On the thermodynamic equilibrium between (R)-2-hydroxyacyl-CoA and 2-enoyl-CoA

Article abstract of DOI:10.1111/j.1742-4658.2010.07597.x

A combined experimental and computational approach has been applied to investigate the equilibria between several α-hydroxyacyl-CoA compounds and their 2-enoyl-CoA derivatives. In contrast to those of their β, γ and α counterparts, the equilibria for the α-compounds are relatively poorly characterized, but qualitatively they appear to be unusually sensitive to substituents. Using a variety of techniques, we have succeeded in measuring the equilibrium constants for the reactions beginning from 2-hydroxyglutaryl-CoA and lactyl-CoA. A complementary computational evaluation of the equilibrium constants shows quantitative agreement with the measured values. By examining the computational results, we arrive at an explanation of the substituent sensitivity and provide a prediction for the, as yet unmeasured, equilibrium involving 2-hydroxyisocaproyl-CoA.

Full text of DOI:10.1111/j.1742-4658.2010.07597.x

On the thermodynamic equilibrium between
(R)-2-hydroxyacyl-CoA and 2-enoyl-CoA
Anutthaman Parthasarathy1, Wolfgang Buckel1 and David M. Smith2
1 Laboratory for Microbiology, Philipps-Universitat, Marburg, Germany
¨
2 Centre for Computational Solutions in the Life Sciences, Rudjer Boskovic Institute, Zagreb, Croatia
Keywords
A combined experimental and computational approach has been applied to
ab initio calculations; enzymes; kinetics;
investigate the equilibria between several a-hydroxyacyl-CoA compounds
and their 2-enoyl-CoA derivatives. In contrast to those of their b, c and d
counterparts, the equilibria for the a-compounds are relatively poorly char-
acterized, but qualitatively they appear to be unusually sensitive to substit-
uents. Using a variety of techniques, we have succeeded in measuring the
equilibrium constants for the reactions beginning from 2-hydroxyglutaryl-
CoA and lactyl-CoA. A complementary computational evaluation of the
equilibrium constants shows quantitative agreement with the measured
values. By examining the computational results, we arrive at an explanation
of the substituent sensitivity and provide a prediction for the, as yet
unmeasured, equilibrium involving 2-hydroxyisocaproyl-CoA.
solvent effects; substituent effects
Correspondence
D. M. Smith, Centre for Computational
Solutions in the Life Sciences, Rudjer
Boskovic Institute, Bijenicka 54, 10000
Zagreb, Croatia
Fax: +385 1 456 1182
Tel: +385 1 456 1182
E-mail: David.Smith@irb.hr
(Received 20 December 2009, revised
25 January 2010, accepted 28 January
2010)
doi:10.1111/j.1742-4658.2010.07597.x
Introduction
Dehydratases catalyze a,b-eliminations of water from
hydroxy compounds, and form large class of
in the resulting enoxy radical intermediate (4) by at
least 26 units [5]. Recycling of the initiatory electron
from the second ketyl intermediate thus produced (5)
yields enoyl-CoA (2) and completes the catalytic
cycle.
a
enzymes; over 100 different types are listed in the
enzyme nomenclature database (EC 4.2.1.–). In most
cases, the hydroxyl group is located in the b-position
of an adjacent carboxylate, CoA-thioester or ketone,
and the a-proton to be removed is thus activated. In
a-amino acid fermentation pathways, however, dehy-
dratases are found, whose substrates are a-hydroxya-
cyl-CoA derivatives [1,2]. In such cases, the
b-hydrogen to be removed during a,b-elimination of
water has an approximate pKa of 40. The requisite
activation of this proton is achieved by transient
addition of one high-energy electron to the thioester
carbonyl, forming a ketyl radical anion (3, Scheme 1).
This allows the elimination of the a-hydroxyl group
[3,4] (3 fi 4) and lowers the pKa of the b-hydrogen
Recently, the mechanism shown in Scheme 1 has
received strong support with the reported observation
of the allylic ketyl radical intermediate (5) in the
enyzmatic dehydration of (R)-2-hydroxyisocaproyl-
CoA [1a, R = CH(CH3)2; Fig. 1].[4] However, no
such observations have yet been possible for the
analogous dehydrations of (R)-2-hydroxyglutaryl-CoA
(1b, R = CH2CO2H) or (R)-lactyl-CoA (1c, R = H).
