Mechanism of inhibition of aliphatic epoxide carboxylation by the coenzyme M analog 2-bromoethanesulfonate

Article abstract of DOI:10.1074/jbc.M110.144410

The bacterial metabolism of epoxypropane formed from propylene oxidation uses the atypical cofactor coenzyme M (CoM, 2-mercaptoethanesulfonate) as the nucleophile for epoxide ring opening and as a carrier of intermediates that undergo dehydrogenation, reductive cleavage, and carboxylation to form acetoacetate in a three-step metabolic pathway. 2-Ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of this pathway, is the only known member of the disulfide oxidoreductase family of enzymes that is a carboxylase. In the present work, the CoM analog 2-bromoethanesulfonate (BES) is shown to be a reversible inhibitor of 2-KPCC and hydroxypropyl-CoM dehydrogenase but not of epoxyalkane:CoM transferase. Further investigations revealed that BES is a time-dependent inactivator of dithiothreitol-reduced 2-KPCC, where the redox active cysteines are in the free thiol forms. BES did not inactivate air-oxidized 2-KPCC, where the redox active cysteine pair is in the disulfide form. The inactivation of 2-KPCC exhibited saturation kinetics, and CoM slowed the rate of inactivation. Mass spectral analysis demonstrated that BES inactivation of reduced 2-KPCC occurs with covalent modification of the interchange thiol (Cys⁸²) by a group with a molecular mass identical to that of ethylsulfonate. The flavin thiol Cys⁸⁷ was not alkylated by BES under reducing conditions, and no amino acid residues were modified by BES in the oxidized enzyme. The UV-visible spectrum of BES-modifed 2-KPCC showed the characteristic charge transfer absorbance expected with alkylation at Cys⁸². These results identify BES as a reactive CoM analog that specifically alkylates the interchange thiol that facilitates thioether bond cleavage and enolacetone formation during catalysis.

Full text of DOI:10.1074/jbc.M110.144410

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 33, pp. 25232–25242, August 13, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Mechanism of Inhibition of Aliphatic Epoxide Carboxylation
by the Coenzyme M Analog 2-Bromoethanesulfonate*
Received for publication, May 12, 2010, and in revised form, June 14, 2010 Published, JBC Papers in Press, June 15, 2010, DOI 10.1074/jbc.M110.144410
Jeffrey M. Boyd‡1, Daniel D. Clark§, Melissa A. Kofoed‡, and Scott A. Ensign‡2
From the ‡Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300 and the §Department of
Chemistry, California State University, Chico, California 95929
The bacterial metabolism of epoxypropane formed from pro-
pylene oxidation uses the atypical cofactor coenzyme M (CoM,
2-mercaptoethanesulfonate) as the nucleophile for epoxide ring
opening and as a carrier of intermediates that undergo dehydro-
genation, reductive cleavage, and carboxylation to form aceto-
acetate in a three-step metabolic pathway. 2-Ketopropyl-CoM
carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of
this pathway, is the only known member of the disulfide oxi-
doreductase family of enzymes that is a carboxylase. In the pres-
ent work, the CoM analog 2-bromoethanesulfonate (BES) is
shown to be a reversible inhibitor of 2-KPCC and hydroxypro-
pyl-CoM dehydrogenase but not of epoxyalkane:CoM transfer-
ase. Further investigations revealed that BES is a time-depen-
dent inactivator of dithiothreitol-reduced 2-KPCC, where the
redox active cysteines are in the free thiol forms. BES did not
inactivate air-oxidized 2-KPCC, where the redox active cysteine
pair is in the disulfide form. The inactivation of 2-KPCC exhib-
ited saturation kinetics, and CoM slowed the rate of inactiva-
tion. Mass spectral analysis demonstrated that BES inactivation
of reduced 2-KPCC occurs with covalent modification of the
interchange thiol (Cys82) by a group with a molecular mass iden-
tical to that of ethylsulfonate. The flavin thiol Cys87 was not
alkylated by BES under reducing conditions, and no amino acid
residues were modified by BES in the oxidized enzyme. The UV-
visible spectrum of BES-modifed 2-KPCC showed the charac-
teristic charge transfer absorbance expected with alkylation at
Cys82. These results identify BES as a reactive CoM analog that
specifically alkylates the interchange thiol that facilitates
thioether bond cleavage and enolacetone formation during
catalysis.
undergo dehydrogenation, reductive cleavage, and carboxyla-
tion (Fig. 1) (1–3). Epoxide catabolism from (R)- and (S)-ep-
oxypropane converges at the intermediate 2-ketopropyl-CoM
(2-KPC), which is a substrate for 2-ketopropyl-CoM carboxyl-
ase/oxidoreductase (2-KPCC), a unique member of the disul-
fide oxidoreductase (DSOR) family of enzymes (3–5).
Like other members of the DSOR family, 2-KPCC uses
NADPH as the reductant to reduce a bound flavin. The bound
flavin in turn reduces a disulfide bond to free thiols designated
as the flavin thiol and the interchange thiol (Fig. 2A) (4–6). For
the canonical members of the DSOR family such as glutathione
reductase and dihydrolipoamide dehydrogenase, the inter-
change thiol facilitates substrate disulfide bond reduction to the
free thiols by forming a mixed disulfide between the inter-
change thiol and a substrate molecule, followed by reduction
and release of the bound substrate with reformation of the
cysteine disulfide (7). Unlike these prototype enzymes,
2-KPCC uses the redox active cysteine pair to facilitate
thioether rather than disulfide bond cleavage, forming a
mixed disulfide of the interchange thiol and CoM along with
enolacetone, which undergoes carboxylation to form aceto-
acetate (Fig. 2C) (4–6). 2-KPCC is the only known DSOR
enzyme to catalyze thioether bond cleavage and substrate
carboxylation and thus is of general interest as a new class of
dual function oxidoreductase/ligase.
Although kinetic, mechanistic, and structural studies of
2-KPCC have revealed important details of the reaction mech-
anism, much remains to be learned about how this enzyme
operates. Central to the mechanism of this enzyme are the
unique properties of CoM, the simplest known organic cofac-
tor, consisting of sulfonate and thiol functional groups sep-
arated by an ethyl linker (Fig. 1). Aside from epoxide carbox-
ylation, the only known function of CoM is in archaeal
methanogenesis, where CoM carries the methyl group that is
subsequently reduced to methane by the nickel-containing
enzyme methyl-CoM reductase (8–11).
The bacterial metabolism of epoxides formed from short-
chain aliphatic alkene epoxidation occurs by a three-step
metabolic pathway that uses the atypical cofactor coenzyme
M (CoM, 2-mercaptoethanesulfonate)3 as a nucleophile for
epoxide ring opening and as a carrier of intermediates that
The CoM analog 2-bromoethanesulfonate (BES) was identi-
fied as a potent inhibitor of methanogenesis (12–14), and found
to specifically inactivate MCR by binding as a CoM analog and
rendering the enzyme inactive by oxidizing the nickel tetrapyr-
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant GM51805.
