The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH-rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and (S)-[(2)H1]fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-(2)H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme-substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts.
The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH-rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and(S)-[(2)H1]fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-(2)H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme-substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts.
Fluoroacetate
is a highly toxic
natural product found in plants in Australia, Brazil, and Africa.[1,2] Its mechanism of toxicity has been attributed to its cellular activation
to fluoroacetyl-coenzyme A (CoA) and further metabolism to fluorocitrate,
a mechanism-based inhibitor of the aconitase enzyme of the tricarboxylic
acid cycle.[3,4] Despite its prevalence in plants, only one
genetic host of the fluoroacetate pathway, the soil bacterium Streptomyces cattleya, has been characterized to date.[5,6] As one mechanism of biological resistance, S. cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK), which can reverse
the activation of fluoroacetate.[7,8] Remarkably, this enzyme
exhibits a 106-fold preference for its cognate substrate,
fluoroacetyl-CoA, over acetyl-CoA, an abundant central metabolite
and cellular competitor that differs only in the absence of the fluorine
substitution.[9] On the basis of its ability
to exploit the unique properties of fluorine to achieve substrate
specificity, FlK represents an ideal model system in which to query
molecular recognition of fluorine and its influence on enzymatic reactivity
in a naturally evolved protein–ligand pair.We have previously
shown that the hydrolytic mechanism of FlK involves
a minimum of three kinetic steps: (i) formation of the enzyme–substrate
complex (KD = k–1/k1), (ii) acylation of FlK to form an
acyl-anhydride intermediate on Glu 50 (k2), and (iii) FlK deacylation (k3) or
breakdown of this intermediate to yield the carboxylic acid product
(Scheme 1).[10] A
large component of fluoroacetyl-CoA (R = F) specificity (104-fold) is based on a change in the rate-limiting step from formation
of the acyl-enzyme intermediate for fluoroacetyl-CoA (ii; k2 = kcat) to its
breakdown for acetyl-CoA (R = H) (iii; k3 = kcat). Despite similarities to canonical
hot dog-fold thioesterases,[11] our previous
mechanistic studies indicate that the chemical basis for the acceleration
of FlK-catalyzed deacylation for the fluorinated substrate is utilization
of an unusual hydrolytic mechanism initiated by Cα deprotonation of the acyl-enzyme intermediate.[10] The resultant enolate can then break down through a putative
ketene intermediate to give fluoroacetate.
Scheme 1
Minimal Kinetic Mechanism
for FlK-Catalyzed Acyl-CoA Hydrolysis (fluoroacetyl-CoA,
R = F; acetyl-CoA, R = H)
The differences in chemical mechanism between fluoroacetyl-CoA
and acetyl-CoA hydrolysis provide a means for FlK to kinetically discriminate
between two substrates that are both present at saturating concentrations
under physiological conditions based on the high intracellular concentration
of acetyl-CoA (2 mM) and the low KM for
fluoroacetyl-CoA (8 μM).[10] However,
it is interesting to note that this kinetic discrimination occurs
after the first committed step in the reaction mechanism, which is
enzyme acylation (k2), and that the specificity
constant kcat/KM depends only on the binding and acylation steps of the reaction
(Scheme 1).[12] We
therefore set out to further explore the function of the active site
triad involved in enzyme acylation, consisting of His 76, Glu 50,
and Thr 42. We first provide additional evidence supporting the formation
of an acyl-enzyme intermediate during the hydrolysis of fluoroacetyl-CoA,
and we show that the rate of formation of this intermediate does not
depend on the key specificity-determining residue, His 76, which is
involved in Cα deprotonation. Instead, the primary
determinant of specificity of Glu 50 acylation appears to be based
on the inductively electron-withdrawing effect of the α-substituent.
We also explore the role of Thr 42 with respect to acylation and deacylation
and conclude that its main function in catalysis is to anchor the
hydrogen bonding network between Glu 50 and His 76.In addition
to investigating enzyme acylation, we also continue
to examine the role of fluorine in controlling the chemical mechanism
of enzyme deacylation. Although the fluoroacetyl-CoA α-protons
are expected to be more acidic than those of acetyl-CoA, their lower
pKa is insufficient to explain why the
Cα deprotonation mechanism for deacylation is inaccessible
to other α-substituted acyl-CoAs. In particular, acetoacetyl-CoA
is predicted to have an α-proton pKa lower than that of fluoroacetyl-CoA based on its ability to form
a resonance-stabilized enolate, but free energy relationship analysis
of its FlK-catalyzed hydrolysis suggests that it cannot access the
Cα deprotonation pathway.[10] Using substrates with stereogenic centers at the substrate α-carbon,
we demonstrate that polarization is not the sole determinant of reaction
through the Cα deprotonation pathway. In this regard,
FlK utilizes chiral recognition of fluoroacetyl-CoA’s prochiral
fluoromethyl group to position the pro-R proton for preferential abstraction by His 76, indicating that specificity
is based not only on the unique reactivity of fluorinated compounds
but also potentially on fluorine molecular recognition. Taken together,
these results show that fluorine impacts both the acylation and deacylation
steps of the FlK reaction mechanism by substrate activation as well
as fluorine-specific interactions in the active site.
Materials and
Methods
Commercial Materials
Acetyl-coenzyme A sodium salt,
coenzyme A hydrate, coenzyme A trilithium salt, anhydrous N,N-dimethylformamide (DMF), 2-(N-morpholino)ethanesulfonic acid (MES), trifluoroacetic
acid (TFA), oxalyl chloride (2 M in dichloromethane), 5,5′-dithiobis(2-nitrobenzoic
acid) (DTNB), methyl(S)-(−)-lactate, methyl(R)-(+)-lactate, methanesulfonyl
chloride, magnesium chloride hexahydrate, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol
(Bis-Tris), 1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis-Tris
propane), and tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)
were purchased from Sigma-Aldrich (St. Louis, MO). Monosodium phosphate
monohydrate, disodium phosphate heptahydrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), hexanes, ethyl acetate, methanol, triethylamine, adenosine
5′-triphosphate trisodium salt, and tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) were purchased from Fisher Scientific (Pittsburgh,
PA). 2-Fluoropropionic acid was purchased from Oakwood Products (West
Columbia, SC).
