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INTRODUCTION |
In animals, the de novo synthesis of long-chain fatty
acids from acetyl- and malonyl-CoA is catalyzed by a single protein, the FAS,1 that consists of
two identical, multifunctional polypeptides (1-3). Although all of the
functional domains are present on a single polypeptide, coupling of the
individual activities necessary for catalysis of the overall reaction
of fatty acid synthesis is performed only by the dimer (4, 5). In 1981, Stoops and Wakil (6) found that the bifunctional reagent
dibromopropanone reacts with the FAS dimer such that the
4'-phosphopantetheine thiol of the ACP domain becomes cross-linked with
the active site cysteine thiol of the
-ketoacyl synthase domain. The
electrophoretic mobility of FAS on SDS-polyacrylamide gels was
dramatically reduced following treatment with dibromopropanone,
indicating that the cross-linking had occurred between the two
subunits. Approximately two molecules of the reagent were incorporated
into the fully cross-linked dimer, and the ability to synthesize
palmitate was lost. These observations inspired a model for the FAS in
which the two subunits are arranged in head-to-tail orientation, so that the
-ketoacyl synthase of one subunit is juxtaposed with the
ACP domain of the opposite subunit and two sites for palmitate synthesis are formed per dimer (7).
Support for the two-site model was provided by experimental evidence
showing that the FAS dimer, in which the chain-terminating reaction had
been blocked, assembles and retains two long-chain fatty acyl moieties
(8, 9). Subsequently, sequencing (10-12) and mutagenesis (13) of the
animal fatty acid synthase established unequivocally the arrangement of
the functional domains in the FAS polypeptide as, starting from the
amino terminus,
-ketoacyl synthase, malonyl/acetyltransferase,
dehydrase, enoyl reductase,
-ketoacyl reductase, ACP, and
thioesterase. The dehydrase and enoyl reductase domains are separated
by a region of approximately 600 amino acids that has not yet been
ascribed a specific function. Initially, only the condensation reaction
was believed to be catalyzed across the subunit interface, but later,
experimental evidence was obtained which demonstrated that the
translocation of acetyl and malonyl moieties from CoA ester to the
4'-phosphopantetheine of the ACP domain also requires the dimeric form
of the protein (14). Thus it has become generally accepted that the two
subunits lie side-by-side in a fully extended, antiparallel
configuration so that each of the two centers for palmitate synthesis
requires cooperation between catalytic domains located in the
amino-terminal half of one subunit with those of the carboxyl-terminal
half of the companion subunit (1, 3, 15).
This side-by-side, head-to-tail model had not been subjected to
rigorous testing until recently when we introduced an in
vitro mutant complementation strategy to map the functional
topology of this multifunctional complex (16-18). The approach
requires the engineering of various modified FASs, in which the
activity of one of the functional domains is specifically compromised
by mutation. Heterodimers formed from subunits containing different, single mutations may be capable of fatty acid synthesis if the two
mutations are located on domains that normally cooperate with each
other across the subunit interface. To date, the results of this
ongoing mutant complementation analysis have confirmed several key
features of the original. For example, mutations in either the
-ketoacyl synthase or malonyl/acetyltransferase domain complement
mutations in either the ACP or thioesterase domains, whereas
mutations in the ACP and thioesterase domains fail to complement each other (17, 18). These findings confirmed that the
-ketoacyl synthase and malonyl/acetyltransferase domains, located in
the amino-terminal half of the FAS polypeptide, can contribute to the
same center of palmitate synthesis as do the ACP and thioesterase
domains of the companion subunit. On the other hand, the
complementation analysis also revealed some unanticipated properties
that could not be explained by the prevailing model. First, conversion
of the
-hydroxyacyl-ACP to the enoyl-ACP was found to be catalyzed
by cooperation of the ACP and dehydrase domains of the same subunit
(17). Because the dehydrase and ACP domains are separated by more than
1000 residues, this finding provided the first experimental evidence
indicating that the two constituent polypeptides are not positioned
side-by-side in a rigid, fully extended, antiparallel orientation but
are coiled in such a way as to permit functional contacts between
domains distantly located on the same polypeptide. Second, both the
substrate translocation and condensation reactions were found to be
catalyzed by cooperation of the malonyl/acetyl transferase and
-ketoacyl synthase domains, respectively, with the ACP domains of
either subunit (18). Because the active centers of
-ketoacyl
synthase and malonyl/acetyl transferase domains are separated from the 4'-phosphopantetheine moiety by more than 1500 residues, this finding
raised further doubts as to the validity of the fully extended, rigid
antiparallel polypeptide model.