1 Present address: Inflammation Program, Depts. of Internal Medicine and role cofactor from the ϩ1 to ϩ2 oxidation state (15). Based on
Microbiology, Roy and Lucille Carver College of Medicine, University of
these studies of BES inhibition of methanogens and MCR, BES
Iowa, Iowa City, IA 53341.
was studied as a possible inhibitor of bacterial alkene metabo-
2 To whom correspondence should be addressed. Tel.: 435-797-3969; Fax:
435-797-3390; E-mail: scott.ensign@usu.edu.
lism and found to specifically inhibit growth of Xanthobacter
3 The abbreviations used are: CoM, coenzyme M (2-mercaptoethanesul-
fonate); 2-KPC, 2-ketopropyl-CoM; 2-KPCC, 2-ketopropyl-CoM carbox-
ylase/oxidoreductase; DSOR, disulfide oxidoreductase; BES, 2-bromo-
ethanesulfonate; EaCoMT, epoxyalkane:CoM transferase; R-HPCDH,
R-hydroxypropyl-CoM dehydrogenase; ␤-HBDH, ␤-hydroxybutyrate
dehydrogenase; R-HPC, R-hydroxypropyl-CoM; ES, ethylsulfonate.
25232 JOURNAL OF BIOLOGICAL CHEMISTRY
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BES Inhibition of CoM-dependent Epoxide Metabolism
autotrophicus Py2 and Rhodococcus rhodochrous B276 with mMϪ1 cmϪ1) was monitored using a Shimadzu UV-2401 spec-
propylene, but not with carbon sources that do not use the trophotometer containing a water-jacketed cell holder main-
CoM-dependent pathway and enzymes of Fig. 1 (16). Treat- tained at 30 °C. Rates were determined from the linear portions
ment of propylene-grown X. autotrophicus with BES resulted in of progress curves (typically within the first 60 s of assays).
loss of epoxide carboxylation activity that could only be
Coupled Spectrophotometric Assay of 2-KPCC-catalyzed 2-KPC
restored by new protein synthesis. The addition of purified Carboxylation—A continuous spectrophotometric assay was
2-KPCC restored epoxide carboxylase activity in cell extracts developed that couples acetoacetate production by 2-KPCC to
inactivated with BES, suggesting that 2-KPCC is the target of acetoacetate reduction and NADH oxidation by ␤-HBDH. Assays
BES inactivation (16).
were conducted in 2-ml anaerobic quartz cuvettes that contained
BES is the first specific irreversible inactivator of epoxide 1 ml of assay components. Buffer and stock solutions were made
carboxylation to be identified, and as such the molecular basis anoxic by sparging or by repeated evacuation and flushing with N2
of inhibition and inactivation is of significant interest. In the on a vacuum manifold, except for potassium bicarbonate solu-
present work, BES is shown to be a reversible rapid equilibrium tions, which were prepared by adding anoxic water to a degassed
inhibitor of 2-hydroyxpropyl-CoM dehydrogenase and 2-KPCC, vial containing KHCO3 solid. Assay components were added from
and a time-dependent irreversible inactivator of 2-KPCC. Inacti- stock solutions to sealed cuvettes as described above. The concen-
vation of 2-KPCC results from the highly specific alkylation of the trations of assay components in the cuvettes were 50 mM Tris
interchange thiol of 2-KPCC, identifying BES as the first affinity buffer, pH7.4, 10mM DTT, 0.2mM NADH, 40mM KHCO3,10mM
label and active site probe of this unusual member of the DSOR CO2, 0–10 mM BES, 0.345 mg of ␤-HBDH (90 units of enzyme
family of enzymes.
activity), varying concentrations of 2-KPC (0 to 0.70 mM), and
2-KPCC (0.05–0.1 mg). The amount of ␤-HBDH included in
assays was severalfold higher than that required for saturation of
EXPERIMENTAL PROCEDURES
Materials—All solid chemicals, solvents, and endoprotein- rates. The cuvettes were equilibrated at 30 °C prior to initiating
ases used for liquid chromatography-mass spectrometry (LC- assays by the addition of 2-KPCC. Rates were determined from the
MS) studies were obtained from Sigma and were LC-MS grade linear portions of progress curves (typically 20–120 s). The change
or the next highest purity available. HPLC resins were obtained in absorbance at 340 nm over time was used to calculate rates of
from Agilent (Santa Clara, CA).
␤-hydroxybutyrate production using the extinction coefficient for
Bacterial Cell Growth and Enzyme Purification—X. autotro- NADH (6.22 mMϪ1 cmϪ1).
phicus strain Py2 and Escherichia coli strains containing ex-
Assay of 2-KPCC-catalyzed 2-KPC Protonation—The
pression vectors were cultured as described previously (17–20). 2-KPCC-catalyzed formation of acetone from 2-KPC was
Recombinant epoxyalkane:CoM transferase (EaCoMT), recombi- determined by gas chromatography using the assay procedure
nant R-hydroxypropyl-CoM dehydrogenase (R-HPCDH), and described previously (4). Buffer and stock solutions were made
native 2-KPCC were purified to homogeneity (Ͼ95% pure by SDS- anoxic and depleted of residual CO2 by sparging and/or
PAGE analysis) as described previously (17, 19, 20). Recombinant repeated evacuation and flushing with N2 that had been passed
␤-hydroxybutyrate dehydrogenase (␤-HBDH) from Rhodobacter through a column of Ascarite II (Thomas Scientific, Swedes-
sp. DSMZ 12077 (21) was expressed in E. coli and purified as boro, NJ). Assays were conducted in a 1-ml total assay volume
described previously (18).
in 9-ml serum vials containing 50 mM Tris buffer, pH 7.4, 5 mM
EaCoMT Activity Assay—The activity of EaCoMT was deter- NADPH, 3 mM 2-KPC, and 0.2 mg of 2-KPCC. The assays were
mined by monitoring the depletion of epoxypropane from the initiated by the addition of 2-KPC.
head space of sealed assay vials by FID gas chromatography as
Analysis of Initial Velocity Data—Initial velocities were plot-
described previously (20). Assays were conducted in a 1-ml ted versus substrate concentration and fit to the standard form
total assay volume in 9-ml serum vials containing 50 mM Tris of the Michaelis-Menten equation as described by Cleland (22)
buffer, pH 7.4, 5 mM CoM, and 0.03 mg of EaCoMT. BES (5 mM) using the software SigmaPlot 11. For analysis of enzyme inhibi-
was included as indicated. The assays were initiated by the addi- tion, data were fit to the following form of the Michaelis-Men-
tion of epoxypropane (4 mM) from a 500 mM stock solution ten equation, which describes the effect of a noncompetitive
prepared in H2O.
(mixed) inhibitor (22).