Protein Expression and Purification
FlK and FlK-T42S
were expressed and purified as described previously.[9] Acetate kinase from Escherichia coli and
phosphotransacetylase from S. cattleya were cloned,
expressed, and purified as described previously.[8]
Synthesis of Substrates
Fluoroacetyl-CoA,
chloroacetyl-CoA,
and bromoacetyl-CoA were synthesized as described previously.[10] Tetrahydrofuran (THF) used in chemical synthesis
was dried using a VAC Solvent Purifier System (Vacuum Atmospheres
Co., Amesbury, MA). Acyl-CoAs were purified by high-performance liquid
chromatography (HPLC) using an Agilent Eclipse XDB-C18 column (9.4
mm × 250 mm, 5 μm) connected to an Agilent 1200 binary
pump and an Agilent G1315D diode-array detector, which was used to
monitor coenzyme A absorbance at 260 nm. Following sample loading,
the column was washed with 0.2% aqueous TFA (3 mL/min) until the absorbance
at 260 nm returned to baseline. A linear gradient from 0 to 100% methanol
over 90 min (3 mL/min) with 0.2% aqueous TFA as the aqueous mobile
phase was then applied. Fractions were collected using an Agilent
1260 fraction collector and then assayed for the desired acyl-CoAs
by liquid chromatography and mass spectrometry using an Agilent 1290
binary pump coupled to an Agilent 6130 single-quadrupole electrospray
ionization mass spectrometer. The fractions were then analyzed using
a Phenomenex Kinetex C18 column (4.6 mm × 30 mm, 2.6 μm)
with a linear gradient from 0 to 100% acetonitrile over 2 min (0.7
mL/min) using 0.1% formic acid as the aqueous mobile phase. Fractions
containing the desired compound were pooled and lyophilized. Acyl-CoAs
were then dissolved in water, quantified by their absorbance at 260
nm, and stored at −80 °C until they were used further.
High-resolution mass spectra were acquired at the QB3/Chemistry Mass
Spectrometry Facility at the University of California (Berkeley, CA).
Nuclear magnetic resonance (NMR) spectra were recorded at the College
of Chemistry NMR Facility at the University of California (Berkeley,
CA) or at the Central California 900 MHz NMR Facility (Berkeley, CA).
One-dimensional spectra were recorded on Bruker AV-600 or AVQ-400
NMR spectrometers at 298 K. Chemical shifts are expressed in parts
per million (δ) downfield from tetramethylsilane (1H, 2H, and 13C) or trichlorofluoromethane (19F) and are referenced to the solvent signal using standard
CoA numbering (Table S1 of the Supporting Information). Heteronuclear multiple-bond correlation (HMBC) experiments were
performed on a Bruker AV-500 NMR spectrometer at 298 K.
(RS)-2-Fluoropropionyl-CoA
2-Fluoropropionic
acid (92 mg, 1 mmol) was placed in an oven-dried round-bottom flask
equipped with a stir bar and a reflux condenser. The flask was placed
under nitrogen pressure, and dry THF (2 mL), DMF (100 μL), and
oxalyl chloride (2 M in dichloromethane, 500 μL) were added
by syringe. The reaction mixture was heated to 65 °C and stirred
for 3–4 h. After the mixture had been cooled, a portion of
the reaction mixture (300 μL) was added to a stirred, ice-cooled
solution of coenzyme A hydrate (50 mg, ∼0.06 mmol) and triethylamine
(41.8 μL, 0.3 mmol) in anhydrous DMF (1 mL). After 1 min, the
reaction was quenched by addition of water (20 mL) and the mixture
lyophilized. The lyophilizate was dissolved in water (1 mL) and purified
by reverse-phase HPLC (15 μmol, 25%): 1H NMR (600
MHz, D2O, 25 °C) δ 8.56 (s, 1H, H8), 8.38 (s, 1H, H2), 6.15 (d, J = 6 Hz,
1H, H1′), 5.12 (dq, JHH = 7.2 Hz, JHF = 49.5 Hz, 1H, CHF), 4.84 (m, 2H, H2′ and H3′), 4.56 (s, 1H, H4′), 4.24 (d, J = 15 Hz, 2H, H5′), 3.97 (s, 1H, H3″), 3.85 (m, 1H, H1″), 3.61 (m, 1H, H1″), 3.41 (t, J = 6.6 Hz, 2H, H5″), 3.31 (t, J = 6.6 Hz, 2H, H8″), 3.00 (t, J = 6 Hz, 2H, H9″),
2.39 (t, J = 6.6 Hz, 2H, H6″),
1.46 (dd, JHH = 6.6 Hz, JHF = 24.9 Hz, 3H, CH3), 0.90 (s, 3H, H10″), 0.78 (s, 3H, H11″); 13C NMR (150.9 MHz, D2O, 25 °C) δ
203.2, 203.0 (FCHCO), 174.6 (C7″), 173.9 (C4″), 149.8 (C6), 144.7 (C2), 142.7 (C4), 142.4 (C8), 118.5 (C5), 87.9 (C1′), 87.5, 86.8 (FCHCO), 86.8 (C4′), 74.4 (C2′),
74.3 (C3″), 74.2 (C3′), 73.7 (C1″), 65.1 (C5′), 38.3 (C8″), 36.9 (C2″) 35.3 (C5″), 35.2
(C6″), 27.1 (C9″), 20.7 (CH3), 18.3 (C10″), 17.9
(C11″); 19F NMR (564.7 MHz, D2O, 25 °C) δ −181.2 (sextet, J =
26.4 Hz); HR-ESI-MS calcd (M – H+) m/z 840.1247, found (M – H+) m/z 840.1236.
(S)- and
(R)-2-Fluoropropionyl-CoA
Sodium (S)-2-fluoropropionate and sodium (R)-2-fluoropropionate
were synthesized from methyl(R)-(−)-lactate
and methyl(S)-(+)-lactate
using literature methods.[13] 2-Fluoropropionyl-CoAs
were then synthesized from the corresponding carboxylic acids (1 mmol
scale) as described for (RS)-2-fluoropropionyl-CoA
(10 μmol, 17%).
(S)-2-Fluoropropionyl-CoA
(Figure S1 of the Supporting Information):
(R)- or (S)-1-(Methoxycarbonyl)ethylmethanesulfonate
Methyl lactate (1.0 g, 9.6 mmol) was dissolved in toluene before
triethylamine (1.2 g, 1.6 mL, 11.5 mmol) was added. The reaction mixture
was cooled in an ice/water bath, and methanesulfonyl chloride (1.2
g, 0.82 mL, 10.6 mmol) was added dropwise over 15 min. The reaction
mixture was stirred on ice for 1 h, warmed to room temperature, and
filtered. The filtrate was concentrated under vacuum to yield a light
pink oil, which was purified on a silica column using an 80:20 hexanes/ethyl
acetate mixture as the mobile phase to give (S)-
or (R)-1-(methoxycarbonyl)ethylmethanesulfonate as
a clear oil (1.43 g, 84%).
