Fig. 5. Effect of cerulenin on malonyl-CoA-CO, exchange and malonyl-acyl carrier protein (ACP) decarboxylase activities of P-ketoacyl-ACP synthetase. The reaction mixtures contained 2.0 pmoles of potassium phosphate buffer, pH 7.0, 5.0 pmoles of z-mercaptoethanol, 0.1 pmole of EDTA, 2.0 fig QI’ /?-ketoscyl-ACP synthetase, and the indicated amounts of cerulenin in 0.1 ml. Incubations were at room temperature for 5 min. Aliquots were removed and assayed for ,%ketoacyl-ACP synthetase (s--e), malonyl-ACP decarboxylase (O--O) and malonyl-CoA-CO, exchange (A--a) activities as described under Materials and Methods. Results are expressed as percentage of reaction rates relative to experiments in which treatment with cerulenin was omitted.
Stoichiometry of the reaction between P-ketoacyl-acyl carrier protein synthetase and cerulenin The possibility that the interaction of P-ketoacyl-acyl carrier protein synthetase and cerulenin leads to the formation of a covalent complex was tested by denaturing the inhibited enzyme with trichloroacetic acid. As shown in Fig. 7, when substrate quantities of enzyme were treated with [3H]cerulenin and subjected to trichloroacetic acid precipitation, the precipitated protein contained bound cerulenin. The amount of cerulenin bound to the enzyme was proportional to the extent of inhibition, and it is apparent that approximately I mole of cerulenin was bound per mole of enzyme when inhibition of the enzyme approached IOO %. DISCUSSION
The results of experiments conducted by Bloch and his coworkers’ with a fatty acid synthetase of M. phlei indicated that cerulenin inhibited this multienzyme complex by inactivating /?-ketoacyl-acyl carrier protein synthetase. The experiments described in this communication support and extend those observations. Cerulenin inhibition of the E. coli fatty acid synthetase was shown to be due to the specific inhibition of the P-ketoacyl-acyl carrier protein synthetase component which could be studied as a homogeneous protein in this system. It was known that E. coli /?-ketoacylacyl carrier protein synthetase contains in its active site a specific cysteine residue’
100
25
0
15
0
30 MINUTES
45
0
I
2 CERULENIN,
3 mhl
4
Fig. 6. Irreversible inactivation of /3-ketoacyl-acyi carrier protein (ACP) synthetase by cerulenin. The reaction mixtures contained 2.0 pmoles of potassium phosphate buffer, pH 7.0, 5.0 pmoles of a-met-captoethanol, 0.1 ymole of EDTA, 4.0 fig of P-ketoacyl-ACP synthetase. 0.4 nmole of cerulenin in 0.1 ml. After 2, 5 and IO min of incubation at room temperature, 10.0 nmoles of acetyi-AC? were added. p-Ketoacyl-ACP synthetase was assayed in the absence (O--O) and in the presence ( of added acetyl-ACP. Results are expressed as percentage of reaction rates relative to experim which treatment with cerulenin was omitted. Fig. 7. Sioichiometry of the reaction between cerulenin and /I-ketoacyl-acyl carrier protein (ACP) synthetase. /?-Ketoacyl-ACP s-ynthetase, 260 pup of protein, was incubated in 0.x M potassium phosphate buffer, pH 7.0, in a volume of 0.05 ml with the indicated amounts of [3HJcerulenin. Foilowing incubation at room temperature for 5 mm, aliquots were removed and assayed for /‘I-ketoacyl-ACP -IB) and ceru!enin binding (O-O) as described under Materials and Methods. Enzymatic activity is expressed as percentage of reaction rates relative to experiments in which treatment with cerulenin was omitted. Cerulenin binding is expressed as moles orC inhibitor bound per mole of enzyme.
which is presumably first partial
involved
reaction
sensitive to alkylation enzyme is protected
in the formation
catalyzed
of an acyl-enzyme
by this enzyme
by ioduacetamide
from iodoacetamide
(Reaction
which inactivates inhibition
intermediate
I.). This
cysteine
the enzyme;
in the is very
however,
the
when an acetyl group is bound
at the active site. The possibihty that cerulenin, a 12 carbon atom fatty acid amide, is bound at the active site of P-ketoacyl-acyl carrier protein synthetase ~2s suggested by the fact that this enzyme can react with fatty acyl thioesters whose chain lengths vary from 2 to 16 carbon The finding
atoms as part of its
that acetyl-acyl
cysteine residue in the active site, protected permitted
the demonstration
the inhibition and
,&ketoacyl-acyl
concentrations
carrier
from
donates
of E. coli fatty acid synthetase is dUe to synthetase.