We have proposed a revised model that retains the concept of
head-to-tail orientation of the two subunits but also allows for
head-to-tail interactions within each subunit (18, 19). At present,
because the evidence in support of the revised model is derived
entirely from functional studies performed with mutant FASs, we are now
exploring experimental approaches that can provide structural data with
which to test the validity of the new model. The results of the mutant
complementation analysis imply that the active-site cysteine residue of
the
-ketoacyl synthase domain of each subunit must lie close to the
4'-phosphopantetheine thiol of both subunits. This finding would appear
to be inconsistent with the conclusion drawn from the original
dibromopropanone cross-linking experiments by Stoops and Wakil (6),
namely that cross-linking occurs exclusively between subunits. However,
the fact that three molecular species with retarded electrophoretic
mobilities are formed by treatment with dibromopropanone has never been
explained. Because an immediate inference of the revised model is that
cross-linking would be expected to occur both within and between
subunits, we have reinvestigated the specificity of the
dibromopropanone reaction with the FAS to determine whether one of
these species might represent an internally cross-linked subunit.
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EXPERIMENTAL PROCEDURES |
Materials--
1,3-Dibromopropanone was purchased from Alpha
Æsar, Ward Hill, MA, and purified by high performance liquid
chromatography as described previously (20).
Construction of cDNAs Encoding His6- and
FLAG-Tagged FASs and Expression of the Proteins in Sf9
cells--
The strategies for construction of cDNAs encoding the
wild-type FAS, single domain-specific mutants and for introduction of His6 or FLAG tags have been described in detail previously
(16, 18, 21, 22). The carboxyl-terminal FLAG-tagged C161A mutant was
constructed by first generating a mutated partial cDNA fragment by
polymerase chain reaction amplification, using a primer set M161T3/54B1152 and pFAS 74.20 (partial FAS cDNA in pUCBM20) as template (21). Authenticity of the amplification product was confirmed
by DNA sequencing, and the fragment was moved stepwise into the
full-length, wild-type carboxyl-terminal FLAG-tagged construct. The
quadruple mutant (C161A,S581A,S2151A,S2302A) was assembled stepwise by
incorporating partial cDNA fragments carrying individual mutations
in to full-length FAS cDNA. At each step, successful introduction
of the new substitution was confirmed by DNA sequencing. The final FAS
cDNA constructs, in the context of the pFASTBAC 1 vector, were used
to generate recombinant baculovirus stocks by the transposition method
employing the BAC-to-BAC baculovirus expression system according to the
manufacturer instructions. Sf9 cells were then infected with
the purified recombinant viruses and cultured for 48 h at
27 °C. The tagged FAS proteins were partially purified from the
cytosols as described earlier (21) and then subjected to final
purification by affinity chromatography (22); glycerol (10%, v/v) was
included in all buffers used for chromatography.
Formation and Purification of Heterodimeric FASs--
Two
different FAS dimer preparations, either wild-type, the single mutants
C161A or S2151A, or the quadruple mutant (C161A,S581A,S2151A,S2302A), one containing the His6-tag the other containing the
FLAG-tag, were mixed, and their subunits were randomized by
dissociation and reassociation (22). The heterodimers consisting of one
His-tagged and one FLAG-tagged subunit were purified by sequential
affinity chromatography on columns of anti-FLAG M2 antibodies (Eastman Kodak Co.) and nickel-nitrilotriacetic acid/agarose (Qiagen, Inc., Santa Clarita, CA) as described previously (22). The FAS activities of
the purified heterodimers were: wild type/wild type 2260 ± 80, C161A/wild type 1080 ± 30, S2151A/wild type 1080 ± 14, C161A/S2151A 142 ± 14 and C161A,S2151A,S581A,S2302A/wild type
102 ± 2 milliunits/mg. The latter heterodimer was used in lieu of
a C161A,S2151A/wild type heterodimer (presently unavailable), because
the S581A and S2302A mutations, which compromise specifically the
malonyl/acetyl transferase and
-ketoacyl synthase activities,
respectively (17, 18), were found to have no effect on the reaction of
the FAS with dibromopropanone (see "Results"). For clarity, this
mutant is identified in the text by the two mutations that are relevant to this study, namely C161A,S2151A.
Modification of FAS with Dibromopropanone--
The FAS storage
buffer was replaced with 0.2 M sodium phosphate, pH 7, containing 1 mM EDTA, either by successive dilution and
concentration in Centricon-50 devices (Amicon. Inc., Beverly, MA), by
centrifugation through a gel filtration column (23), or by dialysis.
Fresh solutions of dibromopropanone were prepared by evaporation of the
benzene solvent from the stock solution and redissolution of the
reagent in phosphate buffer. The concentration of the reagent was
determined before every experiment as described previously (20). The
cross-linking reaction was carried out at 20 °C and quenched by the
addition of mercaptoethanol to 1 or 2 mM. All buffers were
flushed with nitrogen to remove dissolved oxygen.