Formamide (3 mL) was placed in a two-neck round-bottom flask equipped
with a stir bar and heated to 60 °C. Potassium fluoride (1.2
g, 22 mmol) was dissolved in the formamide while the mixture was being
stirred. A short-path distillation head equipped with a thermometer
and a receiving flask cooled with a dry ice/acetone bath was then
attached. (S)- or (R)-1-(methoxycarbonyl)ethylmethanesulfonate
(1 g, 5.5 mmol) was added slowly. The reaction mixture was then placed
under vacuum, and the product was continuously distilled during the
course of the reaction to yield methyl (R)- or(S)-2-fluoropropionate as a clear oil (200 mg, 35%).
Methyl (R)- or(S)-2-fluoropropionate
(100 mg, 0.6 mmol) was dissolved in water, and sodium hydroxide (0.6
mmol from a 10 M stock solution) was added. The reaction mixture was
stirred at room temperature for 1 h, at which time it was judged to
be ∼80% complete based on 19F NMR. The solution
was flash-frozen and lyophilized to yield sodium (R)- or(S)-2-fluoropropionate as a white solid (56
mg, 82%).
Sodium (S)-[2H1]fluoroacetate and sodium (R)-[2H1]fluoroacetate were synthesized
as described
previously.[14] (S)- and(R)-[2H1]fluoroacetyl-CoA were
synthesized from the corresponding sodium [2H1]fluoroacetates enzymatically using acetate kinase (AckA) from E. coli and phosphotransacetylase (PTA) from S.
cattleya. Reaction mixtures containing Tris-HCl (pH 7.5,
100 mM), MgCl2 (5 mM), TCEP (2.5 mM), ATP (50 mM), coenzyme
A (40 mM), sodium [2H1]fluoroacetate (50 mM),
AckA (10 μM), and PTA (5 μM) in a total volume of 1 mL
were incubated at 37 °C for 2 h. [2H1]Fluoroacetyl-CoAs
were then purified by reverse-phase HPLC. Fractions containing the
desired compounds were pooled and lyophilized to yield (S)- or(R)-[2H1]fluoroacetyl-CoA
(6 μmol, 15%).
Pre-steady-state
kinetic experiments were performed using rapid chemical quench followed
by HPLC separation of coenzyme A from unhydrolyzed acyl-CoA. FlK [50
or 150 μM in 20 mM Tris-HCl (pH 7.6) and 50 mM NaCl] or FlK-T42S
[40 μM in 20 mM Tris-HCl (pH 7.6) and 50 mM NaCl] was mixed
with substrate diluted in water [chloroacetyl-CoA and [2H1]fluoroacetyl-CoAs, 500 μM; cyanoacetyl-CoA and
fluoroacetyl-CoA, 750 μM; bromoacetyl-CoA and (R)-2-fluoropropionyl-CoA, 1 mM; (S)-2-fluoropropionyl-CoA,
5 mM; acetyl-CoA, 6 mM] using a Chemical Quench Flow Model RQF-3 (KinTek).
The reaction was stopped at various times by mixing with 50% TFA to
achieve a final concentration of 17% TFA. Quenched samples were analyzed
by HPLC on an Agilent Eclipse XDB-C18 (3.5 μm, 3.0 mm ×
150 mm) column using a linear gradient from 0 to 100% methanol over
15 min (0.6 mL/min) with 50 mM sodium phosphate and 0.1% TFA (pH 4.5)
as the mobile phase. Conversion percentages were calculated on the
basis of the 260 nm peak areas for acyl-CoA substrate and free CoA
product. Plots of coenzyme A formed versus time were fit to the burst-phase
equation:where [E0] is the
enzyme concentration, k2 is the burst-phase
(acylation) rate constant,
and k3 is the steady-state (deacylation)
rate constant.[15] This equation is a reduced
form of the general burst equation:where kcat = (k2k3)/(k2 + k3), the burst height
(β) = k22/(k2 + k3)2, and the
rate of burst formation (λ) = k2 + k3, which results from making the
assumption that k2 ≫ k3.[15] In cases where this assumption
was invalid, only kcat is reported. In
cases that could not be fit with a burst phase, they were fit as pseudo-first-order
reactions according to the equation [CoA] = k[FlK]t + [CoA]0, where the y-intercept,
[CoA]0, is the concentration of CoA released before the
first data point, k is the rate constant, and t is time. For fluoroacetyl-CoA and [2H1]fluoroacetyl-CoAs, a sum-of-squares F test was
used to determine whether the data were best fit with a line or with
the burst-phase equation. The null hypothesis was a linear fit, and
the alternative hypothesis was a burst-phase fit with the burst amplitude
fixed at 25 μM. The p value threshold was set
at 0.01, meaning that if the F test gave a p value of >0.01, there was no compelling reason to reject
the null hypothesis, whereas if the p value was <0.01,
the null hypothesis was rejected in favor of a burst-phase fit. The F test gave p = 0.0309 for [1H2]fluoroacetyl-CoA, p = 0.0866 for (S)-[2H1]fluoroacetyl-CoA, and p = 0.0022 for (R)-[2H1]fluoroacetyl-CoA. Therefore, data for [1H2]fluoroacetyl-CoA and (S)-[2H1]fluoroacetyl-CoA were fit with a line, and data for (R)-[2H1]fluoroacetyl-CoA were fit with the burst-phase
equation.For pre-steady-state kinetic experiments at pH 6,
FlK was exchanged into 100 mM Bis-Tris (pH 6.0) prior to the experiment
using a MicroBiospin 6 column (Bio-Rad) that had been pre-equilibrated
with the appropriate buffer. FlK was then quantified by absorbance
and diluted to 50 μM with the same buffer prior to use in rapid
quench experiments.
Taft Free Energy Relationship Analysis of
FlK Acylation Rates
Taft plots were constructed by plotting
the log of the acylation
rate constant (log k2) versus the Taft
polar substituent constant σ* for the α-substituent of
each acyl-CoA tested.[16] Acylation rate
constants and the corresponding error bars were derived from nonlinear
curve fitting of triplicate pre-steady-state kinetic data sets. Taft
plots were fit to the equation log k = ρ*σ*
+ c, where ρ* is the polar sensitivity factor
and c is a constant.
Steady-State pH–Rate
Profiles
Steady-state kinetic
experiments for determining the pH–rate profile of FlK were
conducted on a Beckman Coulter DU800 spectrophotometer by continuously
monitoring the absorbance of the acyl-CoA thioester bond at 232 nm
(ε = 4500 M–1 cm–1) in a
0.2 cm path length quartz cuvette at 25 °C. Reaction mixtures
contained the appropriate buffer [100 mM; Bis-Tris propane (pH 6.3–7),
Tris-HCl (pH 7–9), or Bis-Tris propane (pH 9)], fluoroacetyl-CoA
(10, 25, 50, or 100 μM), and FlK (1 nM) or FlK-H76A (1 μM).