Whereas the equilibrium constants (K) of b-hydrox-
yacyl-CoA, c-hydroxyacyl-CoA or d-hydroxyacyl-CoA
typically lie around unity [6], the situation is much less
clear for the a-hydroxyacyl-CoA derivatives. For
Abbreviations
Nbs2, 5,5¢-dithiobis(2-nitrobenzoate); TFA, trifluoroacetic acid; THF, trifluoroacetic acid.
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A. Parthasarathy et al.
Equilibrium between hydroxyacyl-CoA and enoyl-CoA
equilibria shown in Fig. 1. Specifically, using a bidirec-
tional kinetic analysis, we present experimental deter-
minations of Kb and Kc. The obtained values are
supported and rationalized by high-level ab initio
molecular orbital calculations, which are also used to
obtain a value for the elusive Ka.
Results and Discussion
For the determination of the experimental value of Kb,
we performed activity measurements in both the for-
ward and reverse directions, requiring the preparation
of both 1b and 2b. Although an enzymatic method
was potentially applicable for the production of 1b,
the incubations in that case also yielded the thermo-
dynamically more favorable (R)-4-hydroxyglutaryl-
CoA, which is not dehydrated [11]. To avoid this
complication, a chemical method for the preparation
of 1b was used; namely, (R)-2-hydroxyglutaryl-CoA
(1b) was prepared by the direct reaction of CoASH
and (R)-butyrolactone-5-carboxylchloride in aqueous
NaHCO3 [11]. After the thiol had been consumed, the
reaction was acidified to pH 0.5 and incubated at
25 ꢀC for 3 h until the equilibrium between lactone-
CoA and 2-hydroxyglutaryl-CoA (1 : 4) was estab-
lished. Prior to use, the mixture was neutralized,
whereby equilibration was terminated. It was shown
that the remaining lactone-CoA did not interfere with
the dehydration. In the case of glutaconyl-CoA (2b)
preparation, there were no complicating factors, and
the target compound was prepared by incubation of
glutaconate with acetyl-CoA, catalyzed by glutaconate-
CoA transferase from Acidaminococcus fermentans
produced in Escherichia coli [12].
Scheme 1. Electron recycling mechanism for the conversion of
a-hydroxyacyl-CoA derivatives to the corresponding enoyl-CoA
compounds.
example, it has recently been determined that the enzy-
matic dehydration of 1a (also known as 2-hydroxy-
4-methylpentanoyl-CoA), derived from the amino acid
(S)-leucine, to isocaprenoyl-CoA (4-methyl-2-pente-
noyl-CoA, probably the E-isomer, 2a), occurs irrevers-
ibly, within the limits of detection [7]. In contrast, the
equilibrium of the dehydration of (R)-lactyl-CoA to
acryloyl-CoA (1c fi 2c) strongly favors the hydroxy
compound. Under physiological conditions, (R)-lactyl-
CoA is only effectively dehydrated, because the very
small equilibrium concentration of the unsaturated
compound (2c) is irreversibly trapped by the consecu-
tive reductase, resulting in propionyl-CoA [8,9]. With
(R)-2-hydroxyglutaryl-CoA (1b) and (E)-glutaconyl-
CoA (2b) as substrates, the equilibrium appears to lie
more in the middle [10], although the value of Kb, like
those of Ka and Kc, is presently unknown.
For our source of 2-hydroxyglutaryl-CoA dehydra-
tase, we employed the strictly anaerobic bacterium
Clostridium symbiosum, which is involved in the
fermentation of glutamate to ammonia, CO2, acetate,
butyrate, and H2. The heterodimeric enzyme
In order to place the known qualitative results on a
more solid footing, we adopted a combined experimen-
tal and computational approach to characterize the
H
[2] [H2O]
[1]
H OH
K
Kconc.
=
R
O
R
O
H2O
H H
H
SCoA
SCoA
[2]
[1]
Kconc.
K =
=
55.5
1
2
Ka >> 1
2-hydroxyisocaproyl-CoA (1a)
2-hydroxyglutaryl-CoA (1b)
lactyl-CoA (1c)
E-isocaprenoyl-CoA (2a) + H2O
E-glutaconyl-CoA (2b) + H2O
acryloyl-CoA (2c) + H2O
R=CH(CH3)2
Kb ~ 1
Fig. 1. Dehydrations of the a-hydroxyacyl-
CoA derivatives discussed in this work. As
the biochemical experiments were
performed in dilute aqueous conditions, we
employ K, as opposed to Kconc., for
convenience.