SDS-Polyacrylamide Gel Electrophoresis and Western
Analysis--
FAS preparations were analyzed by SDS-polyacrylamide
electrophoresis using 2% polyacrylamide in the stacking gel and 4% in the running gel. Kaleidoscope standards (Bio-Rad Labs., Hercules, CA)
were routinely applied to each gel and electrophoresis was continued
until the myosin marker (blue) was about 0.5 cm from the bottom of the
gel; thus, these run times are significantly longer than those
typically used for SDS-polyacrylamide electrophoresis. Phosphorylase
b cross-linked standards (Sigma) were also included in some
experiments. Pro-Blue (Owl Separation Systems, Woburn, MA) was used as
a general protein stain, and the proportion of the various stained
zones was estimated by densitometric scanning with the aid of NIH Image
software (Version 1.60). For Western analysis, proteins were
electroblotted onto nitrocellulose membranes and exposed successively
to either murine anti-FLAG M2 monoclonal antibody (Eastman Kodak Co.)
or murine anti-PentaHis monoclonal antibody (Qiagen Inc.), and then to
alkaline phosphatase-coupled goat anti-mouse IgG antibodies (Bio-Rad
Labs, Hercules, CA), following the manufacturer protocols.
Mass Spectrometry--
The MALDI-TOF experiments were performed
under conditions that result in protein denaturation and obliteration
of specific noncovalent interactions. Mass spectra were recorded on a
Voyager DE STR instrument (PerSeptive Biosystems, Framingham, MA),
equipped with a two-stage ion source, delayed extraction (24), and a high current detector. The Voyager/Grams software supplied with the
instrument was used for instrument control and data analysis. All
measurements were carried out in the linear mode. The linear path-length in this instrument is 2.0 m. Desorption ionization was
initiated with a nitrogen laser operating at 337 nm with a repetition
rate of 3 Hz. The overall acceleration voltage used was 25 kV. A
sampling rate of 20 ns (50 MHz) and an input filter of 20 MHz were used
for data collection. Each spectrum represents the sum of 150-250
individual laser shots, and mass measurements were done automatically
using the instrument default calibration. FAS samples (0.5 µl in 5 mM ammonium acetate, pH 7.8) were mixed with an equal
volume of matrix solution directly on the stainless steel sample target
plate and allowed to dry at room temperature. The matrix solution
consisted of 10 g/liter of sinapinic acid dissolved in 0.3% aqueous
trifluoroacetic acid:acetonitrile (2:1, v/v).
Gel Filtration--
Proteins were chromatographed on a
SigmaChromTM GFC-1300 column (0.75 × 30 cm) in 0.25 M
potassium phosphate, pH 7 and 1 mM EDTA at a flow rate of
0.5 ml/min and were detected by monitoring absorbance at 280 nm. Blue
dextran, FAS dimers, and monomers were eluted with retention times of
9.0, 10.5, and 11.5 min, respectively.
Enzyme Assays--
Overall fatty acid synthesizing activity was
measured spectrophotometrically (25); one unit of FAS activity is
defined as 1 µmol of NADPH oxidized per min at 37 °C.
-Ketobutyryl-CoA reductase activity of FAS was determined by
monitoring the accompanying oxidation of NADPH as described earlier
(17). The interthiol decanoyl transferase activity of the
-ketoacyl
synthase domain FAS was monitored by high performance liquid
chromatographic separation of substrates and products as described
previously (26).
Hydrolysis of Propanonyl Bond by High pH--
The cross-linked
products were first separated on 1.5-mm SDS-polyacrylamide gels.
Protein bands, revealed using an imidazole-zinc negative stain (27),
were cut out; and metal ions were removed by incubation of the gel
pieces successively with 0.5 M EDTA, pH 8, and water, each
for 10-15 min. The proteins were then electroeluted in a Centrilutor
(Amicon) using Centricon-50 or -100 units. During electroelution, the
Tris-glycine-0.1% SDS buffer was cooled and continuously recirculated
between the upper and lower chambers. The solution containing the
purified cross-linked FAS species was adjusted to pH 11-11.5, by the
addition of 1 M NaOH, and incubated at 37 °C for 1 or
2 h.
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RESULTS |
Reaction Conditions for Inactivation and Cross-linking of FAS by
Dibromopropanone--
In agreement with the earlier findings of Stoops
and Wakil (6, 28), as a consequence of treatment of the FAS with
dibromopropanone, we observed the formation of three new molecular
species with retarded electrophoretic mobilities on SDS-polyacrylamide
gels (Fig. 1A,
species ii, iii, and iv). Typically,
when a centrifuged gel filtration column was used to remove
dithiothreitol from the preparation and the FAS was exposed to a
1.5-fold molar excess of reagent for 60 s, the species exhibiting
retarded electrophoretic mobilities accounted for more than 80% of the
total FAS species present. Formation of the three new molecular species
occurred extremely rapidly and was accompanied by a loss in the ability of the protein to synthesize fatty acids; only 4% of the FAS activity remained after 60 s exposure to dibromopropanone (Fig.