R-HPCDH Assay—The activity of R-HPCDH was measured
with R-hydroxypropyl-CoM (R-HPC) as substrate by monitor-
␷ ϭ Vmax͓S͔/͑Km͑1 ϩ ͓I͔/Kis͒ ϩ ͓S͔͑1 ϩ ͓I͔/Kii͒͒
(Eq. 1)
ϩ
ing the reduction of NAD spectrophotometrically (19). Assays
were conducted in 2-ml anaerobic quartz cuvettes that con-
Removal of BES from Aqueous Solutions with AG1-X8 Ion-
tained 1 ml of assay components. Buffer and stock solutions exchange Resin—AG1-X8 (Bio-Rad, chloride form, mesh size
were made anoxic by sparging or by repeated evacuation and 100–200) resin was rehydrated in 10 volumes of 50 mM Tris
flushing with N2 on a vacuum manifold. Assay components buffer, pH 7.4, and filtered under vacuum through 7-cm diam-
were added from stock solutions to sealed cuvettes (2-ml) that eter Whatman No. 1 filter paper. This process was repeated 3
had been sparged with nitrogen using gas-tight syringes. Assays times before the resin was finally vacuum dried for 10 min. To
ϩ
contained 50 mM Tris buffer, pH 7.4, 10 mM NAD , 1 mM assess the ability of AG1-X8 resin to quantitatively remove BES
EDTA, and varying concentrations of R-HPC (23.3–1000 ␮M) from solutions, BES (150 ␮l of a 125 mM stock solution) was
and BES (0–7.5 mM). Assays were initiated by the addition of added to 15 mg of dried AG1-X8 resin in a microcentrifuge tube
enzyme (1.4 ␮g of R-HPCDH). NADH formation (⑀340 ϭ 6.22 followed by incubation for 3 min at room temperature. The
AUGUST 13, 2010•VOLUME 285•NUMBER 33
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BES Inhibition of CoM-dependent Epoxide Metabolism
sample was briefly spun in a microcentrifuge (5 s pulse) to pellet recording UV-visible spectra on a Shimadzu UV-2401PC spec-
the resin. Portions of the AG-1X8-treated BES solution (0–25 trophotometer. The concentrations of samples were normal-
␮l) were then transferred to assay cuvettes to assess their effects ized by extracting and quantifying FAD using sodium dodecyl
on R-HPCDH and 2-KPC assays, conducted as described above. sulfate as described by Aliverti and co-workers (24).
No inhibition of 2-KPCC or R-HPCDH activity was observed,
Preparation of Acrylamide Protein Plugs for LC-MS Analysis—
demonstrating that the AG1-X8 resin removed a sufficient 2-KPCC (30.3 mg/ml stock) was diluted with 100 mM Tris, pH
amount of BES to prevent rapid equilibrium inhibition effects 7.4, to a concentration of 50 ng/␮l. Three 1-ml samples of the
in 2-KPCC and R-HPCDH assays.
diluted 2-KPC were prepared in sealed 4-ml serum vials as fol-
Assay of Time-dependent Inactivation of 2-KPCC by BES— lows: the first sample was prepared aerobically and contained
Samples of 2-KPCC (2.28 mg) were incubated anoxically at no additional components, the second sample was prepared
30 °C in 3-ml serum vials containing a total volume of 1.25 ml of aerobically and contained 5 mM BES, and the third sample was
buffer (50 mM Tris, pH 7.4) either containing or lacking DTT prepared anaerobically and contained 10 mM DTT and 5 mM
(10 mM) and BES (0–10 mM). Incubations were initiated by the BES. This sample was reduced with DTT before the addition of
addition of BES from a stock solution. At the desired time BES. All samples were allowed to incubate at room temperature
points, 0.15-ml samples were removed with gas-tight syringes, for 6.5 h. After incubation, the samples were transferred to
transferred to 1.5-ml microcentrifuge tubes containing 15 mg 1.5-ml microcentrifuge tubes containing 15 mg of AG1-X8
of AG1-X8 ion-exchange resin to remove residual BES, and resin. The resin was pelleted and the enzyme solution was then
then incubated for 3 min and pulse-centrifuged as described decanted away from the resin and prepared for SDS-PAGE.
above. Fifty-␮l samples were then removed from the superna-
Four ␮l of the 50 ng/␮l solution was diluted to 14 ␮l with 2ϫ
tant and used as the source of 2-KPCC for coupled spectropho- sample loading mixture. These samples were boiled and loaded
tometric assays of 2-KPCC activity, conducted as described onto a 12% acrylamide gel that had been poured 24 h in advance
above. 2-KPCC activity assays were conducted in duplicate for and allowed to sit at room temperature. Individual bands, con-
each incubation time point. The apparent first-order rate con- taining 200 ng of protein, were cut into 4 equal slices and placed
stants (kobs) for each individual BES concentration were deter- into 1.5-ml microcentrifuge tubes. Two hundred ␮l of neat
mined by fitting the data to Equation 2,
HPLC grade acetonitrile was added to the tubes and the protein
samples were incubated at room temperature for 5 min. Aceto-
nitrile was decanted from the dehydrated SDS-PAGE gel slices,
and the samples were dried in a SpeedVac concentrator at room
temperature.
␷/␷0 ϫ 100 ϭ exp͑Ϫkobs ϫ t͒
(Eq. 2)
where ␷ is the observed velocity at any given time, ␷ is the
0
velocity at incubation t ϭ 0 (␷/␷ is the fractional velocity
0
Preparation of Liquid 2-KPCC Samples for LC-MS Analysis—
2-KPCC (30.3 mg/ml) was diluted with 100 mM Tris buffer,
pH 7.4, to a concentration of 2 mg/ml. Three samples (0.15
ml each) were prepared aerobically with 5 mM BES, and
anaerobically with 10 mM DTT and 5 mM BES as described
above. The samples were incubated for 4 h at room temper-
ature before the addition of 15 mg of AG1-X8 resin. The
resin was pelleted, and the samples were decanted and
desalted by passing them over a 1 ϫ 5-cm column of Seph-
remaining after a given pre-incubation), and t is time in min.
The maximal rate of enzyme inactivation (kinact) and the con-
centration of inhibitor required to reach the half-maximal rate
of inactivation (Ki) were determined by fitting the kobs values
for individual BES concentrations ([I]) to Equation 3 (23) using
the software SigmaPlot.
k
obs ϭ kinact͓I͔/͑Ki ϩ ͓I͔͒
(Eq. 3)
Assay of Time-dependent Inactivation of R-HPCDH by BES— adex G-25 (PD-10 Amersham Biosciences), which had pre-
Samples of R-HPCDH (0.273 mg) were tested for possible time- viously been equilibrated with 10 mM NH4HCO3. The sam-
dependent inactivation by BES, essentially as described above ples were eluted with 10 mM NH4HCO3 (volume ϳ3 ml) and
for 2-KPCC, with the following modifications. The total vol- placed in a spin column concentrator (Centricon YM 30,
ume of the assay mixture was reduced to 0.15 ml, and the entire Amicon), which had been previously washed with 10 mM
reaction mixture was removed and subjected to AG1-X8 treat- NH4HCO3. The samples were concentrated to 0.15 ml and
ment after a 200-min incubation time. Fifty ␮l of the AG-1X8- flash frozen in liquid N2.
treated sample was then diluted 8-fold by the addition of buffer.