Each rate was measured in triplicate. Experiments at saturation (kcat conditions) were repeated with similar results
in a series of sulfonate buffers [MES (pH 6–7), HEPES (pH 7–8),
and TAPS (pH 7.5–9)] to ensure the observed activity resulted
from changes in pH and not buffer composition. Our ability to measure
FlK-catalyzed hydrolysis rates below pH 6 was limited by protein instability,
while our ability to measure enzymatic rates above pH 9 was limited
by rapid nonenzymatic substrate hydrolysis. Kinetic parameters (kcat and KM) were
determined by fitting the initial rate data to the equation V0 = (Vmax[S])/(KM + [S]), where V0 is the initial rate and [S] is the substrate concentration, using
Origin version 6.0 (OriginLab Corp., Northampton, MA). The pH–rate
profile for wild-type FlK in which decreases in activity were observed
at both low and high pH was fit to the equationwhere kobs is
the observed kcat at a given pH, kmax is the pH-independent rate constant, pKa1 is the pKa of
the group that ionizes at basic pH, and pKa2 is the pKa of the group that ionizes
at acidic pH. A comparison between the pH–rate profile for
wild-type FlK and pH dependence studies of the FlK-H76A mutant led
to the conclusion that the mutant lacks an ionization at acidic pH.
Steady-State Kinetic Analysis of 2-Fluoropropionyl-CoAs
Steady-state rates of FlK-catalyzed 2-fluoropropionyl-CoA hydrolysis
were measured by monitoring the increase in absorbance at 412 nm from
the reaction of 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) with
enzymatically produced coenzyme A in a Beckman Coulter DU800 spectrophotometer.
Assays (100 μL) were performed at 25 °C in Tris-HCl (pH
7.6, 100 mM) containing DTNB (0.5 mM), the appropriate 2-fluoropropionyl-CoA,
and FlK (5 nM). Each rate was measured in triplicate. Absorbance values
were converted to coenzyme A concentration using a standard curve.
Kinetic parameters (kcat and KM) were determined by fitting the initial rate data to
the equation V0 = (Vmax[S])/(KM + [S]), where V0 is the initial rate and [S] is the substrate
concentration, using Origin version 6.0.
Results and Discussion
Probing
the Role of the Catalytic Residues in Acylation Specificity
Because acylation of Glu 50 represents the committed step in FlK-catalyzed
hydrolysis, we were interested in further exploring the contribution
of the other catalytic residues, His 76 and Thr 42, to this step (Figure 1A). Our previous pre-steady-state kinetic analyses
indicated that the acylation rate constant differs by at least 2 orders
of magnitude between the fluoroacetyl-CoA and acetyl-CoA substrates
and that mutation of His 76 to Ala led to a loss of specificity in
both the acylation and deacylation steps.[10] While the defect in deacylation can be attributed to the loss of
the key base required for the Cα deprotonation pathway,
the 10-fold reduction in the acylation rate constant for the H76A
mutant is more difficult to interpret because of the participation
of the His 76 side chain in a hydrogen bond network involving Thr
42 and Glu 50 that has been shown to be important for their positioning
(Figure S3 of the Supporting Information).[9,17] However, active roles for His 76 in determining
substrate specificity for enzyme acylation beyond simple structural
function are also possible. One possibility is that His 76 could be
involved in the deprotonation of the Glu 50 nucleophile to promote
nucleophilic attack on the fluoroacetyl-CoA carbonyl group. Another
potential although unlikely role could involve deprotonation of the
fluoroacetyl-CoA α-carbon by His 76 to initiate an E1Cb-like
(elimination–addition) mechanism to generate a ketene, which
could then be trapped by Glu 50 to form the acyl-enzyme intermediate
in a manner analogous to its breakdown. We therefore set out to clarify
whether general base catalysis involving His 76 is essential for controlling
the observed specificity in acylation.
Figure 1
Steady-state and pre-steady-state
kinetic analysis to investigate
the role of the FlK catalytic residues in acylation specificity. (A)
The catalytic triad of FlK consists of Thr 42, Glu 50, and His 76
(PDB entry 3P2Q). (B) pH–rate profile for wild-type FlK. (C) pH–rate
profile for FlK-H76A. Values are reported as means ± the standard
deviation (n = 3). (D) Pre-steady-state kinetics
of FlK-catalyzed fluoroacetyl-CoA hydrolysis of fluoroacetyl-CoA at
pH 6. (E) Pre-steady-state kinetics of FlK-T42S-catalyzed acetyl-CoA
hydrolysis. (F) Pre-steady-state kinetics of FlK-T42S-catalyzed fluoroacetyl-CoA
hydrolysis. Dotted lines indicate 1 equiv of enzyme.
Steady-state and pre-steady-state
kinetic analysis to investigate
the role of the FlK catalytic residues in acylation specificity. (A)
The catalytic triad of FlK consists of Thr 42, Glu 50, and His 76
(PDB entry 3P2Q). (B) pH–rate profile for wild-type FlK. (C) pH–rate
profile for FlK-H76A. Values are reported as means ± the standard
deviation (n = 3). (D) Pre-steady-state kinetics
of FlK-catalyzed fluoroacetyl-CoA hydrolysis of fluoroacetyl-CoA at
pH 6. (E) Pre-steady-state kinetics of FlK-T42S-catalyzed acetyl-CoA
hydrolysis. (F) Pre-steady-state kinetics of FlK-T42S-catalyzed fluoroacetyl-CoA
hydrolysis. Dotted lines indicate 1 equiv of enzyme.We initiated these studies by examining FlK acylation
under conditions
in which His 76 is protonated and therefore unable to participate
in general base catalysis, which should minimize structural perturbations
in the active site compared to those occurring during mutagenesis.
To determine appropriate conditions, we first constructed a pH–rate
profile of FlK and observed an ionization event at acidic pH (Figure 1B). The apparent absence of this ionization in the
pH–rate profile of the FlK-H76A mutant is consistent with its
assignment to His 76 (Figure 1C). Although
a second ionization event at basic pH was also observed, it is not
clearly attributable to any of the residues known to be involved in
catalysis. The existence of a similar ionization in the H76A mutant
(Figure 1C), as well as in other hot dog-fold
thioesterases,[18] suggests that the origin
of this deprotonation event is more complex and not directly related
to His 76. We then performed pre-steady-state kinetic studies of FlK
at pH 6, where His 76 should be predominantly protonated based on
pH dependence studies. Under these conditions, we observed a burst
phase of product formation consistent with enzyme acylation followed
by a slower steady-state rate, which we interpret to be caused by
the inaccessibility of the Cα deprotonation pathway
in the absence of general base catalysis by His 76 (Figure 1D). The observed burst-phase rate constant (270
± 120 s–1) demonstrates the kinetic competence
of the protonated His 76 in forming the acyl-enzyme intermediate.