1B). The rate of loss in ability to reduce
-ketobutyryl-CoA to butyryl-CoA was slower; about 15% of the
activity remained after 60 s (Fig. 1B). For the overall
FAS reaction, complete integrity of both the
-ketoacyl synthase
active-site cysteine and the phosphopantetheine of the ACP domain are
crucial. However, for the reduction of
-ketobutyryl-CoA by the FAS,
the
-ketoacyl synthase active-site cysteine is not required.
Translocation of the
-ketobutyryl moiety from CoA to the ACP domain,
reduction of the keto group and translocation of the product back to
CoASH involves only the malonyl/acetyltransferase, the
phosphopantetheine thiol and the
-carbon processing enzymes. The
difference in rate of loss of these two activities suggested that
perhaps the active-site cysteine residue is the primary target of the
reagent, and indeed the rate of disappearance of the original species
i paralleled closely the rate of loss of
-ketobutyryl-CoA reductase activity rather than the rate of loss of overall fatty acid
synthesizing activity.

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Fig. 1.
Modification of FAS by dibromopropanone.
The storage buffer was replaced with the reaction buffer using two
consecutive centrifuged gel filtration columns. The enzyme during
reaction was at 0.9 mg/ml, and dibromopropanone was at 1.5-fold molar
excess over FAS subunit concentration. A, SDS-polyacrylamide
gel electrophoresis analysis of dibromopropanone-modified FAS.
Lane 1, wild-type FAS; lane 2, wild-type FAS
after 60 s of exposure to dibromopropanone. Phosphorylase
b cross-linked dimers (195 kDa) and hexamers (584 kDa),
which exhibit near ideal electrophoretic behavior under the conditions
employed, migrate close to the bottom and top of the gel, respectively.
B, inhibition of fatty acid synthesis and -ketobutyryl
CoA reduction activities of FAS by dibromopropanone and quantitation of
the residual species i (all expressed as percent of 0 time
values).
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This inference was further supported by the results of an experiment
that compared the loss in
-ketobutyryl-CoA reductase and interthiol
acyltransferase activities of FAS treated with different molar ratios
of dibromopropanone. The interthiol acyltransferase reaction is
catalyzed exclusively by the
-ketoacyl synthase domain and uses
model substrates to assess the ability of the
-ketoacyl synthase to
transfer acyl moieties between substrate and acceptor thiols via the
cysteine nucleophile (26). Two different homodimeric FAS mutants were
employed for this experiment, C161A, in which the
-ketoacyl synthase
nucleophile is replaced, and S2151A, in which the site of
posttranslational insertion of the phosphopantetheine is removed. In
the presence of a two-fold molar excess of reagent, 90% of the
interthiol acyltransferase activity associated with the S2151A mutant
was eliminated (Fig. 2). Under identical
conditions, less than 20% of the
-ketobutyryl-CoA reductase
activity of the C161A mutant was lost. To achieve 90% loss in
-ketobutyryl-CoA reductase activity with this mutant, more than a
25-fold molar excess of reagent was required. In contrast, only a
1.5-fold molar excess of reagent was required to eliminate 85% of the
-ketobutyryl-CoA reductase activity associated with the wild-type
FAS (Fig. 1B). Clearly in the absence of Cys-161, the
phosphopantetheine thiol attached to Ser-2151 is a relatively poor
target for dibromopropanone.

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Fig. 2.
Modification of S2151A and C161A FAS mutants
by dibromopropanone. FAS mutants, 1 mg/ml, were modified with
dibromopropanone, at the concentration indicated in the figure, for 3 min. Reaction was stopped with 1 mM dithiothreitol, and
activities were measured as described under "Experimental
Procedures."
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These results are consistent with Cys-161 being the primary target for
the reagent in the wild-type FAS. Reaction of the second alkylating
group of the bifunctional reagent with the phosphopantetheine thiol
likely is facilitated by the proximity of the phosphopantetheine to the
cysteine nucleophile, rather than by the inherent reactivity of the
phosphopantetheine thiol.