Instrumentation Used for Mass Spectral Sample Analysis—
Five ␮l of the diluted enzyme sample was used for assaying All samples were analyzed with an Agilent 1100 HPLC con-
R-HPCDH activity using 1 mM R-HPC as substrate as described nected to a ThermoFinnigan LCQ Advantage ion-trap mass
above.
spectrometer equipped with a nanospray ion source. Samples
UV-Visible Spectral Analysis of 2-KPCC—Samples of 2-KPCC were loaded onto nanospray columns either offline via a pres-
(1.8 mg/ml) were incubated anoxically as described above in the sure bomb (ProXeon), or online via the autosampler with the
presence of 10 mM DTT and with or without 10 mM BES. After aid of an inline C8 sample trap (Michrom Bioresources micro-
a 4-h incubation, the sample with BES had lost 98% activity cap trap) fitted to the LCQ divert valve. All columns were
based on activity assays conducted as described above. At that sprayed at 250 nl/min with the spray voltage set at 2.2 kV and
time, the samples were desalted using prepacked columns of the heated capillary set at 200 °C.
Sephadex G-25 (Pharmacia, PD-10) equilibrated in 100 mM
Determination of BES Modification Stoichiometry by Mass
lysine buffer, pH 11, containing 100 mM NaCl and 0.1 mM Spectrometry—2-KPCC was diluted 1:100 into 2% (v/v) aceto-
EDTA. Desalted samples were air oxidized for 30 min before nitrile containing 0.1% formic acid (Buffer A). Approximately
25234 JOURNAL OF BIOLOGICAL CHEMISTRY
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BES Inhibition of CoM-dependent Epoxide Metabolism
50 ng was loaded offline onto a 75-␮m inner diameter ϫ
10-cm PicoFrit column (New Objective) packed with Zorbax
300SB-C8 (Agilent). The column was developed with a 10-min
hold of buffer A, followed by a 90-min linear gradient from
0–90% buffer B (neat acetonitrile, 0.1% formic acid), and a
20-min hold at 90% buffer B. Native molecular mass was deter-
mined by averaging all MS spectra (across a width of one-half of
the peak height) for a 2-KPCC polypeptide. The charge enve-
lope was deconvoluted using both ProMass Deconvolution 2.0
and Bioworks 3.1 algorithms to give two separate average mass
values for 2-KPCC.
In-Gel Digestion of 2-KPCC—Two hundred nanograms of
2-KPCC, for each condition, was run in triplicate on 12%
SDS-PAGE gels. Gels were stained with Coomassie Blue and
destained using standard protocols. Each protein band was
excised, cut into four pieces, and placed into a microcentri-
fuge tube. To each tube, 200 ␮l of neat acetonitrile was
added. After 5 min acetonitrile was discarded and samples
were dried in a SpeedVac for storage at 4 °C until processing.
Gel samples were processed using a standard in-gel digestion
procedure incorporating tris(2-carboxyethyl)phosphine
reduction and cysteine alkylation with iodoacetamide. To
achieve sequence coverage of all six 2-KPCC cysteine residues,
three parallel digests were performed for each condition. To the
processed and dried gel cores, proteases were added from
stocks in digestion buffer (50 mM NH4HCO3, 5% (v/v) acetoni-
trile) as follows: trypsin (600 ng), trypsin ϩ Pro-C (300 ng each),
and Glu-C ϩ Lys-C (300 ng each). Gel cores were allowed to
swell with the added protease(s) for 15 min, followed by addi-
tion of 25 ␮l of digestion buffer, and incubation at 37 °C for ϳ18
h. Peptides were extracted by shaking with 100 ␮l of 5% (v/v)
formic acid for 30 min at 30 °C. Samples were then microcen-
trifuged at 14,000 ϫ g for 2 min. Twenty-five microliters of the
supernatant from the trypsin digest was loaded on-line, with 5
␮l of the other digests loaded off-line; in an attempt to capture
less hydrophobic peptides that could have been lost by use of
the C8 sample trap.
FIGURE 1. Pathway of bacterial propylene metabolism.
Mapping Location of BES Modification—All protein and pep-
tide identifications were made using SEQUEST as part of Bio-
works 3.1. The protein data base used for searching MS/MS
spectra contained five protein sequences: 2-KPCC (Q56839),
Lys-C (Q7M135), Glu-C (1814271A), porcine trypsin (P00761),
and endoproteinase Pro-C (P27195) nested within 100 random
decoy sequences. The generation of random decoys was accom-
plished using ExPASy RandSeq using the amino acid sequence
of 2-KPCC. Using SEQUEST, the criteria for DTA file genera-
tion was set as follows: group scan ϭ 20, group count ϭ 2,
minimum ion count ϭ 20. All other parameters were default.
Peptide hits to the data base were filtered by charge state and
RESULTS
Development of a Continuous Spectrophotometric Assay for
Measurement of 2-KPCC Carboxylation—The physiologically
important reaction for 2-KPCC is the carboxylation of 2-KPC
using NADPH as reductant as shown in Fig. 1 and summarized
in Equation 4.
2-KPC ϩ CO2 ϩ NADPH 3 acetoacetate ϩ NADPϩ ϩ CoM
(Eq. 4)
In the absence of CO2, 2-KPCC catalyzes the protonation of
2-KPC to form acetone in a fortuitous reaction that occurs at a
Xcorr score (ϩ1, Ն3.0; ϩ2 Ն 4.0; ϩ3, Ն 5.0). Additionally, pep- rate that is about 25% of the rate of 2-KPC carboxylation,
tide identifications were required to have a dCN score of Ն0.1.
Six differential modifications were used in the data base
searches and included oxidized methionine, iodoacetamide-
modified cysteine, acrylamide-modified cysteine, bromoeth-
anesulfonate-modified cysteine, and oxidized bromoethanesul-
fonate-modified cysteine.
2-KPC ϩ Hϩ ϩ NADPH 3 acetone ϩ NADPϩ ϩ CoM (Eq. 5)
Both of these reactions have been exploited in previous studies
of 2-KPCC to measure enzyme activity. For the carboxylation
assay, activity was determined by measuring the incorporation
of radiolabeled 14CO2 into acetoacetate, whereas for the proto-
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BES Inhibition of CoM-dependent Epoxide Metabolism
the need for NADPH and the inter-
mediacy of FADH2 (see Fig. 2, A and
B). In the presence of DTT the car-
boxylation of 2-KPC thus occurs as
shown in Equation 6.
2-KPC ϩ CO2 ϩ DTTred
3
acetoacetate ϩ DTTox ϩ CoM
(Eq. 6)
For the development of the coupled
assay, excess ␤-HBDH and NADH
were included in the assay to
remove the acetoacetate, in the
ϩ
process forming NAD as shown in
Equation 7.
Acetoacetate ϩ NADH ϩ Hϩ
3
␤-hydroxybutyrate ϩ NADϩ
(Eq. 7)
The success of this coupled assay
relies on the fact that 2-KPCC does
not use NADH as a reductant (i.e.
the enzyme is specific for NADPH,
or the alternate reductant DTT).