In comparison, no burst phase is observed in FlK at pH 7.6 and fluoroacetyl-CoA
is turned over at a steady-state rate of 220–270 s–1.[9,10] These results demonstrate that general base catalysis
involving His 76 is not required for acylation activity or fluoroacetylation
specificity.Because His 76 appears to play a structural role
in FlK acylation,
we turned our attention to exploring the contribution of Thr 42 to
Glu 50 acylation. Indeed, steady-state kinetic studies have shown
that the FlK-T42S mutant retains only ∼200-fold selectivity
for fluoroacetyl-CoA over acetyl-CoA, an effect that is partially
attributable to a 30-fold decrease in the KM for acetyl-CoA.[9] Using pre-steady-state
measurements, we determined that the acylation rate constant (k2) for the T42S mutant for acetyl-CoA was within
error of the rate constant measured for wild-type FlK while the deacylation
rate constant (k3) was reduced by 6-fold
(Figure 1E). Kinetic modeling suggests that
the observed decrease in KM for acetyl-CoA
in FlK-T42S can be explained by this defect in the deacylation rate
in the absence of any changes in substrate affinity (Figure S4 of
the Supporting Information). For fluoroacetyl-CoA,
the T42S mutation leads to a 3-fold decrease in acylation rate (k2) and a 25-fold decrease in deacylation rate
(k3) (Figure 1F).
From the different behavior of the two substrates with respect to
acylation, it seems as if Thr 42 may make a small contribution to
fluoroacetyl-CoA acylation specificity. Crystallographic studies have
shown that Thr 42 is important in orienting Glu 50 and His 76 for
reactivity. In the crystal structure of the T42S mutant, the Glu 50
side chain is rotated by 90°, both Ser 42 and His 76 populate
two rotamers, and the hydrogen bonding network among the catalytic
residues is disrupted (Figure S3 of the Supporting
Information).[17] The observed acylation
defect is therefore likely to be related to structural effects that
result in misorientation of the catalytic residues. Furthermore, the
lack of an acylation defect for acetyl-CoA in both the H76A and T42S
mutants suggests that the catalytic site is not optimized for utilization
of this substrate.
Determining the Impact of Substrate Polarization
on the Acylation
Rate
On the basis of our studies, the catalytic residues,
His 76 and Thr 42, appear to play a limited role in controlling the
specificity of Glu 50 acylation. We therefore set out to probe the
participation of the substrate itself in determining acylation specificity.
On the basis of the electron-withdrawing nature of the fluorine atom,
the fluoroacetyl-CoA carbonyl group is expected to be more activated
toward nucleophilic attack than acetyl-CoA. We have previously compared
the rates of chemical hydrolysis of these two substrates and showed
that the rate of fluoroacetyl-CoA hydrolysis is at least 10-fold higher
than the rate for acetyl-CoA.[10] This ratio
increases with an increasing pH up to 100-fold, which is similar to
the observed magnitude of discrimination between fluoroacetyl-CoA
and acetyl-CoA in the acylation step. To probe the role of carbonyl
inductive polarization in acylation specificity, we examined the pre-steady-state
kinetics of FlK-catalyzed hydrolysis of a series of acyl-CoAs with
different functional groups at the α-position to access a wide
range of values of the Taft polar substituent constant (σ*)
to define the impact on this particular step.[16]The rate of acylation (k2) for
each substrate was measured using pre-steady-state kinetic experiments.
For acetyl-CoA, acetoacetyl-CoA (33 ± 3 μM), bromoacetyl-CoA
(30 ± 3 μM), and chloroacetyl-CoA (28 ± 3 μM),
a burst phase of product formation corresponding to 1 equiv of enzyme
(25 μM) was observed, followed by a slower steady-state rate
identical to the previously measured steady-state rate (Figure 2A–C), indicating that hydrolysis of these
substrates is likely to proceed through the same acyl-enzyme intermediate
that was trapped for acetyl-CoA (Scheme 1).
For FlK-catalyzed cyanoacetyl-CoA hydrolysis, plots of CoA formed
versus time could not be fit with an equation describing burst-phase
kinetic behavior and were instead best fit by a simple linear fit
(Figure 2D). However, the dead time of the
rapid quench instrument is 2 ms, precluding the measurement of rate
constants approaching or exceeding 500 s–1. To distinguish
between the absence of a burst phase and the presence of a burst phase
that is over before the first quenched time point, we examined the
amount of CoA formed after 2 ms. In the absence of a burst phase,
the CoA concentration would be given by [CoA] = kcat[FlK]t, corresponding to a line with
a y-intercept of zero. On the other hand, if a burst
phase had occurred before the first quenched time point, the CoA concentration
would be given by [CoA] = kcat[FlK]t + [FlK], corresponding to a line with a y-intercept of [FlK]. Linear fitting of the data gave a y-intercept of 77 ± 7 μM, within error of the FlK concentration
of 75 μM used in the assay (Figure 2D).
We therefore conclude that cyanoacetyl-CoA is also hydrolyzed through
a mechanism involving an acyl-enzyme intermediate, although we could
not include this substrate in our free energy relationship analysis
because the acylation rate could not be accurately measured.
Figure 2
Pre-steady-state
kinetic and Taft analysis of a series of acyl-CoAs.
The dashed line indicates 1 equiv of enzyme: (A) acetoacetyl-CoA (R
= Ac), (B) bromoacetyl-CoA (R = Br), (C) chloroacetyl-CoA (R = Cl),
and (D) cyanoacetyl-CoA (R = CN). (E) Taft plot for the acylation
step of FlK-catalyzed acyl-CoA hydrolysis. Acylation rate constants
(k2) are derived from nonlinear curve
fitting of pre-steady-state kinetic time courses. Values are reported
as means ± the standard deviation (n = 3). The
linear fit gave a ρ* value of 1.7 ± 0.2 (R2 = 0.986).
Pre-steady-state
kinetic and Taft analysis of a series of acyl-CoAs.