Identification of the Three Molecular Species Formed by Reaction of
Dibromopropanone with the FAS--
Three separate approaches were used
to identify the three electrophoretic species formed uniquely as the
result of exposure of the FAS to dibromopropanone. First, the
electrophoretic species formed by treatment of a panel of mutant homo-
and heterodimers were analyzed by general protein staining. Second, the
presence or absence of individual subunits in each electrophoretic
species was assessed by detection of the specific tags using Western
blotting. This procedure clearly distinguishes between FAS polypeptides containing the His6 and FLAG tags because neither are the
His6-tagged homodimeric FASs recognized by the anti-FLAG
antibodies nor are the FLAG-tagged homodimers by the anti-His
antibodies (22). Third, the molecular masses of the various species
formed as the result of dibromopropanone treatment of the mutant FASs
were assessed by gel filtration and mass spectrometry.
The relative proportions of species i-iv present in the
dibromopropanone-treated FAS were dependent on the method used to remove dithiothreitol from the preparations. Considerably more of the
unmodified FAS species i remained when either the
centrifuged gel filtration column or dialysis procedures were employed
than when the Centricon-50 device was used. The proportion of species iii formed was also higher when the latter procedures were
used. We attribute these quantitative differences to the susceptibility of the Cys-161 thiol to oxidation. The dialysis procedure is inherently slow, and the centrifuged gel filtration column procedure potentially exposes the FAS protein solution to a very large surface area after
loading on the column and spinning out the solvent. The Centricon
procedure, on the other hand, is relatively speedy and does not expose
the FAS solution unduly to air. Typically then, using the Centricon
procedure to remove dithiothreitol, more than 95% of the wild-type FAS
can be cross-linked by dibromopropanone. Under these conditions, the
major products formed are species iv, 45 ± 5%, and species ii,
35 ± 2%; species iii and species i account
for only 15 ± 5 and 5 ± 2%, respectively. All subsequent experiments were performed using FASs that were freed of dithiothreitol using the Centricon procedure.
On treatment with dibromopropanone, neither FAS dimers containing the
C161A mutation in both subunits nor those containing the S2151A in both
subunits formed any of the unique electrophoretic species produced by
the wild-type FAS, as revealed by Pro-Blue protein staining (Fig.
3, compare A and
B). This finding confirmed the original observation of
Stoops and Wakil (28) that only a cysteine and a pantetheine thiol are
involved in the reaction of dibromopropanone with the chicken FAS and
demonstrated that these thiols are located at positions 161 and 2151 in
the rat FAS. Because mutants lacking the thiol at residue 2151 are
still able to react with dibromopropanone at the Cys-161 thiol (see Fig. 2), it is clear that derivatization of FAS at a single site, i.e. without cross-linking, does not alter its
electrophoretic mobility.

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Fig. 3.
SDS-polyacrylamide separation and Western
analysis of products resulting from the treatment of various FAS mutant
heterodimers with dibromopropanone. Three identification methods
were used to identify products of dibromopropanone cross-linking of
different heterodimers formed from C161A, S2151A, and wild-type FAS
subunit: lane 1, Pro-Blue general protein stain; lane
2, anti-FLAG; and lane 3, anti-His antibody binding. The
C161A,S2151A FAS mutant utilized in this study contained two additional
mutations that had been introduced for use in unrelated
experiments: neither the S581A (malonyl/acetyltransferase
domain) nor S2302A (thioesterase domain) replacements, when introduced
as single mutations, had any influence on the formation of cross-linked
FAS species (details not shown), so that, for the purpose of the
cross-linking study, this FAS can be regarded as a double
mutant, C161A, S2151A. The origin of each of the new molecular
species formed by reaction with dibromopropanone is illustrated
schematically in the cartoons adjacent to each of the
electrophoretograms; the "X" indicates replacement of
the key residue with alanine, and the solid bar identifies
residues participating in cross-linking.
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Dibromopropanone-treatment of a heterodimer consisting of one
FLAG-tagged wild-type subunit and one His6-tagged subunit
carrying both the C161A and S2151A mutations yielded only one new
electrophoretic species, as revealed by Pro-Blue staining (Fig.
3C, panel 1). This product, identified as species
ii, the fastest moving of the three species formed by treatment of the
wild-type dimers with dibromopropanone, accounted for about 50% of the
FAS protein present, and the remaining 50% was accounted for as
species i, having unaltered electrophoretic mobility. Western analysis
revealed that species ii contained only the FLAG-tagged
wild-type subunit, and species i contained almost
exclusively the His6-tagged subunit carrying the double
mutation (Fig. 3C, panels 2 and 3).
Because formation of species ii is absolutely dependent on
the integrity of the thiol groups at both Cys-161 and Ser-2151 (Fig.
3B), it follows that species ii must result from
the reaction of dibromopropanone with the thiol group at Cys-161 and
the phosphopantetheine at Ser-2151 within the wild-type subunit. The
companion subunit carrying the double mutation is not modified by the
reagent and migrates in the position of the normal FAS subunit, species i.