R=CH2CO2H
Kc << 1
R=H
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1739

Equilibrium between hydroxyacyl-CoA and enoyl-CoA
A. Parthasarathy et al.
(48 + 43 kDa) contains, per mole (91 kDa), 1 mol of
FMNH2 and 8 mol of iron + 8 mol of sulfur, proba-
bly as two [4Fe–4S] clusters. It has to be activated by
from A. fermentans [13]. The CoA-thioesters were pre-
pared from acrylate, (R)-lactate and 3-pentynoic acid
[16] by the carbonyl-diimidazole method [17], and
analyzed enzymatically and by MALDI-TOF MS
[18]. The kinetic parameters for the hydration of
acryloyl-CoA (2c) to (R)-lactyl-CoA (1c) were
incubation with a reducing agent, ATP, and Mg2+
,
mediated by a homodimeric protein (2 · 27 kDa) with
one [4Fe–4S]+ cluster between the two subunits.
Thereby, one electron is transferred from the activator
protein to the dehydratase, driven by hydrolysis of two
molecules of ATP. Prior to use in our activity mea-
surements, 2-hydroxyglutaryl-CoA dehydratase, puri-
fied from C. symbiosum, was activated under strict
anaerobic conditions with ATP, MgCl2 and dithionite,
mediated by catalytic amounts of the activator from
A. fermentans produced in E. coli [13].
measured as Km = 0.150
85 yielding kcat = 126
6 UÆmg)1
[kcat ⁄ Km (2c)] = 0.84
0.05 · 106 s)1Æm)1
0.004 mm and Vmax
10 s)1 and
Because
=
,
.
the equilibrium concentration of acryloyl-CoA (2c) is
very low, it was more difficult to determine the kinetics
of (R)-lactyl-CoA dehydration (1c fi 2c). Reasonable
estimates are given by: Km = 0.32
0.02 mm and
Vmax = 3.0
0.4 UÆmg)1, with kcat = 4.5
0.6 s)1
The dehydration (1b fi 2b) and hydration
(2b fi 1b) reactions were followed spectrophotometri-
cally at 290 nm (e290 nm = 2.1 mm)1Æcm)1). Although
the absorbance maximum of enoyl-CoA (2b) lies at
260 nm (De260 nm = 6.0 mm)1Æcm)1), the longer wave-
length was chosen to avoid interference with the high
absorbance of the adenine moiety of CoA
(e260 nm = 16 mm)1Æcm)1) [7].
and [kcat ⁄ Km (1c)] = 1.41
0.1 · 104 s)1Æm)1). Substi-
tution of these data into the Briggs–Haldane equation
yields:
Kc ¼ ½kcat=Kmð1cÞꢀ=½kcat=Kmð2cÞꢀ ¼ 0:017 ꢁ 0:007
This low value of Kc corroborates the high redox
potential of the acryloyl-CoA ⁄ propionyl-CoA pair
(E0¢ = + 69 mV) as compared with those of the higher
homologs of 2-enoyl-CoA ⁄acyl-CoA (E0¢ = )10 mV)
[19].
The relative magnitudes of Kb and Kc confirm an
unexpectedly large (ꢂ 20 kJÆmol)1) substituent effect
on the dehydration equilibrium, arising from the pres-
ence of a carboxymethyl group on the b-carbon in 1b
in place of a hydrogen atom in 1c. This fact, combined
with the similarly large effect that was apparent upon
further substitution by the isopropyl group in 1a, led
us to perform ab initio molecular orbital calculations
in order to seek an explanation.
The activity measurements between 0 and 1 mm (R)-
2-hydroxyglutaryl-CoA (1b) and 0 and 5 mm glutaco-
nyl-CoA (2b) gave smooth Michaelis–Menten curves,
from which Km and kcat values could be calculated
by simulation. In experiments starting with
(R)-2-hydroxyglutaryl-CoA (1b), we obtained a Km of
0.052
0.003 mm and a kcat of 83
8 s)1, resulting
in
1600
a
specificity constant [kcat ⁄ Km (1b)] of
300 s)1Æmm)1. Beginning the reaction with,
instead, (E)-glutaconyl-CoA (2b) resulted in a Km of
0.25
with
28
0.02 mm and a kcat of 7.0
specificity constant [kcat ⁄ Km (2b)] of
6 s)1Æmm)1. Kb was subsequently calculated from
0.7 s)1, associated
a
To enable high-accuracy calculations, we elected to
replace the adenylphosphopantetheine chain of CoA
by the S-CH3 group, resulting in the model systems
shown in Fig. 2. We expected this substitution to have
only a minor effect on the individual equilibrium con-
stants and, because it is adopted uniformly, there
should be virtually no effect on the relative equilibrium
constants. Although our final goal was to compute the
free energies of the reactions shown in Fig. 1 under
aqueous conditions, we elected to present the gas-
phase results as well. The rationale behind this is that
the gas-phase calculations encompass the fundamental
electronic effects governing the differences in the equi-
librium constants. By decomposing the final aqueous
energy differences into a gas-phase component and a
component related to solvation, we are thus able to
comment on the extent to which each of these aspects
influences the final result.