The dashed line indicates 1 equiv of enzyme: (A) acetoacetyl-CoA (R
= Ac), (B)bromoacetyl-CoA (R = Br), (C) chloroacetyl-CoA (R = Cl),
and (D) cyanoacetyl-CoA (R = CN). (E) Taft plot for the acylation
step of FlK-catalyzed acyl-CoA hydrolysis. Acylation rate constants
(k2) are derived from nonlinear curve
fitting of pre-steady-state kinetic time courses. Values are reported
as means ± the standard deviation (n = 3). The
linear fit gave a ρ* value of 1.7 ± 0.2 (R2 = 0.986).A plot of log k2 versus σ*
for
the series of acyl-CoAs revealed a linear relationship (Figure 2E), indicating that acylation of all of the substrates
proceeds through a common mechanism and transition state, which is
in contrast to what is observed when kcat is plotted similarly.[16] On the basis
of this linear relationship, specificity in FlK acylation appears
to be based on inductive carbonyl polarization rather than a change
in mechanism, rate-limiting step, or transition-state structure induced
by the fluorine substituent. The slope of the line, ρ*, is the
polar sensitivity factor that compares the reaction under study to
the reference reaction, which is methyl ester hydrolysis.[16] A slope of >1 shows that the reaction under
study is more sensitive to substituents than the reference reaction,
while a slope of <1 shows that it is less sensitive. The Taft plot
for acylation gave a ρ* value of 1.7 ± 0.2, indicating
that FlK acylation is more sensitive to the influence of substituents
that alter induced carbonyl polarization than the chemical hydrolysis
of methyl esters on which the Taft scale is based. This sensitivity
may be attributable to the inductive polarization of thioesters being
higher than that of esters, or to additional activation of the carbonyl
group on the enzyme. In combination with the mutagenesis studies discussed
above, our Taft analysis of FlK substrates suggests that specificity
in the rate of enzyme acylation is determined mainly by the intrinsic
polarization of the fluoroacetyl-CoA thioester compared to the acetyl-CoA
thioester rather than by features of the enzyme that specifically
accelerate reaction with the fluorinated substrate.
Investigating
the Role of Fluorine Molecular Recognition in
FlK-Catalyzed Thioester Hydrolysis
Although our studies suggest
that the main selectivity determinant for FlK acylation is the intrinsic
reactivity of the fluoroacetyl group, this finding does not exclude
the possibility that the C–F bond is recognized in the enzyme
active site. If so, interactions between FlK and the fluoroacetyl-CoA
substrate could reduce free rotation of the fluoromethyl group such
that each of the two prochiral α-protons would occupy a different
position in three-dimensional space. In this case, we might expect
that FlK could exhibit a kinetic preference for substrates with an
available pro-R or pro-S proton. To test this hypothesis, we prepared
(RS)-2-fluoropropionyl-CoA, a substrate in which
one hydrogen at the α-carbon of a fluoroacetyl-CoA has been
replaced by a methyl group (Figure 3A). Upon
incubation of this substrate with FlK, we observed two steady-state
kinetic phases (Figure 3B and Figure S5 of
the Supporting Information), suggesting
that the two stereoisomers may be hydrolyzed at different rates specified
by their relative values of kcat/KM. A plot of the fast and slow rates as a function
of substrate concentration revealed that both phases exhibit saturation
kinetic behavior (Figure 3C). Although the
true kcat and KM for the two individual stereoisomers cannot be extracted from this
data, we hypothesized that these two phases might correspond to different
rates of hydrolysis for the different stereoisomers.
Figure 3
Kinetic analysis of 2-fluoropropionyl-CoA
hydrolysis. (A) 2-Fluoropropionyl-CoA
stereoisomer structures. (B) Time course for hydrolysis of 100 μM
(RS)-2-fluoropropionyl-CoA. (C) Observed rate constants
(kobs) for the slow phases and fast phases
plotted over a range of substrate concentrations. (D) Steady-state
kinetic analysis of (S)-2-fluoropropionyl-CoA. Values
are reported as means ± the standard deviation (n = 3). (E) Steady-state kinetic analysis of (R)-2-fluoropropionyl-CoA.
Values are reported as means ± the standard deviation (n = 3). (F) Pre-steady-state kinetic analysis of FlK-catalyzed
hydrolysis of (S)-2-fluoropropionyl-CoA. (G) Pre-steady-state
kinetic analysis of FlK-catalyzed hydrolysis of (R)-2-fluoropropionyl-CoA. (H) Analysis of FlK acylation by 2-fluoropropionyl-CoA
in terms of the Felkin–Ahn model. The steric and electronic
preferences of the substrate in combination with the structural constraints
of the enzyme may explain the defect in acylation rate for (S)-2-fluoropropionyl-CoA compared to (R)-2-fluoropropionyl-CoA.
Kinetic analysis of 2-fluoropropionyl-CoA
hydrolysis. (A) 2-Fluoropropionyl-CoA
stereoisomer structures. (B) Time course for hydrolysis of 100 μM
(RS)-2-fluoropropionyl-CoA. (C) Observed rate constants
(kobs) for the slow phases and fast phases
plotted over a range of substrate concentrations. (D) Steady-state
kinetic analysis of (S)-2-fluoropropionyl-CoA. Values
are reported as means ± the standard deviation (n = 3). (E) Steady-state kinetic analysis of (R)-2-fluoropropionyl-CoA.
Values are reported as means ± the standard deviation (n = 3). (F) Pre-steady-state kinetic analysis of FlK-catalyzed
hydrolysis of (S)-2-fluoropropionyl-CoA. (G) Pre-steady-state
kinetic analysis of FlK-catalyzed hydrolysis of (R)-2-fluoropropionyl-CoA. (H) Analysis of FlK acylation by 2-fluoropropionyl-CoA
in terms of the Felkin–Ahn model. The steric and electronic
preferences of the substrate in combination with the structural constraints
of the enzyme may explain the defect in acylation rate for (S)-2-fluoropropionyl-CoA compared to (R)-2-fluoropropionyl-CoA.To test this idea, we prepared the individual (S)- and(R)-2-fluoropropionyl-CoA stereoisomers (Figure 3A) from the corresponding chiral methyl lactates
and measured the steady-state kinetic parameters for each stereoisomer
(Figure 3D,E). Indeed, we observed that each
stereoisomer was hydrolyzed at a different rate. Interestingly, when
these substrates are added to the steady-state Taft plot for log kcat versus σ*,[10] the S isomer falls more on the same line as fluoroacetyl-CoA
and chloroacetyl-CoA while the R isomer appears to
behave more like the less inductively polarized substrates (Figure
S6 of the Supporting Information). This
result can be interpreted to indicate that there is a change in mechanism
or rate-limiting step between the S and R isomers and that hydrolysis of the S isomer proceeds
through the same kinetic and chemical mechanism as fluoroacetyl-CoA,
while hydrolysis of the R isomer proceeds through
the same kinetic and chemical mechanism as acetyl-CoA. However, we
cannot rule out the possibility that a rate-limiting rearrangement
of the R isomer acyl-enzyme intermediate produces
differing kinetic behavior but allows this substrate to access the
same chemical mechanism for hydrolysis as the S isomer.