Dibromopropanone treatment of a heterodimer consisting of one
FLAG-tagged subunit containing the C161A mutation and a
His6-tagged subunit carrying the S2151A mutation also
resulted in the formation of only one new species, this time species
iii, as revealed by staining with Pro-Blue (Fig.
3D, panel 1). However, in this case, both the
FLAG-tagged (C161A) and His6-tagged (S2151A) subunits were
present in species iii (Fig. 3D, panels
2 and 3). Because the heterodimer containing the C161A
and S2151A mutations on opposite subunits can form neither intrasubunit
cross-links nor double intersubunit cross-links, and because species
iii contains both subunits, it follows that species
iii must be a singly cross-linked dimer.
Dibromopropanone-treatment of a heterodimer consisting of one
FLAG-tagged subunit containing the C161A mutation and one
His6-tagged wild-type subunit yielded both species
ii and iii, but not species iv, as
revealed by Pro-Blue staining (Fig. 3E, panel 1).
Again species ii contained only the wild-type subunit,
whereas species iii contained both the wild-type and the
C161A mutant subunits (Fig. 3E, panels 2 and
3). These results support the earlier inference that species
ii is a cross-linked intrasubunit, whereas species iii is a singly cross-linked dimer.
A similar result was obtained by dibromopropanone treatment of a
heterodimer consisting of one FLAG-tagged wild-type subunit and one
His6-tagged subunit carrying the S2151A mutation. Species ii and iii were formed but not species
iv, as evidenced by staining with Pro-Blue (Fig.
3F, panel 1) and again species ii
contained only the FLAG-tagged wild-type subunit, whereas species
iii contained both the FLAG-tagged wild-type subunit and the
His6-tagged S2151A subunit (Fig. 3F,
panels 2 and 3).
For comparison, the analysis of the products formed by
dibromopropanone-treatment of a wild-type dimer containing
independently tagged subunits was also included in this series of
experiments. All three species formed characteristically as a result of
exposure to dibromopropanone (species ii, iii, and
iv) are in evidence, as revealed by staining with Pro-Blue
(Fig. 3A, panel 1), and all three contain both
wild-type subunits, as revealed by Western blotting (Fig.
3A, panels 2 and 3). In this case,
either of the wild-type subunits is able to form intrasubunit
cross-links so that species ii contains both FLAG-tagged and
His6-tagged subunits. The observation that species
iv, which contains both subunits, is formed only when the
Cys-161 and phosphopantetheine thiols are preserved on both subunits is
consistent with this species representing a doubly cross-linked dimer
in which the Cys-161 thiols of each subunit are cross-linked to the
phosphopantetheine thiols of the opposite subunit.
Estimation of the Molecular Masses of the Cross-linked FAS Species
by Gel Filtration and Mass Spectrometry--
When the products of
dibromopropanone-treatment of the wild-type FAS were subjected to gel
filtration, under nondenaturing conditions, a single protein zone
emerged from the column with an elution time characteristic of the FAS
dimer (details not shown), indicating that no oligomers larger than the
dimer are formed by cross-linking.
The three molecular species produced by dibromopropanone treatment of
the wild-type FAS were isolated from the acrylamide gel by
electroelution, and most of the free SDS was removed using SDS-OutTM (Pierce). However, all attempts to remove the
residual protein-bound SDS, by dialysis against 0.25% octyl
-glucoside or by extraction with organic solvent, produced a largely
insoluble residue that yielded no detectable signal in MALDI-TOF mass
spectrometry. We therefore resorted to determining the masses of the
various cross-linked species by subjecting to mass spectrometry the
entire mixtures of products formed by each of the FAS mutants, without
prior exposure to SDS.
Given that species in the MALDI ion source can generate ions only by
addition of an integral number of charged particles (protons in this
case), one can distinguish between monomers and dimers. FAS dimers (546 kDa) can theoretically give rise to peaks at m/z 546 K (1+), m/z 273 K (2+),
m/z 182 K (3+), m/z
136 K (4+), m/z 109 K (5+),
m/z 91 K (6+), etc., whereas FAS monomers
can give rise to peaks at m/z 273 K (1+),
m/z 136 K (2+), m/z
91 K (3+) etc. Thus peaks representing odd multiply charged states
of the dimer provide a unique signature for the presence of the
cross-linked dimeric form of the protein. None of these species is
produced in any significant amount from the native wild-type FAS,
consistent with there being no covalent links between the subunits
(Fig. 4A).

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Fig. 4.
Estimation of molecular masses of wild type
and dibromopropanone-cross-linked FAS heterodimers. Wild-type FAS
(A) and dibromopropanone-cross-linked wild-type
(B), C161A,S2151A/wild type (C), and C161A/S2151A
(D) heterodimeric preparations were analyzed by MALDI-TOF
mass spectrometry as described under "Experimental
Procedures."