the Briggs–Haldane equation:
Kb ¼ ½kcat=Kmð1bÞꢀ=½kcat=Kmð2bÞꢀ ¼ 1600=28 ¼ 57 ꢁ 1:5
For the measurement of Kc, the equilibrium constant
for (R)-lactyl-CoA (1c) and acryloyl-CoA (2c), we
purified lactyl-CoA dehydratase from the strict anaer-
obe Clostridium propionicum to apparent homogeneity.
Like 2-hydroxyglutaryl-CoA dehydratase, lactyl-CoA
dehydratase is a heterodimer (a, 48 kDa; b, 41 kDa)
containing two [4Fe–4S] clusters and substoichiometric
amounts of FMN and riboflavin [9,14]. The purified
enzyme was treated with 3-pentynoyl-CoA to abolish a
slight acryloyl-CoA reductase activity [9,15]. Activation
of the dehydratase occurred under conditions similar
to that used for the C. symbiosum dehydratase in the
presence of Mg-ATP, dithionite, and the activator
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A. Parthasarathy et al.
Equilibrium between hydroxyacyl-CoA and enoyl-CoA
Kcaalc.= 1610
+
Keaxpt.> 1000
1a
2a
Kcbalc.= 8.42
+
Kebxpt.= 57
1b
1c
2b
Kccalc.= 0.02
+
Fig. 2. Comparison of the calculated
and experimental equilibrium constants
determined in this work.
Kecxpt.= 0.017
2c
Table 1. Experimentally determined and calculated values for the
Table 2. Calculated free energies of solvation (DGsꢄ) for the species
shown in Fig. 1.
equilibria represented by Fig. 1.
Calculated [G3(MP2)]
Experimental
1a
2a
1b
2b
1c
2c
H2O
)24.7
for x(g)
a,b
a,c
a,c
a
Equilibrium
DGðꢃgÞ
DGðꢄaqÞ
Kd
DGðꢄaqÞ
Kd
DGsꢄ
)4.4
)6.8
)43.7
)40.6
)16.4
)2.5
The calculated free energy of solvation (in kJÆmol)1
(1 molÆL-1)
are corrected by RT ln(RT) for each species. See text.
)
a
A
B
C
)9.0
)1.6
2.9
)28.3
)15.2
0.1
1610
8.42
0.02
< )27.1
)19.9
0.1
> 1000
57
fi
x(aq) (1 molÆL)1). The final DGðꢄaqÞ values in Table 1
0.017
~
a
b
c
kJÆmol)1 at 298 K.
1 atm reference.
Dimensionless, K = Kconc. ⁄ 55.5 (see Fig. 1).
1 molÆL)1 reference.
d
solution-phase equilibrium constants. It thus appears
that the explanation of the unusually large substituent
effects is not related to fundamental electronic factors
at the molecular level.
Table 1 shows the standard (referenced to 1 atm of
pressure) free energy change for each reaction in the
gas phase [1(g) fi 2(g) + H2O(g); DGðꢃgÞ]. (Although 1
and 2 strictly represent the CoA thioester, for simplic-
ity, we keep the same notation for the methyl thioest-
ers used in the calculations.)
The standard free energy changes in aqueous solu-
tion [1(aq) fi 2(aq) + H2O(aq); DGðꢄaqÞ, referenced to
1 molÆL)1] could be expected to preferentially favor
the products, simply because of the sizeable solvation
free energy of water [experimentally, DGsꢄ (H2O) =
)26.5 kJÆmol)1 [21]]. This preference for the bimo-
lecular products is reduced by the reference state
In the gas phase, the dehydration of 1c was found
to be mildly (2.9 kJÆmol)1) endergonic. The introduc-
tion of a carboxymethyl substituent at the b-carbon
was found to preferentially stabilize the 2-enoyl spe-
cies (2b), such that dehydration of 2a was exergonic
correction (from 1 atm to 1 molÆL)1) of RT ln(RT)
~
of 7.9 kJÆmol)1 (at 298 K) for each species to
by 1.6 kJÆmol)1
.