Nonetheless, the kinetic data indicate that fluorine-based polarization
of the substrate alone is insufficient to dictate the reaction pathway
through which thioester hydrolysis proceeds.In addition to
the difference in kcat, the two stereoisomers
diverge with regard to their KM values,
with the R isomer exhibiting
a KM lower than that measured for the S isomer. This observation could be explained by either
preferential binding of the R isomer, faster acylation
by the R isomer, or slower deacylation of the R isomer. To determine the basis of the observed KM difference, we examined the pre-steady-state
kinetic behavior of each isomer under saturating conditions (Figure 3F,G). For (S)-2-fluoropropionyl-CoA,
we observed single-phase kinetics with a rate constant equal to kcat for the S isomer, which
indicates that acylation is rate-limiting for this substrate as it
is for the native substrate (Figure 3F). In
contrast, we observed a burst of 1 equiv of CoA formed with (R)-2-fluoropropionyl-CoA, followed by a slower steady-state
rate with a rate constant matching the kcat of the R isomer (Figure 3G). We interpret this kinetic behavior to demonstrate that rate-liming
step for the R isomer has changed to breakdown of
the acyl-enzyme intermediate (k3 = kcat). Taken together, the results of the pre-steady-state
kinetic experiments are also consistent with the behavior of the isomers
with regard to the linear free energy plot (Figure S6 of the Supporting Information). Additional kinetic modeling
using these rate constants shows that the differences in the rates
of chemical catalysis between the two substrates are sufficient to
explain their differing steady-state kinetics without the need to
invoke differential binding affinities (Figure S7 of the Supporting Information).Because the only
significant difference between the S and R isomers is expected to be the orientation
of the α-substituents, the differences in the rate constants
of the acylation (2-fold) and deacylation (4-fold) steps between the
two stereoisomers suggest that the fluorine atom is specifically recognized
by the enzyme and that the α-carbon does not freely rotate.
Indeed, the difference observed in the deacylation rate for each isomer
can be explained by a requirement for the α-proton to be accessible
to His 76 for the substrate to access the faster Cα deprotonation pathway.[10] The orientation
of the substrate via the fluorine atom could result in the methyl
group occupying the space where an α-proton would normally be
poised for abstraction by His 76, which could lead to a change in
the mechanism from Cα deprotonation to addition of
water followed by elimination of the Glu 50 leaving group. On the
basis of the kinetic data, we hypothesize the His 76 preferentially
abstracts the pro-R proton, leading
to a compromised deacylation rate when the methyl group occupies this
position instead. However, the basis for the difference between the
acylation rates is more difficult to explain. Considering an addition–elimination
mechanism for enzyme acylation, the observed difference in the acylation
rates is likely due to the increased steric bulk of the additional
methyl substituent, which kinetic data suggest is positioned unfavorably
for acylation in the S isomer. One possible source
of this kinetic selectivity is that recognition of the fluorine substituent
by FlK could force nucleophilic attack by Glu 50 to occur along a
trajectory that is disfavored based on the Felkin–Anh model
for carbonyl addition (Figure 3H).[19,20]
Stereochemical Course of the FlK Reaction
To directly
test the stereochemical course of acylation and deacylation without
the need for the enzyme to accommodate an additional α-substituent
in its active site, we prepared (S)-[2H1]fluoroacetyl-CoA and (R)-[2H1]fluoroacetyl-CoA from the corresponding carboxylic
acids.[14] Our previous kinetic studies with
[2H2]fluoroacetyl-CoA showed that there is a
kinetic isotope effect (KIE) of 2.4 ± 0.1 on the deacylation
step of the reaction mechanism, consistent with abstraction of an
α-proton from the substrate in this step.[10] We therefore predict that if the enzyme utilizes chiral
recognition of the prochiral fluoromethyl group in the deacylation
step, one of the [2H1]fluoroacetyl-CoA stereoisomers
will have its 2H positioned for proton abstraction and
will exhibit a primary KIE, while the other stereoisomer will show
a much smaller secondary KIE, which may be negligible compared to
the error in the rates. In contrast, based on the addition–elimination
mechanism that we have proposed for the acylation step, we would expect
both substrates to exhibit a secondary inverse KIE on acylation, which
may be too small to detect by direct comparison of rates.To
interrogate both the acylation and deacylation steps using the chiral
deuterated substrates, we examined the pre-steady-state kinetics of
their FlK-catalyzed hydrolysis (Figure 4).
Similar to the unlabeled fluoroacetyl-CoA, (S)-[2H1]fluoroacetyl-CoA exhibited single-phase pre-steady-state
kinetic behavior with the observed kcat within error of that for the undeuterated substrate. While this
result does not conclusively demonstrate the presence of an inverse
secondary KIE, it not inconsistent considering the propagated error.
In contrast, (R)-[2H1]fluoroacetyl-CoA
exhibited burst-phase kinetic behavior. While the acylation rate constant
for this substrate was within error of the kcat observed for the undeuterated substrate, a kinetic isotope
effect (KIE) of 1.7 ± 0.2 was observed on the steady-state rate,
consistent with the previously reported KIE of 2.4 observed with the
doubly deuterated [2H2]fluoroacetyl-CoA substrate.
This result provides further evidence that while the acylation rate
is influenced mainly by polarization, consistent with an addition–elimination-type
mechanism, deacylation requires specific recognition of the prochiral
fluoromethyl group to position the α-carbon for proton abstraction
catalyzed by His 76, which preferentially abstracts the pro-R proton. Notably, the mechanistically related
hot dog-fold dehydratases, which utilize a mechanism initiated by
Cα deprotonation to catalyze elimination of water
from 3-hydroxyacyl-CoAs, also conduct stereoselective deprotonation.[21,22] While the pro-2S proton is removed
by dehydratases, it has the same relative stereochemistry as the pro-2R proton in fluoroacetyl-CoA because
the S designation is derived from a change in substituent
priorities based on the Cahn–Ingold–Prelog rules. While
the proton abstraction occurs on an acyl-enzyme intermediate in FlK
instead of on the substrate itself, the commonality in mechanism between
the hot dog-fold dehydratases and FlK is consistent with their surprisingly
close evolutionary relationship,[10] as well
as the structural similarity of their active sites.[9,23]
Figure 4
Pre-steady-state
kinetic analysis of monodeuterated fluoroacetyl-CoAs.
FlK-catalyzed hydrolysis of (A) undeuterated fluoroacetyl-CoA (left)
is compared to hydrolysis of (B) (S)-[2H1]fluoroacetyl-CoA (center) and (C) (R)-[2H1]fluoroacetyl-CoA (right).
Pre-steady-state
kinetic analysis of monodeuterated fluoroacetyl-CoAs.
FlK-catalyzed hydrolysis of (A) undeuterated fluoroacetyl-CoA (left)
is compared to hydrolysis of (B)(S)-[2H1]fluoroacetyl-CoA (center) and (C) (R)-[2H1]fluoroacetyl-CoA (right).