␤-HBDH, when present in large
excess, efficiently removes acetoac-
etate at an NADH concentration of
FIGURE2. Reactionscatalyzedby2-KPCC. A, reductionoftheredoxactivedisulfidebyNADPH. B, reductionof
the redox active disulfide by DTT. C, mechanism of 2-KPC carboxylation. D, mechanism of inactivation of
2-KPCC by BES. The reduced form of the flavin thiol is shown as the thiolate to highlight the charge transfer,
with increased absorbance at 540 nm, resulting from interaction with FAD. To experimentally observe maximal
charge transfer in BES-modified 2-KPCC it was necessary to increase the pH from a value of 7.4 to 11.0.
ϩ
0.2 mM, allowing NAD formation
to be measured spectrophotometri-
cally according to Equation 7.
TABLE 1
␤-HBDH has also been successfully used as a coupling enzyme
for measuring the activity of acetone carboxylase, which cata-
lyzes the ATP-dependent carboxylation of acetone to form ace-
toacetate (18).
BES inhibition of enzymes involved in (R)-epoxypropane metabolism
Enzyme assays were performed as described under “Experimental Procedures.”
Activities are percentages relative to assays conducted in the absence of BES Ϯ
S.E. The concentrations of substrate in each assay were as follows: (R)-ep-
oxypropane, 4 mM; R-HPC, 4 mM; and 2-KPC, 3 mM. Assays are the averages of
triplicate measurements.
Using the newly developed coupled enzyme assay, the
observed rates of NADH oxidation were linear over the course
of the assays and directly proportional to the amount of
2-KPCC present in the assays. The rates at near saturating con-
centrations of 2-KPC were the same as in the discontinuous
assay with DTT as reductant, which are about 60% of the dis-
continuous rates when NADPH is used as reductant.
Activity in presence
Enzyme
of 5 mM BES
%
EaCoMT
R-HPCDH
2-KPCC
100
32.7 Ϯ 3.6
61.2 Ϯ 4.3
BES Inhibition of the Enzymes of (R)-Epoxypropane
Metabolism—BES was tested as a possible inhibitor of the three
individual enzymes that together carboxylate the (R)-isomer of
epoxypropane to acetoacetate (Fig. 1). As shown in Table 1,
when present at a concentration comparable with that of the
substrate, BES was an inhibitor of both R-HPCDH (70% inhi-
bition) and 2-KPCC (40% inhibition) but not of EaCoMT.
For both R-HPCDH and 2-KPCC, which were assayed using
continuous spectrophotometric assays over short time
frames (20–120 s), the progress curves for product forma-
tion were largely linear in the absence and presence of BES,
demonstrating that the inhibition is largely rapid equilib-
nation assay, activity was determined by measuring acetone
production by gas chromatography. Both of these assays are
discontinuous, making them laborious for the kinetic analyses
necessary for the studies described below. 2-KPCC activity can
be measured by monitoring the loss of absorbance from oxida-
tion of NADPH, but this requires observable changes in
NADPH concentration, resulting in changes of rate during the
assay time courses. It was thus of interest to develop a contin-
uous spectrophotometric assay for 2-KPCC where reductant
could be present at a saturating level throughout the course of
the assay. To achieve this, we relied on the fact that DTT can
serve as an alternative reductant for 2-KPCC by directly reduc- rium over these short time courses. For 2-KPCC there was a
ing the redox active disulfide required for catalysis, bypassing small amount of time-dependent loss of activity (loss of pro-
25236 JOURNAL OF BIOLOGICAL CHEMISTRY
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BES Inhibition of CoM-dependent Epoxide Metabolism
FIGURE 4. Noncompetitive rapid equilibrium inhibition of 2-KPCC-cata-
lyzed 2-KPC oxidation by BES. Assays of 2-KPC carboxylation were per-
formed as described under “Experimental Procedures.” The double-recipro-
cal plots for assays performed in the presence of different concentrations of
BES are shown. Data points represent the average of duplicate experiments.
The solid lines were generated by nonlinear least-square fits of the v versus S
data to the equation for a rectangular hyperbola using Sigmaplot. Symbols:
F, 0 mM BES; f, 2.5 mM BES; Œ, 5 mM BES; ꢀ, 7.5 mM BES.
FIGURE 3. Noncompetitive rapid-equilibrium inhibition of R-HPCDH-cat-
alyzed R-HPC oxidation by BES. Assays of R-HPC oxidation were performed
as described under “Experimental Procedures.” The double-reciprocal plots
for assays performed in the presence of different concentrations of BES are
shown. Data points represent the average of duplicate experiments. The solid
lines were generated by nonlinear least-square fits of the v versus S data to the
equation for a rectangular hyperbola using Sigmaplot. Symbols: F, 0 mM BES;
f, 2 mM BES; Œ, 3 mM BES; ࡗ, 5 mM BES; ꢀ, 7 mM BES.
gress curve linearity) that was more pronounced when assays then removed by microcentrifugation. This procedure was
were allowed to proceed for longer time periods (more than found to rapidly and quantitatively remove all non-protein-
2 min), suggesting that 2-KPCC might be subject to time-de- bound BES from samples. For R-HPCDH, preincubation of the
pendent inactivation as well as rapid equilibrium inhibition. enzyme with BES for 200 min resulted in no loss of enzymatic
It should be noted that BES did not inhibit the activity of the activity once BES was removed, demonstrating that BES is not
coupling enzyme ␤-HBDH when assayed directly with ace- an irreversible inactivator.
toacetate as the substrate, demonstrating that the inhibition
is directed toward 2-KPCC.
Kinetic Characterization of Rapid Equilibrium Inhibition of
2-KPCC by BES—As noted above, the progress curves for
Kinetic Characterization of BES Inhibition of R-HPCDH—A 2-KPCC were largely linear over short time courses in the
kinetic analysis of the inhibition of R-HPCDH by BES was con- absence or presence of 5 mM BES, demonstrating that there is a
ducted by varying the concentrations of R-HPC and BES at a rapid equilibrium component to the inhibition. As shown in
ϩ
fixed, saturating concentration of NAD (10 mM). As shown in Fig. 4, for these short time courses BES behaved as a mixed
Fig. 3, BES behaved as a mixed (noncompetitive) inhibitor of inhibitor of 2-KPC carboxylation at a fixed saturating concen-
R-HPC oxidation with a fit of the experimental data providing tration of DTT and CO2, providing the following kinetic
the following kinetic parameters: kcat ϭ 29.0 Ϯ 0.6 sϪ1, Km ϭ parameters: kcat ϭ 12.7 Ϯ 0.3 minϪ1, Km ϭ 35.0 Ϯ 3.7 ␮M, Kis ϭ
124 Ϯ 10 ␮M, Kis ϭ 1,710 Ϯ 280 ␮M, and Kii ϭ 2,300 Ϯ 200 ␮M. 2980 Ϯ 710 ␮M, and Kii ϭ 4460 Ϯ 330 ␮M.