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The conditions under which MALDI-TOF experiments are carried out almost
always result in near complete disruption of any noncovalent associations that may be present. Nevertheless, the trace of
m/z 182 K species detected was reproducible
and persisted after incubation of the FAS with 3 mM
dithiothreitol at 20 °C for 15 min, suggesting that it did not
represent an artifact resulting from disulfide bond formation between
subunits. This phenomenon, termed "nonspecific protein ion
clustering" in MALDI mass spectra has been observed previously2 and reported by
others (29, 30). In contrast, wild-type FAS that had been exposed to
dibromopropanone generated strong peaks at both
m/z 182 K and m/z
109 K species (Fig. 4B), confirming that cross-linking
had occurred between subunits.
The mass spectrum of the dibromopropanone-treated wild-type FAS
exhibits a molecular ion distribution skewed toward the doubly charged
species and the singly charged molecular ion (m/z
546 K, in this case), which is typically found in MALDI-TOF mass
spectra, is missing. The observed molecular ion distribution is a
product of both the propensity of a molecule for acquisition of charges under the conditions of ionization and sensitivity of the detector for
molecular ions at various m/z values. Given, that
there is little data available on MALDI-TOF analysis of proteins larger than 200 kDa, it is not clear at the moment whether the absence of the
singly charged species (m/z 546 K) is
characteristic of large protein molecules, or is an artifact resulting
from detector sensitivity bias.
The dimer signature species, m/z 182 K and
m/z 109 K, were also formed from
heterodimers consisting of one subunit bearing the C161A mutation and
the other bearing the S2151A mutation (Fig. 4D). Because
this heterodimer can only form a single intersubunit cross-link
(species iii, Fig. 3D), the mass spectrometric
data confirm that the electrophoretic species iii (present
at the level of ~35%) is indeed a cross-linked dimer. Heterodimers
consisting of a wild-type subunit paired with a C161A,S2151A doubly
mutated subunit, after dibromopropanone treatment, did not generate
either the m/z 182 K or
m/z 109 K species (Fig. 4C). Thus
the electrophoretic species ii (present at the level of
~30%) derived from this heterodimer (Fig. 3C) cannot be a
dimer and must therefore represent an internally cross-linked subunit.
Because the electrophoretic species i and ii as
well as species iii and iv have identical
molecular masses, 272 and 544 kDa, respectively, it is clear that the
various FAS species exhibit anomalous electrophoretic behavior. The
reason is not known but likely results from differences in SDS-binding
capacities and/or shape of the various species.
Reversal of Cross-linking by Exposure to Elevated pH--
Given
that only the Cys-161 and phosphopantetheine thiols participate in
formation of the cross-linked species iv and that both
thiols must be available on both subunits in order that species iv can be formed, species iv must contain two
cross-links. However, because cross-linking can occur both inter- and
intrasubunit, it is possible that a doubly cross-linked dimer could be
formed by two intrasubunit cross-links that interlock the subunits like two links in a chain. Were this the case, then cleavage of one or both
of the cross-links would generate only single subunit species, and
species ii, the single subunit cross-linked internally, would be expected to be formed as an intermediate. On the other hand,
if species iv is in fact a dimer containing two intersubunit cross-links, then cleavage of one of the cross-links ought to generate
an intermediate corresponding to the singly cross-linked dimer,
identified as species iii. To distinguish between these two
possibilities, we made use of the inadvertent discovery that propanone
cross-links are susceptible to cleavage by mild alkaline treatment.
This treatment has no effect on the integrity of FAS that has not been
exposed to dibromopropanone (Fig. 5,
lanes 2 and 3). Species iv, formed by
treatment of the wild-type FAS with dibromopropanone and isolated by
preparative SDS-polyacrylamide electrophoresis is approximately
70-75% pure (Fig. 5, lane 4). Exposure of species
iv to high pH for 1 and 2 h resulted in the appearance
of increasing amounts of species iii but no species ii (Fig. 5, lanes 5 and 6). This
result ruled out the possibility that cross-linked dimers could be
formed by linking the two subunits through two intrasubunit
cross-links. Thus species iv, which accounts for 45% of the
cross-linked products, results from two intersubunit cross-links, and
species ii, which accounts for 35%, results from a single
intrasubunit cross-link. The singly cross-linked dimer, species
iii, which accounts for 15% of the products may be formed as a consequence of oxidation of a small fraction of the participating thiols at residues 161 and/or 2151, and/or from the presence of a small
amount of apo-ACP in the preparation, and/or reaction of the second
bromine atom with water.

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Fig. 5.