Conclusions
Our data suggest a chemical,
kinetic, and structural mechanism
for the selectivity of FlK for fluoroacetyl-CoA (Scheme 2). Following substrate binding, fluoroacetyl-CoA reacts with
Glu 50 to acylate this side chain. Taft free energy relationship analysis
of the acylation step (Figure 2E) indicates
that the observed enhancement of 2 orders of magnitude in the rate
of acylation for fluoroacetyl-CoA compared to that of acetyl-CoA is
provided by the intrinsic inductive polarization of the fluoroacetyl-CoA
carbonyl group. Kinetic studies of (R)- and(S)-2-fluoropropionyl-CoA demonstrated that although these
substrates differ only by the orientation of their α-substituents,
their rates of acylation differ by 2-fold. Given the differences in
acylation rate between the two 2-fluoropropionyl-CoA stereoisomers,
we propose that the C–F bond is specifically recognized by
FlK, allowing it to distinguish the two enantiotopic faces of the
carbonyl group during the acylation step.
Scheme 2
Model for the Influence
of Fluorine Recognition on the Course of
the FlK-Catalyzed Reaction
On the basis of
the kinetic
analysis of substrates with a stereogenic center at the α-carbon,
we propose that Glu 50 attacks the fluoroacetyl-CoA thioester bond
from the re face. The pro-R proton of the resultant intermediate is then removed by
His 76. Following hydration of the putative intermediate, the carboxylic
acid enolate is reprotonated on the same face from which the proton
was removed.
Model for the Influence
of Fluorine Recognition on the Course of
the FlK-Catalyzed Reaction
On the basis of
the kinetic
analysis of substrates with a stereogenic center at the α-carbon,
we propose that Glu 50 attacks the fluoroacetyl-CoA thioester bond
from the re face. The pro-R proton of the resultant intermediate is then removed by
His 76. Following hydration of the putative intermediate, the carboxylic
acid enolate is reprotonated on the same face from which the proton
was removed.After FlK acylation, a proton
is abstracted by His 76 to generate
an enolate that can break down through a proposed ketene intermediate.
A 4-fold difference in the rates of deacylation of (S)- and(R)-2-fluoropropionyl-CoA suggested that
FlK might utilize chiral recognition of the prochiral fluoromethyl
group of fluoroacetyl-CoA in the deacylation step to specifically
position one proton for abstraction. Indeed, kinetic studies of (S)- and(R)-[2H1]fluoroacetyl-CoA
demonstrated a primary KIE of 1.7 ± 0.2 on only the R stereoisomer, suggesting that the pro-R proton is specifically abstracted. Taken together, our studies of
FlK substrates with a stereogenic center at the α-carbon provide
evidence that the C–F bond is specifically recognized by FlK
to orient the α-carbon for proton abstraction catalyzed by His
76. The previously reported lack of exchange between the fluoroacetyl-CoA
α-protons and solvent[10] along with
the pH–rate profiles of FlK and FlK-H76A (Figure 1B,C) suggests that FlK may utilize a one-base mechanism to
achieve this transformation, rather than separate catalytic residues
that function in general acid catalysis and general base catalysis.
Therefore, following hydration of the putative ketene intermediate,
we propose that the resultant enolate is reprotonated by the His 76
imidazolium side chain on the same face from which deprotonation occurred.
This mechanistic proposal is similar to the one-base mechanism elucidated
for the hot dog-fold dehydratases.[21,22]Taken
together, our results suggest that molecular recognition
of fluorine, rather than simple inductive polarization and enolate
stability, is involved in making the Cα deprotonation
pathway accessible to the fluorinated substrate. Thus, substrate polarization
appears to be necessary, but not sufficient, for rate acceleration.
This observation provides a possible explanation for the inability
of substrates like acetoacetyl-CoA, which is expected to form a more
stable enolate than fluoroacetyl-CoA but is much larger in size, to
access the Cα deprotonation pathway. In the absence
of a high-resolution structure of the FlK–substrate complex,
it is difficult to precisely define the structural determinants of
chiral fluoromethyl group recognition. However, previously reported
structural and mutagenesis studies of FlK have suggested multiple
possible mechanisms through which molecular recognition of the C–F
bond may be achieved. Computational docking studies have suggested
that the guanidinium group of Arg 120 and the backbone amide of Gly
69 may be involved in C–F recognition through dipolar interactions,[9,17] while mutagenesis data suggest that the hydrophobic nature of the
FlK active site may contribute to molecular recognition of the C–F
bond based on its polar hydrophobicity.[9]Although fluorine’s ability to initiate unusual enzymatic
reaction pathways involved in mechanism-based inhibition is well-known,
FlK represents a rare example in which an enzyme exploits the unique
properties of fluorine to enhance its reaction rate and to drive substrate
selectivity. The results reported here demonstrate that discrimination
of the fluoroacetyl-CoA substrate relies not only on the enhanced
inductive polarization afforded by fluorine’s high electronegativity
but also on the molecular recognition of fluorine to position the
substrate for optimal reactivity. These studies of FlK provide insights
into how enzymes evolve to recognize unusual functional groups, with
implications for fluorine molecular recognition and the design of
mechanism-based inhibitors.
Authors: Fanglu Huang; Stephen F Haydock; Dieter Spiteller; Tatiana Mironenko; Tsung-Lin Li; David O'Hagan; Peter F Leadlay; Jonathan B Spencer Journal: Chem Biol Date: 2006-05
Authors: Feng Song; Zhihao Zhuang; Lorenzo Finci; Debra Dunaway-Mariano; Ryan Kniewel; John A Buglino; Veronica Solorzano; Jin Wu; Christopher D Lima Journal: J Biol Chem Date: 2006-02-07 Impact factor: 5.157
Authors: M Sanada; T Miyano; S Iwadare; J M Williamson; B H Arison; J L Smith; A W Douglas; J M Liesch; E Inamine Journal: J Antibiot (Tokyo) Date: 1986-02 Impact factor: 2.649
Authors: Patrick Bentler; Klaus Bergander; Constantin G Daniliuc; Christian Mück-Lichtenfeld; Ravindra P Jumde; Anna K H Hirsch; Ryan Gilmour Journal: Angew Chem Int Ed Engl Date: 2019-07-03 Impact factor: 15.336
Authors: Alexander Axer; Ravindra P Jumde; Sebastian Adam; Andreas Faust; Michael Schäfers; Manfred Fobker; Jesko Koehnke; Anna K H Hirsch; Ryan Gilmour Journal: Chem Sci Date: 2020-11-23 Impact factor: 9.825
Scheme 1
Figure 1
Figure 2
Figure 3
Figure 4
Scheme 2