By comparison, CoM and ethanethiol (a CoM analog lacking
Time- and Concentration-dependent Inactivation of 2-KPCC
the sulfonate group) were shown in a previous study to be by BES—A previous study showed that the addition of BES to
mixed inhibitors of R-HPC oxidation by R-HPCDH as well (19). cell extracts of X. autotrophicus resulted in an irreversible
The inhibition constants for CoM were very similar to those loss of epoxide carboxylation activity that could only be
determined here for BES (Kis ϭ 2,250 ␮M; Kii ϭ 3,620 ␮M), restored by adding purified active 2-KPCC back to the cell
whereas the inhibition constants for ethanethiol were ϳ10-fold extracts, suggesting that 2-KPCC is irreversibly inactivated
higher (Kis ϭ 26,700 ␮M; Kii ϭ 38,900 ␮M) (19).
by BES (16). To investigate this in more detail, 2-KPCC was
To determine whether R-HPCDH was subjected to time-de- incubated anoxically in the presence of DTT with various
pendent inactivation by BES under non-turnover conditions, concentrations of BES, and samples were removed at vari-
R-HPCDH samples were incubated with BES for 200 min and ous time points and assayed for activity after removal of BES
then assayed for activity after removal of BES. To remove BES as described above. To ensure that no significant abiotic
prior to conducting enzyme assays, AG1-X8, an anion ex- reaction of BES and DTT occurred (i.e. alkylation of one or
change resin with a high affinity for small molecules containing both of the thiols of DTT), the following experiment was
a sulfonate functional group, was added to protein samples and performed. DTT (10 mM) was incubated anoxically with and
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BES Inhibition of CoM-dependent Epoxide Metabolism
k1
L; E⅐I
kϪ1
kinact
¡ E Ϫ I*
E ϩ I
|
O
SCHEME 1
A fit of the data to Equation 3 provided the following kinetic
parameters: kinact (the theoretical maximal rate of inactiva-
tion) ϭ 0.012 Ϯ 0.001 minϪ1 and Ki (the concentration of BES
giving the half-maximal rate of inactivation) ϭ 1.4 Ϯ 0.3 mM.
The fact that inactivation of 2-KPCC exhibits saturation
kinetics indicates that BES is an affinity label mimicking the
CoM portion of the substrate 2-KPC, forming a Michaelis com-
plex with the enzyme prior to irreversible inactivation. A hall-
mark of affinity labels is partial protection from inactivation by
substrates and substrate analogs that bind reversibly. Under the
reducing conditions used for these incubations 2-KPC under-
goes turnover to produce acetone as the product (see Equation
5; DTT can be used in place of NADPH for this reaction as well).
Therefore, CoM, the product of the reaction, was tested for its
ability to protect 2-KPCC from BES inactivation. In the pres-
ence of 5 mM BES and 5 mM CoM, 2-KPCC retained 55% activ-
ity after a 190-min incubation, whereas 2-KPCC incubated
identically but in the absence of CoM retained only 21%
activity. A variety of other brominated organic compounds,
including bromoethane, 1-bromopropane, and 3-bromopropi-
onate, were not inhibitors or inactivators of 2-KPCC, highlight-
ing the importance of the sulfonate in the specificity of BES.
Reduced but Not Oxidized 2-KPCC Is Inactivated by BES—
The target of inactivation by BES is potentially one or more of
the reduced cysteine thiols of 2-KPCC, which could undergo
alkylation by transfer of the ethylsulfonate moiety (ES) as
shown in Equation 8.
FIGURE 5. 2-Bromoethanesulfonate is a time- and concentration-depen-
dent inactivator of 2-KPCC. Panel A, time- and concentration-dependent
inactivation of 2-KPCC by BES. 2-KPCC was incubated with various concentra-
tions of BES, followed by removal of residual BES and assay of 2-KPCC activity
as described under “Experimental Procedures.” Symbols: F, no BES; f, 0.125
mM BES; Ⅺ, 0.50 mM BES; ⌬, 1.0 mM BES; 224, 2.0 mM BES; Œ, 3.0 mM BES; ꢀ, 5.0
mM BES; E, 9.43 mM BES. Pseudo first-order rate constants (kobs) were deter-
mined by fitting the data in panel A to Equation 2. Panel B, plot of pseudo
first-order rate constants for inactivation (kobs) versus BES concentration. The
line through the data points was generated by nonlinear least-squares fit to
the rectangular hyperbola in Equation 3. Panel B, inset, double-reciprocal plot
of the data. The line through the data points was derived from the non-linear
fit of kobs versus [BES].
RSH ϩ BES 3 RS-ES ϩ Hϩ ϩ BrϪ
(Eq. 8)
To provide evidence for this, air-oxidized 2-KPCC was incu-
bated with 5 mM BES under conditions identical to those
described above, but aerobically and in the absence of DTT.
Under these conditions, no inactivation of 2-KPCC activity was
observed over a 4-h time course, suggesting that one or more
without BES (20 mM) at room temperature for 30 h in 50 mM thiols that can be oxidized to the disulfide form is the target of
Tris, pH 7.4. Samples were then removed and added directly BES.
to 2-KPCC activity assays. The BES from the samples prein-
The target of BES inactivation was further refined by exam-
cubated with and without DTT inhibited 2-KPCC identi- ining BES inactivation of 2-KPCC using NADPH rather than
cally, and to an extent consistent with the concentrations of DTT as the reductant. The addition of 5 mM NADPH resulted
BES present at the outset of the incubations, showing that in inactivation of 2-KPCC at rates identical to those observed in
DTT and BES do not react abiotically to any significant the presence of DTT. Because NADPH specifically reduces the
degree under these conditions.
redox active disulfide at the active site of 2-KPCC through the
As shown in Fig. 5A, in the presence of DTT 2-KPCC exhib- intermediacy of FAD, this suggests that either the interchange
ited a time-dependent loss of activity that followed apparent thiol, the flavin thiol, or both are targets of BES inactivation.
first-order kinetics and was proportional to the amount of BES
Determination of BES Modification Stoichiometry—Table 2
present in the samples. Fig. 5B shows the relationship between summarizes the results for the 2-KPCC average molecular
the apparent first-order rate constants (kobs) for inactivation mass determinations under oxidized and DTT-reduced con-
derived from the data in Fig. 5A and the BES concentration. The ditions, and in the absence and presence of BES, using two
inactivation exhibited saturation kinetics, as would be expected different deconvolution algorithms. All trends appeared
for an affinity label that rapidly forms a reversible EI complex consistent between the results of the two algorithms, with
that undergoes slow, irreversible inactivation according to ProMass 2.0 typically generating lower mass values. Ana-
Scheme 1.
lytical reproducibility was demonstrated for un-treated
25238 JOURNAL OF BIOLOGICAL CHEMISTRY
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BES Inhibition of CoM-dependent Epoxide Metabolism
TABLE 2
Average molecular mass determinations of 2-KPCC using LC-MS and two spectral deconvolution algorithms
Under the conditions utilized for analysis, 2-KPCC was observed as the FAD-depleted monomer.