Fatty
were protected
acid
in parallel
noted previously
carrier protein
synthetase
with iodoacetamide.
by cerulenin
Iodoacetamide
specific cysteine residue and inhibits the first part1‘al reaction catalyzed (Reaction
I). The
fatty acyl transferase component
ed in the fatty acyl-GoA-acyl by iodoacetamide synthetase
of the
carrier protein transacylase
and accounted
by iodoacetamidell.
spnthetase wrions
by
carrier protein.
of /I-ketoacyl-acyl
the inhibition
synthetase
in fatty acid biosynthesisxl,
an acetyl group to the enzyme from inzctivation by ceruienin
carrier protein
protein
of acetyl-acyl
Inhibition
which
the
that inactivation
of ,&ketoacyl-acyl
function
normal
carrier protein,
for the inhibition
enzymatic
dif?ers
alkylates
a
by the enzyme
reaction,
measur-
model reaction, was inhibited
of /3-ketoacyl-acyl
carrier protein
On the other hand, the malonyl-acyl
carrier protein
CERULENIN
EFFECT ON FATTY ACID SYNTHESIS
165
decarboxylase reaction, which represents a component of Reaction 2, was not inhibitboth partial ed by iodoacetamidell. As shown above, model reactions representing Reactions I and 2 (Figs 4 and 5) were equally inhibited by cerulenin. Thus cerulenin differs from iodoacetamide since the former inhibits malonyl-acyl carrier protein decarboxylation whereas the latter does not. /3-Ketoacyl-acyl carrier protein synthetase contains a fatty acyl and a malonyl-acyl carrier protein binding siteg-I’. The relationship between these sites is not known. Alkylation by iodoacetamide of the cysteine in the fatty acyl site apparently does not affect the malonyl-acyl carrier protein site, whereas inactivation by cerulenin affects the activity of both sites. An explanation for this observation might be that both iodoacetamide and cerulenin alkylate the same cysteine residue in the fatty acyl site but that cerulenin inhibits the malonyl site activity because it has a long hydrophobic chain. The hydrophobic chain of cerulenin presumably could prevent the binding of malonyl-acyl carrier protein at the ad_jacent site. The only evidence suggesting that cerulenin reacts with the cysteine at the fatty acyl site is the protection afforded by acetyl-acyl carrier protein. It is conceivable, but not likely, that cerulenin attacks the malonyl-acyl carrier protein site. Experiments attempting to rule out this possibility, by demonstrating that malonyl-acyl carrier protein can protect against inactivation by cerulenin, are not feasible since the enzyme readily decarboxylates malonyl-acyl carrier protein to form acetyl-acyl carrier protein, and the enzyme would be protected by the latter. Thus, specific reaction of cerulenin at the malonyl-acyl carrier protein site cannot be ruled out. It has been established that inhibition of /3-ketoacyl-acyl carrier protein synthetase by iodoacetamide is due to the alkylation of one residue of cysteine per mole of enzymeg. The demonstration that inhibition by cerulenin is irreversible and that it is accompanied by the binding of a single mole of cerulenin per mole of enzyme suggests a highly specific interaction between the enzyme and the inhibitor. It is likely that cerulenin, like iodoacetamide, inhibits /?-ketoacyl-acyl carrier protein synthetase by alkylating the cysteine residue in the active site, but this has not yet been investigated. The cysteine thiol, which is very reactive with other alkylating agentsg-11, could act as a nucleophile1g,20 and initiate an attack upon one of the epoxide ring carbon atoms. The resulting thioether would be expected to be very stable. Such covalent modification of the enzyme could explain the observed inhibition of all the catalytic properties of this enzyme. Although the epoxide group offers an attractive and versatile protein side chain