The small associated substituent
)18.6 kJÆmol)1 (R = 0.082053 K)1). In the case of
~
effect (4.5 kJÆmol)1) can be rationalized by the mild
(net) capability of the alkyl substituent to donate
electrons to the electron-deficient b-carbon in 2c.
Indeed, acrylamide, with no substituent at the b-posi-
tion, has been shown to act as a toxic electrophilic
agent [20], an effect that should be more pronounced
in acryloyl-CoA (2c). The more electron-donating iso-
propyl substituent results in a larger preferential sta-
bilization for the enoyl species (2a), such that the
dehydration of 1a is exergonic by 9.0 kJÆmol)1. Both
of these relatively small, inherent (gas-phase) substitu-
ent effects are more in line with qualitative expecta-
tions than the values apparent from the measured
(R)-lactyl-CoA, the inherent product preference in
solution is partially compensated for by the relatively
large (absolute) value of DGsꢄ(1c) as compared with
DGsꢄ(2c) (Table 2). The result of these competing
effects is that DGðꢄaqÞ(C) is only 2.8 kJÆmol)1 less than
the corresponding gas-phase value. Despite the poten-
tial uncertainties involved, the final calculated value
for DGðꢄaqÞ(C) (0.1 kJÆmol)1) is in very good agreement
with that derived from the measured equilibrium con-
stant of 0.017 (0.1 kJÆmol)1).
The absolute magnitude of the free energies of
solvation of 1b and 2b are much larger than those of
1c and 2c, because of the presence of the hydrophilic
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Equilibrium between hydroxyacyl-CoA and enoyl-CoA
A. Parthasarathy et al.
carboxylic acid groups. Even though the hydroxyacyl
species (1b) species is again solvated more strongly
than the enoyl one (2b), the differenceꢄ between them,
and hence the associated effect on DGðaqÞ(B), is much
smaller than for reaction C. The favorable contribu-
tion from DGsꢄ (H2O) is therefore counteracted to a
muꢄch smaller extent, resulting in the value for
DGðaqÞ(B) being 13.6 kJÆmol)1 more negative than
DGðꢃgÞ (B). The corresponding calculated value of
Kb = 8.42 deviates somewhat from the measured
value of Kb = 57. In terms of energy, however, the
discrepancy of only 4.7 kJÆmol)1 is certainly within
acceptable limits for a solution-phase property.
Experimental procedures
Chemical synthesis of CoA thioesters
Acrylyl-CoA was synthesized under a gentle stream of N2
by reacting a three-fold molar excess of acrylyl chloride in
dry acetonitrile with free CoASH dissolved in 0.5 mL of
aqueous 0.5 m NaHCO3. The solution was stirred at room
temperature until no yellow color of free thiol was obtained
with 5,5¢-dithiobis(2-nitrobenzoate) (Nbs2) [22,23,24]. The
pH was adjusted with 5 m HCl to 2 and the solution was
stored at )20 ꢀC. In view of the instability of the com-
pound, it was prepared and purified the day before use.
2-Hydroxyglutaryl-CoA was synthesized from commer-
cial (R)-2-oxo-tetrafuran carboxylic acid that was converted
to the corresponding acid chloride by reacting with an
excess of oxalyl chloride at 60 ꢀC for 3 h. The excess oxalyl
chloride was removed by evaporation under reduced pres-
sure. Then, a three-fold excess of the acid chloride was
dissolved in dry acetonitrile and reacted with CoASH in
0.5 m NaHCO3 at room temperature, and the pH was low-
ered to 6 to obtain the lactone-CoA. Finally, the lactone-
CoA was equilibrated with (R)-2-hydroxyglutaryl-CoA at
pH 1 and 25 ꢀC for 3 h. The equilibration was stopped by
raising the pH to 8.0. The resulting (R)-2-hydroxyglutaryl-
CoA contained about 3% lactone as analyzed by MALDI-
TOF MS.