Mass accuracy
(Meas-Theor)
(for BIOMASS
3.1)
Mass change with BES
modification (2-KPCC ؉
BES ؉ DTT) ؊ (2-KPCC
؉ BES) (for BIOMASS 3.1)
Measured average molecular mass
Theoretical average
molecular mass
by deconvolution algorithm
Sample
BIOMASS 3.1
ProMass 2.0
M
M Ϯ S.D.
⌬Da
Dppm
⌬Da
Oxidized 2-KPCC (run 1)
Oxidized 2-KPCC (run 2)
Oxidized 2-KPCC ϩ BES
DTT-reduced 2-KPCC ϩ BES
57,345.6a
57,345.6a
57,345.6b
57,454.7c
57,365.0
57,358.0
57,361.0
57,470.0
57,362.1 Ϯ 12.9
57,355.7 Ϯ 6.9
57,358.4 Ϯ 11.0
57,463.3 Ϯ 10.2
ϩ19.4
ϩ12.4
ϩ15.4
ϩ15.3
338
216
268
266
109.0
a Calculated using the ExPASy Compute pI/MW tool using the 2-KPCC protein sequence (Q56839) minus the average atomic mass of two hydrogens.
b Calculated awith no BES modification assumed.
c The average mass with BES modification of a single cysteine (ϩC2H4SO3 ϭ 108.1; calculated using the average atomic masses of the elements, with the sum being rounded to
the nearest 0.1 Da). The calculation assumed the SO3 moiety of BES and the Cys87 thiol of 2-KPCC to be deprotonated and protonated, respectively, under the conditions used
for the analysis (in a solvent containing 0.1% (v/v) formic acid).
TABLE 3
Part A, cysteine containing peptides identified from MS/MS spectra with selected SEQUEST parameters shown for each peptide. Part B,
Bioworks ShowOut view for peptide #1 in panel A, depicting the next two closest matches to the database. Subscripts denote the position of
the cysteine within the 2-KPCC sequence. Symbols represent modifications to cysteine and are as follows: #, iodoacetamide; ϳ, acrylamide;
and @, BES
a Calculated charge state based on the observed m/z value.
b The Xcorr value is the cross-correlation value from the search, or a measure of how closely an experimental MS/MS spectrum matches to a theoretical MS/MS spectrum for a
given peptide in the database.
c Delta Correlation value, dCn indicates how different the first hit is from the second hit in a database search. It is calculated as follows: (Xcorr#1 Ϫ Xcorr#2)/Xcorr#1. A general
rule of thumb is that a dCn of 0.1 or greater is good.
d Relative Sp score. Sp is the preliminary score. After finding all peptides that match within the selected mass tolerance, SEQUEST dose a preliminary scoring of these candidates
to reduce the list down to 500 for the final XScorr analysis. Ideally the RSp value should be 1 on the best matches.
2-KPCC by running the analysis two separate times (runs 1 sented among proteolytic peptides for subsequent MS/MS
and 2, Table 2). When 2-KPCC was pre-treated with BES in analysis.
the absence of DTT, no significant mass change was
Using the proteolytic enzyme combinations described under
observed over that of untreated 2-KPCC. However, when “Experimental Procedures,” 73.2% total sequence coverage (%
2-KPCC was pre-treated with BES in the presence of DTT, a by # amino acids) was achieved for 2-KPCC pre-treated with
corresponding average mass increase of 109.0 Da, compared BES and DTT. This coverage resulted in representation of all
with BES-treated oxidized 2-KPCC, was observed (Table 2). six cysteines. Interestingly, no proline-specific cleavages of
As the average molecular mass for a BES modification was 2-KPCC were observed, despite the abundance of proline resi-
expected to be 107.1 Da, the observed increase was highly dues in 2-KPCC and identification of several Pro-C autolytic
suggestive of a single BES modification.
Mapping the Location of the BES Modification—With evi- information beyond that of the digestion with trypsin alone.
dence for a single BES modification, experiments were per- Table 3, part A, lists the cysteine-containing peptides identi-
peptides. As such, the trypsin ϩ Pro-C digest yielded no new
formed to map the position of this modification. Because the fied from at least two separate MS/MS spectra. As expected,
modification only appeared to take place under reducing most of the cysteines were modified by iodoacetamide as part of
conditions, the modification was presumed to be occurring the in-gel digestion procedure. In one case, an acrylamide-
on a reactive cysteine residue, as noted above. Thus, all six of modified cysteine was observed, which was likely the result of
the cysteine residues present in 2-KPCC must be repre- using an insufficiently polymerized acrylamide gel.
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BES Inhibition of CoM-dependent Epoxide Metabolism
FIGURE 6. A, amino acid sequence with ϩ1 and ϩ2 ion identifications for the peptide containing a BES modification. Symbols, @, BES modification; #,
iodoacetamide modification. All underlined ions were experimentally observed, whereas ions also in bold are those that allowed discrimination between
assignment of the BES modification at Cys82 or Cys87; note that one of these discriminating ions (y2–22 at m/z 1274.1) is the most prominent ion in the spectrum.
B, MS/MS spectra of the BES modified peptide. Inset, full MS spectrum at 95.47 min, with the arrow showing the [M ϩ 3H]3ϩ BES-modified peptide at m/z 1168.4
that was automatically selected for MS/MS.
The single BES modification was localized to cysteine 82, 2-KPCC) and the oxidized flavin in the two-electron reduced
which is the interchange thiol of 2-KPCC (Table 3, part A). enzyme (Fig. 2A, product of step 4). Covalent modification of
Additional scrutiny of the modification at Cys82 was carried out the interchange thiol of 2-KPCC by BES should irreversibly lock
by examining the ShowOut view for the peptide (Table 3, part oxidized 2-KPCC into the form in which the charge transfer can
B). BES modification at Cys82 gives a significantly higher Xcorr be observed, as shown in Fig. 2D. To test this, 2-KPCC was
value than for the modification being placed at Cys87 (the flavin incubated with DTT, with or without BES, followed by gel fil-
thiol). Furthermore, the ion identifications and MS/MS spectra tration chromatography, air oxidation, and UV-visible spectral
of the BES-modified peptide, shown in Fig. 6, A and B, respec- analysis. As shown in Fig. 7, 2-KPCC that had been preincu-
tively, indicated that several of the most prominent ions bated in the absence of BES showed the normal oxidized flavin
observed are those that favor BES modification at position spectrum, whereas 2-KPCC preincubated in the presence of
Cys82 and not Cys87. Of these ions, the most notable is y2–22, BES showed the characteristic spectral changes associated with
observed with an m/z value of 1274.1 (Fig. 6B). Additionally, the charge-transfer complex. These results confirm the mass
when oxidized 2-KPCC that had been pre-treated with BES spectral results showing that the interchange thiol and not the
was digested with trypsin and analyzed by MS/MS, iodo- flavin thiol of 2-KPCC is the specific target of BES inactivation
acetamide and not BES was found to be the modification at and alkylation.
position 82.