modifying reagent, the broad spectrum of groups that it can potentially modifyZ1 -23 makes it impossible to predict with certainty which amino acid residue of /Sketoacyl-acyl carrier protein synthetase is modified. The structure of cerulenin presents several features of general interest. As the hydrocarbon backbone of the molecule is composed of 12 carbon atoms, cerulenin is a fatty acid of intermediate chain length; however, the chemical features associated with this compound appear to be unique. The presence of two double bonds in a fatty acid is not uncommon, but the fact that both are of tram configuration and that one is 02 is novel. The finding that an internal cis oxirane ring is necessary for biological activity and that a carbonyl function is adjacent to this structure also appears to be unique. Although other epoxy-containing compounds have been used as inhibitors or modifiers of enzymatic processes or proteins21-23, these have always been compounds with terminal oxirane rings, as opposed to those reported here. The epoxy ring is of distinct chemical significance in cerulenin, and it is possible that synthesis of similar
compounds may yield other agents acid and lipid metabolism
specifically
reactive
with 0th
enzymes
uf fa;cp
A(‘KNOWLED
The excellent technical assistance of Mr Tadataka Kcsadfi is gratefully z_cknowiedged. This investigation was supported in part by the National Institutes of Health ( I-Ror-I-IL-10406) and the Nationai Science Foundation (GB-38676X:. 1.S.R. is a Nation4 Institutes of Health Postdoctoral Fellow (5-Fo2AM5236~02). REFERENCES I Sane, Y.. Xomura, S.. Kamio, Y.. &nura, S. and Hata. I‘. (1967) J. ,4rrti!~iot.. Ser. A 20. ?4_+-~@ z &ura. S., Katagiri. &I.. Nakagaxva. A., Sane. Y.. Nomura, S. and llata. T. (1967) j, An~ihi~!. Ser. .ft. 20.349 -354 3 Gmura. S.. Nakagawa. A.. Sekikawa. K.. Otani. \I. and Hata. 1.. (; 969) C’hen~. Ph~~r~~~. h/L 7bkvo i:. 2761-2364 4 Ma&mae.‘A., Nomura. S. and Hata. T. (1963) .I. Anrihior., Scar. A 16. 239-243 5 Matsumae. A., Nomura. S. and Ha:a, T. : 1964) J. Arrrihior., Ser. A I 7. I 7 6 Nomura. S., Horiuchi, T.. Gmara, S. a!ld I-faia, -1‘. (1972.1 J. Bbcit~,u. ‘i’~!~),) 71. 783-796 7 Nomura. S., Iloriuchi, ‘f.. FIata. T. and Gmura, S. (1972) J. Aurihio/. 25. 365 358 8 Vance. l)., Goldberg. I.. Mitsuhashi, 0.. Bloch, K.. Bmura. S. and Nomura. S. (1972) L?kx/~e~r~. .b’iop/q~. RCS. Con~/n/rn. 48, 649 6 j6 9 Greenspan, M. I).. Albcrts. A. W. and Vagelos, I’. R. (196y1 J. Rio/. (I/~/PI. 2~4~ 6f77 6485 10 I’rcscott, D. J. and Vagclo~. I’. R. (1970) J. Biol. C/I~/~I. ~45, 5484-5490 I I Albcrts. A. W., Bell. R. M. and Vagelos. I’. R. (1972) J. Bid. Chem. 247. 3190-3198 II Rosenfcld. I. S., I>‘Agnolo. G. and Vngclos. P. R. (1073) J. f?io/. C‘lww. 248, 2452 2460 r3 Majerus. P. W.. A!berts, A. W. and Vagetos, P. R. i1964) Proc. r\rnrl. .~!cad. SC,;. L;.S.y. iz31 -1338 14 Albcrts. A. W.. Majerus. I’. W. and Vagelos, I’. R. ( I$&)) in Mr/hodv in Enr,:~mo/ogy (l.owcnsre~n. 3. 41.. cd.). Vol. 14. pp. 57 60. Academic Frcss. New York 15 Simon, E. J. and Shemin, D. (1y53) ./. Avr. C‘lwnr. Sot. 75. 2520 16 Goldman, t’. and Vagc!os. I’. R. (1961) J. Bio!. Chem. 236. ~OZO--Z~Z~ 17 Ruth, Jr, F. E. and Vagclos. F. R. (1y73) J. Bid. Chew.. in the press IX Vagelos. I’. R., Albcrts. A. W. and Majcrus. I’. W. (1969)ir: ;~fethou~s in Jlll:,:.~wkq>. (Lowcnstcin. J. M., xl.), Vol. 14, pp. bo--63. Academic Press. New York II) Ross, W. C. J. (1~50) J. (‘hcjm Sot. 2257-2272 20 Farkcr. R. E. and Isaacs, N. S. (IC)~Q) Chew. Rev. ~9. 737-799 21 Thomas, E. W., McKelvy, J. F. and Sl-aron. N. (196~) Mnrure ‘2:: 483. 486 22 Waley. S. G.. Miller. J. C., Rose, I. A. and O’Connel. t. L. (1970) !V’r~!wr 227. r8: 2.3 Chen. K. C. S. and Tang. J. (1972) J. Bioi. (.‘lrr~. 247. 2566 2575