The more hydrophobic nature of 1a and 2a is
reflected in their less favorable solvation free energies
(Table 2). In this case, however, it is the enoyl species
(2a) that is better solvated than the hydroxyacyl one
(1a). This serves to slightly reinforce the favorable
effect of DGsꢄ (H2O) rather than to counteract it, as
occurred for equilibria B and C. The final result is that
equilibrium A is predicted to lie very far to the right,
with associated values of DGðꢄaqÞ(A) = )28.3 kJÆmol)1
and Ka = 1610. Given the good agreement between
theory and experiment obtained for equilibria B and
C, we are confident that these values provide a reason-
ably accurate description of the thermodynamics of
2-hydroxyisocaproyl-CoA dehydration. They are cer-
tainly consistent with the fact that the equilibrium
concentration of 1a was not detectable in experiments
concerning its conversion into 2a [7]. The precise
experimental determination of Ka, however, still
remains a challenge for the future.
3-Pentynoyl-CoA was synthesized by the procedure used
for making 3-pentynoyl pantetheine [15,9]. CoASH
(40 lmol) was suspended in 5 mL of dry acetone (CaSO4).
Another flask contained 60 lmol of dicyclohexylcarbodii-
mide in 5 mL of dry tetrahydrofuran (THF). To the THF
solution was added 0.1 g of 3-pentynoic acid. The THF and
acetone solutions were mixed quickly and stirred overnight
in a sealed flask at 4 ꢀC. The solution was filtered on a sin-
tered glass filter to remove dicyclohexylurea. The solvent was
removed with a rotary evaporator to yield oil, which was
treated with diethyl ether and dried under vacuum.
Conclusion
In summary, and in agreement with previous qualita-
tive observations, the measurements presented here
confirm an unusually large effect of the substituent at
the b-carbon on the equilibrium constants of the dehy-
dration reactions shown Fig. 1. Molecular orbital cal-
culations show that the inherent substituent effects, as
reflected in the gas-phase data, are less drastic. The
condensed-phase calculations reproduce the measured
values and reveal that the large effects are primarily
due to a complex interplay of competing effects con-
nected to the solvation process. The calculations pre-
dict that the, as yet, unmeasured equilibrium involving
(R)-2-hydroxyisocaproyl-CoA very strongly favors the
product. Further work is required to determine to
what extent this is connected with the successful obser-
vation of the proposed penultimate intermediate (5) in
the dehydration mechanism (Scheme 1) for reaction A
and not for reactions B or C.
Butyryl-CoA, propionyl-CoA and acetyl-CoA were
synthesized from their anhydrides [25]. To CoA (25 lmol)
in 1 mL of 0.5 m KHCO3, 35 lmol of the respective anhy-
dride in 0.5 mL of acetonitrile was added. After dilution to
5 mL with water, the mixture was reacted at room tempera-
ture until the Nsb2 test was negative, and then acidified to
pH 2. (R)-Lactyl-CoA was synthesized from (R)-lactate and
CoASH using 1,1¢-carbonyldiimidazole [17].
Enzymatic synthesis of glutaconyl-CoA with
glutaconate CoA-transferase
The incubation contained, in 5 mL of 50 mm potassium
phosphate (pH 7.0), 20 lmol of acetyl-CoA, 300 lmol of
glutaconic acid, and 5 U of recombinant transferase. After
1 h at 37 ꢀC, the mixture was acidified to pH 2 and
filtered through a 1 kDa membrane (Amicon; Amersham
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A. Parthasarathy et al.
Equilibrium between hydroxyacyl-CoA and enoyl-CoA
Biosciences, Freiburg, Germany; now part of GE Health-
care, Munich, Germany) to remove precipitated protein.
volume 0.5 mL at 25 ꢀC) with 50 mm Tris ⁄ HCl (pH 8.0),
5 mm MgCl2, 5 mm dithiothreitol, 0.4 mm ATP, and
0.1 mm dithionite, as well as the activator from A. fermen-
tans and dehydratase from C. symbiosum. After incubation
for 5 min, the reaction was started by addition of (R)-2-hy-
droxyglutaryl-CoA. The formation of (E)-glutaconyl-CoA
was measured at 290 nm (e290 nm = 2.2 mm)1Æcm)1) [7]. In
the reverse direction, (E)-glutaconyl-CoA was used as sub-
strate. The kinetic constants were determined with 2.0 lg of
dehydratase (specific activity of 54 lmol min)1Æmg)1 pro-
tein) and 0.6 lg of activator, using either 0.02–1.0 mm (R)-
2-hydroxyglutaryl-CoA or 0.1–5.0 mm glutaconyl-CoA.
Under these conditions, the minimum substrate ⁄ enzyme
ratio was 450 : 1. The data were fitted to the Michaelis–
Menten equation using the excel program. In the routine
assays during purification of the dehydratase, (R)-2-hy-
droxyglutaryl-CoA was replaced by (R)-2-hydroxyglutarate,
acetyl-CoA, and glutaconate CoA-transferase.