Products of hydrolysis of propanonyl bond in
the isolated cross-linked species iv by exposure to
high pH. Lane 1, dibromopropanone-cross-linked FAS;
lane 2, wild-type FAS; lane 3, wild-type FAS
exposed to high pH for 2 h; lane 4, isolated species
iv; lane 5, isolated species iv
exposed to high pH for 1 h; lane 6, isolated species
iv exposed to high pH for 2 h; and lane 7,
isolated species ii. In this experiment, a 4.25% acrylamide
gel was used, and electrophoresis was terminated when the bromphenol
blue dye reached the bottom of the gel. Further details are given under
"Experimental Procedures."
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These findings provide a simple explanation for the previously
perplexing observation that, whereas cross-linking of the native FAS
yields three unique species, cross-linking of FAS that has been nicked
by trypsin yields only one new species. FAS that has been treated with
trypsin is essentially a "nicked dimer" consisting of pairs of
125-kDa amino-terminal and 95-kDa carboxyl-terminal polypeptides (31);
treatment with dibromopropanone gives only a single molecular species
of 220 kDa (9). Clearly, the species formed by cross-linking between
these two nicked subunits and the species formed by cross-linking of
the 125- and 95-kDa species originating from the same subunit will be indistinguishable.
 |
DISCUSSION |
The two residues implicated by Stoops and Wakil (6, 28) as
participants in the reaction of the chicken FAS with dibromopropanone correspond in the rat FAS to Cys-161, the nucleophile for the
-ketoacyl synthase reaction, and the phosphopantetheine at Ser-2151. When either of these residues is mutated to alanine, functionality of
the targeted domain is compromised (16, 19). In the series of
heterodimers engineered for this study, one or both subunits contained,
at positions 161 and 2151, either the wild-type residues or the C161A
or S2151A mutations, or both. Thus, dimers consisting of a wild-type
subunit paired with either a C161A or S2151A subunit exhibited
approximately half of the fatty acid synthesizing activity characteristic of the wild-type FAS as would be anticipated. On the
other hand, dimers consisting of a C161A mutant subunit paired with a
S2151A mutant subunit and those consisting of a wild-type subunit
paired with a subunit containing both the C161A and S2151A mutations,
exhibited only about 5-6% of the activity of the wild-type FAS (see
"Experimental Procedures"). We have previously noticed that
mutations in the
-ketoacyl synthase domain, particularly at the
active-site cysteine appear to influence catalytic activity at the
remaining functional center (16-18). The reason for these lower than
expected activities is not immediately obvious and is the subject of
current investigation. Nevertheless, it should be emphasized that in
this study the FAS mutants are used solely as tools to identify each of
the three cross-linked species formed by treatment of the wild-type FAS
with dibromopropanone. Quantitative assessments of the proportions of
the three species formed by the wild-type FAS can be made independently
of any consideration of the specific activities of the various mutant FASs.
The classical model for the FAS that has prevailed since the early
1980s visualized the two subunits as lying side-by-side in a rigid,
extended, antiparallel configuration. The first indication that this
model might be inadequate came from a mutant complementation analysis
that was designed to map the functional interactions occurring between
domains of the FAS dimer. Several lines of evidence presented in this
report support the hypothesis that, in the wild-type FAS dimer, the
active-site cysteine residue of the
-ketoacyl synthase domains are
positioned close to the phosphopantetheine thiols of both subunits: 1)
species ii has a unique electrophoretic mobility that
distinguishes it from unmodified subunits, subunits that are
derivatized at only the Cys-161 site, and intersubunit cross-linked
species; 2) species ii has a molecular mass consistent with
that of a single subunit; and 3) species ii is formed only from a wild-type subunit; the absence of either of the thiols at
residues 161 or 2151 precludes its formation.
This inference is entirely consistent with the conclusions drawn from
mutant complementation analysis that mapped the functional interactions
occurring between domains of the FAS dimer. These experiments indicated
that functional contacts could be made between the
-ketoacyl
synthase and ACP domains associated with the same or the companion
subunits. Thus reinterpretation of the specificity of the reaction of
the FAS with dibromopropanone provides the first structural evidence in
support of conclusions derived from the mutant complementation
analysis. Based on the relative proportion of the FAS species that
undergo inter- and intrasubunit cross-linking, one can conclude that in
the resting wild-type FAS, as much as 35% of the dimers adopt a
conformation in which the active site cysteine residue Cys-161 is
juxtaposed with the phosphopantetheine moiety attached to residue
Ser-2151 of the same subunit. Thus the old model for the FAS (1-3)
must be revised to accommodate the finding that head-to-tail structural
and functional contacts are possible both between and within subunits.
This new model implies a much greater flexibility in the interaction of
domains within and between subunits than was implied by the earlier
side-by-side, head-to-tail model.