UV-Visible Spectroscopic Evidence for Alkylation of Cys82 by
DISCUSSION
BES—A distinguishing feature of DSOR enzymes is the forma-
The present work expands on our previous work (16) show-
tion of a charge-transfer complex resulting from the interaction ing BES to be an inactivator of bacterial epoxide metabolism by
of the deprotonated flavin thiolate (Cys87 in the case of determining the targets and mechanisms of BES inhibition and
25240 JOURNAL OF BIOLOGICAL CHEMISTRY
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BES Inhibition of CoM-dependent Epoxide Metabolism
is to coordinate the thiol of CoM, thereby lowering its pKa for
nucleophilic attack on epoxypropane (20). A calorimetric study
of the binding of CoM, ethanesulfonate, and ethanethiol to
EaCoMT revealed that the zinc-thiol interaction contributes
much more binding energy to the formation of the enzyme-
CoM complex than the interaction of the sulfonate with the
enzyme (20). It is therefore not surprising that BES is not a CoM
analog (and inhibitor) for EaCoMT because the substitution of
the thiol group by bromine will result in the loss of this key
interaction with zinc.
The kinetics for the time-dependent inactivation of 2-KPCC
by BES show that inactivation occurs by a two-step process, the
first step of which is rapid, reversible and saturable (Scheme 1).
As noted under “Results,” other brominated compounds lack-
ing the sulfonate group did not inhibit 2-KPCC. Thus, as illus-
trated in Fig. 2D, the interaction of the sulfonate of BES with the
two arginine residues that coordinate the sulfonate of the sub-
strate 2-KPC is believed to be key to the selectivity of BES as an
alkylating agent.
FIGURE 7. UV-visible spectra of untreated 2-KPCC and BES-modified
2-KPCC. 2-KPCC samples were prepared as described under “Experimental
Procedures.”Thereducedspectrawererecordedafterremovalofoxygenand
the addition of 1 mM dithionite. Trace 1, air-oxidized 2-KPCC; trace 2, air-oxi-
dized 2-KPCC modified by BES; trace 3, dithionite-reduced 2-KPCC; trace 4,
dithionite-reduced 2-KPCC modified by BES.
The highly selective alkylation of the interchange thiol of
2-KPCC makes BES an excellent probe for investigating novel
features of this unique member of the DSOR family of enzymes,
the only one known to function as a carboxylase. Structural
studies of 2-KPCC have revealed a smaller and more hydropho-
bic active site architecture relative to all other DSOR enzymes
due to amino acid insertions (5). Binding of 2-KPC induces a
unique conformational change that encapsulates the substrate
inactivation. R-HPCDH and 2-KPCC were reversibly inhibited
by BES, whereas 2-KPCC was additionally inactivated by the
highly specific and irreversible alkylation of the interchange
thiol that interacts with the substrate 2-KPC within the enzyme
active site. The latter result is particularly interesting in that it
identifies a fundamental new mechanism for inactivation of a
CoM-dependent enzyme by BES. By comparison, in the case of
methanogenic methyl-CoM reductase, BES inactivation occurs 2-KPC, at the same time opening a hydrophobic channel
via oxidation of Ni(I) in the nickel tetrapyrrole cofactor F430 to
Ni(II), in the process producing a debrominated alkylsulfonate
product (15). Bromopropanesulfonate, which is an even more
potent inhibitor than BES, oxidizes Ni(I) in F430 as well, but by
a different mechanism (15, 25–27).
believed to allow CO2 entry (5). It will be interesting to see if the
ethylsulfonate group bound to the enzyme induces a similar
conformational change.
In addition to catalyzing the carboxylation and protonation
of 2-KPC, 2-KPCC also catalyzes the reverse of the physiologi-
cal carboxylation reaction at a slow rate as well as the decarbox-
ylation of acetoacetate and exchange of 14CO2 into acetoace-
tate, reactions that provided evidence for enolacetone as an
intermediate in the reaction mechanism (Fig. 2C) (4). The latter
two reactions are interesting in that they are stimulated 72- and
38-fold, respectively, by the presence of a saturating concentra-
tion of CoM (4). It will be interesting to see if bound BES mimics
the stimulatory effect of CoM on the decarboxylation and
exchange reactions.
Although half-maximal inhibition of methyl-CoM reductase
was observed at low micromolar concentrations of BES (15),
ϳ1000-fold higher concentrations were required for the inhi-
bition of R-HPCDH and 2-KPCC. Interestingly, BES was a
mixed (noncompetitive) rapid equilibrium inhibitor of both
R-HPCDH and 2-KPCC. The inhibition constants reflecting
competitive inhibition (Kis) were 14- and 85-fold higher than
the Km values for substrates R-HPC and 2-KPC for R-HPCDH
and 2-KPCC, respectively. The inhibition constants reflecting
binding to the ES complex (Kii) were slightly higher, although
similar in magnitude to, the Kis values. These results support
the results of x-ray crystallographic studies of R-HPCDH and
2-KPCC showing that the hydroxypropyl and ketopropyl moi-
eties, in addition to the sulfonate of CoM, are important in high
affinity substrate binding (5, 28). For methyl-CoM reductase,
where the group bound to CoM is the nonpolar methyl group,
the ethanesulfonate could be expected to be the major binding
determinant, perhaps helping to explain why BES is a much
higher affinity inhibitor for this enzyme.
Finally, the utility of BES alkylation as a mechanistic probe is
seen in the charge-transfer absorbance of alkylated 2-KPCC
presented in Fig. 7. No charge transfer was observed for the
alkylated enzyme at pH 7.4, indicating that the flavin thiol
remains protonated at this normal pH value. To observe the
maximal charge transfer, it was necessary to increase the pH to
a value of 11. In contrast, the charge transfer for “classic” DSOR
enzymes such as mercuric reductase alkylated at the inter-
change thiol by iodoacetamide can be seen at pH 7.4 (29). This
observation is compatible with the unique, hydrophobic active
site architecture of 2-KPCC that presumably results in a higher
pKa value for the flavin thiol, at least in the conformation in
which the interchange thiol has been alkylated by the ethylsul-
Interestingly, EaCoMT, the only enzyme of the pathway of
epoxide carboxylation for which CoM is the substrate (Fig. 1),
was not inhibited by BES, even at BES concentrations more
than 2 orders of magnitude higher than the Km for CoM (34
␮M). EaCoMT is a zinc metalloenzyme, in which the role of zinc fonate group.
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BES Inhibition of CoM-dependent Epoxide Metabolism
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25242 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 33•AUGUST 13, 2010

Enzymology:
Mechanism of Inhibition of Aliphatic
Epoxide Carboxylation by the Coenzyme M
Analog 2-Bromoethanesulfonate
Jeffrey M. Boyd, Daniel D. Clark, Melissa A.
Kofoed and Scott A. Ensign
J. Biol. Chem. 2010, 285:25232-25242.
doi: 10.1074/jbc.M110.144410 originally published online June 15, 2010
Access the most updated version of this article at doi: 10.1074/jbc.M110.144410
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