Purification of CoA thioesters by reverse-phase
chromatography
All CoA thioesters were purified by reverse-phase chroma-
tography through Sep-Pak C18 columns (Waters, Milford,
MA, USA). The reaction mixtures at pH 2 were freed from
solvents under reduced pressure and from precipitated
proteins by ultrafiltration. They were then loaded onto
C18 columns washed with methanol and equilibrated with
0.1% (v ⁄ v) trifluoroacetic acid (TFA). After washing with
three volumes of the same solution, elution was performed
with 0.1% TFA in 50% acetonitrile (v ⁄ v). The eluted CoA
esters were freed from acetonitrile on a Speed-Vac concen-
trator (Bachofer, Reutlingen, Germany) and vacuum-dried
on a lyophilizer (Alpha1-4; Christ Instruments, San Diego,
CA, USA). The lyophilized powders were stored at )80 ꢀC
until further use.
Prior to the assay of lactyl-CoA dehydratase from C. pro-
pionicum, the crude enzyme fractions or the purified enzyme
were incubated for 30 min under anaerobic conditions with
5 mm 3-pentynoyl-CoA, which is a reported inactivator of
acrylyl-CoA reductase [9], whose activity interferes with the
assay under the applied reducing conditions [26]. The pro-
tein fraction was freed from the inhibitor by passing it over
a 1 mL PD-10 Spintrap G-25 column (GE Healthcare)
equilibrated with anaerobic buffer, and concentrating via a
Centricon 30 kDa filter (Millipore Corporation, Billerica,
MA, USA). The assay was then performed exactly as that
for 2-hydroxyglutaryl-CoA dehydratase, except that acrylyl-
CoA or lactyl-CoA was used as substrate. The recombinant
activator from A. fermentans could be used instead of the
activator from C. propionicum, which is very unstable and
has never been purified completely [9]. The kinetic constants
were determined with 2.0 lg of dehydratase (specific activity
of 85 lmolÆmin)1Æmg)1 protein) and 0.6 lg of activator,
using either 0.2–10 mm lactyl-CoA or 0.01–2.0 mm acrylyl-
CoA, and evaluated as above. Under these conditions, the
minimum substrate ⁄ enzyme ratio was 370 : 1. Acrylyl-CoA
reductase activity was measured with propionyl-CoA and
ferricenium hexafluorophosphate as electron acceptor [26].
The concentrations of CoASH, acetyl-CoA and glutaco-
nyl-CoA (or any other CoA-ester substrate of glutaconate-
CoA transferase) were determined in a single assay using
Nbs2, oxaloacatate, citrate synthase, and transferase
[23,24,27]. Similarly, lactyl-CoA and acrylyl-CoA were
determined in the same assay, with glutaconate-CoA trans-
ferase being replaced by propionate CoA-transferase [28].
MALDI-TOF MS
The CoA thioester samples were purified as described
above, and the lyophilized samples were dissolved in
10–40 lL of water. Acetyl-CoA or free CoA was used as
internal standard. The matrix was a-cyano-4-hydroxycin-
namic acid (Sigma) dissolved in 70% acetonitrile ⁄ 0.1%
TFA. One microliter of each sample was mixed with 1 lL
of a-cyano-4-hydroxycinnamic acid or a-cyano-3-hydroxy-
cinnamic acid as matrix, and spotted onto a gold plate in a
dilution series. Measurements were performed with
a
355 nm laser in positive reflector mode with a delayed
extraction and a positive polarity on the Proteomics Ana-
lyzer 4800 mass spectrometer (Applied Biosystems, Fra-
mingham, MA, USA) at the MPI for Terrestrial
Microbiology, Marburg, Germany. The acceleration voltage
was 20 000 V, the grid voltage was 58%, and the delay time
was 50 ns. The ratio of reflector voltage was 1.00–1.12. An
average of 0.5% of acceleration was laid on the guidewire.
The mass range measured was 700–1000 Da. For each spec-
trum, more than 1000 shots were accumulated.
Enzymatic assays
All spectrophotometric assays were performed on Ultro-
spec 1100 pro spectrophotometers from Amersham Bio-
sciences, installed under aerobic or anaerobic conditions as
needed, or a Uvikon 943 double-beam spectrophotometer
from Kontron Instruments (Zurich, Switzerland). Quartz
cuvettes were used for measurements below 320 nm, and
disposable plastic cuvettes for measurements above 320 nm.
2-Hydroxyglutaryl-CoA dehydratase activity was mea-
sured under strict anaerobic conditions (d = 1 cm, total
Enzyme purification
Prior to use, columns, Centricon filters, centrifuge tubes,
pipette tips and other plastic materials were stored in a
glovebox (Coy Labs, Ann Arbor, MI, USA) for at least
FEBS Journal 277 (2010) 1738–1746 ª 2010 The Authors Journal compilation ª 2010 FEBS
1743

Equilibrium between hydroxyacyl-CoA and enoyl-CoA
A. Parthasarathy et al.
24 h. All operations during the purifications were per-
formed in this box. Buffers and solutions were degassed
under reduced pressure, purged with nitrogen, and prere-
duced with 2 mm dithiothreitol. Protein was determined by
the Bradford method [29].
0–0.33 m NaCl with 100 mL of buffer B. Two brownish
peaks were obtained. The first eluting peak contained the
activator (5 mm ATP ⁄ MgCl2 was added, and the fractions
were stored on ice), and the second peak was found to be
lactyl-CoA dehydratase. The relevant fractions were com-
bined, desalted, and stored on ice. After addition of solid
ammonium sulfate to a final concentration of 1.5 m, the
solution was sterile-filtered and applied to a Source 15Phe
column (1.0 · 10 cm) equilibrated with buffer C. The col-
umn was washed with 20 mL of buffer C, and the proteins
were eluted in a gradient of 1.5–0 m ammonium sulfate.
The brownish fractions were pooled, and the sample was
concentrated to about 400 lL with a 100 kDa cut-off
Centricon membrane. The concentrated sample (200 lL)
was applied to a Superdex 200 column (HR 1.0 ⁄ 30) equili-
brated with buffer D, and 0.5 mL fractions were collected.
The pure dehydratase (yield 32%) and the still impure acti-
vator were not frozen. The dehydratase preparation lost its
activity at a rate of 10–15% per day when stored in the
glovebox on ice water. Owing to the brownish color of the
dehydratase, a ‘blind’ purification could be performed in
order to save time and specific activity. The purity of the
enzymes was checked by SDS ⁄ PAGE [33].
C. symbiosum HB25 was grown on glutamate, yeast
extract, thioglycollate, and biotin [30], whereas C. propioni-
cum DSM 1682 required alanine, yeast extract, and cysteine
[26]. Production and purification of the recombinant activa-
tor from A. fermentans has been described elsewhere [31].
Purification of 2-hydroxyglutaryl-CoA
dehydratase from C. symbiosum [32]
Frozen cells were suspended in 50 mL of buffer A (50 mm
Mops, pH 7.2) under anaerobic conditions and disrupted
by ultrasonication in the anaerobic chamber. The cell-free
extract was clarified by centrifugation at 100 000 g for 1 h
at 4 ꢀC on an Optima L-90K Ultracentrifuge (Beckman
Coulter, Brea, CA, USA), and applied to a DEAE–Sepha-
rose column equilibrated with buffer A. The column was
washed with buffer A, and elution was performed by run-
ning a linear gradient of 0–0.7 m NaCl. The active fractions
eluted around 0.35 m NaCl. The pooled fractions were
combined, and desalted by filtration through a Centricon
membrane (30 kDa cut-off); solid ammonium sulfate was
then added to 1 m final concentration. This solution was
loaded onto a phenyl–Sepharose column pre-equilibrated
with buffer A containing 1 m ammonium sulfate. After
washing, the proteins were eluted with a gradient of 1–
0.3 m ammonium sulfate. The active fractions starting from
0.5 m ammonium sulfate were combined, desalted, and
loaded onto a Q-Sepharose column pre-equilibrated with
buffer A. After washing of the column, elution was per-
formed with a gradient of 0–0.5 m NaCl. The most active
and pure fractions, which eluted around 0.3 m NaCl, were
combined, desalted, concentrated, and stored at )80 ꢀC
until further use; the yield was 34% (based on the activity
of the cell-free extract).
Computational methods
Calculations were carried out with gaussian 03 [34]. The
geometry of each of the model systems shown in Fig. 2 was
optimized with the B3-LYP ⁄ 6-31G(d) level of theory.
Frequency calculations were performed to derive appropriate
thermochemical corrections. Improved relative energies, in
the gas phase, were obtained using the G3(MP2) model
chemistry [35]. The free energies of solvation [21] were
obtained using a polarizable continuum model, with Uni-
ted Atom Topological Model cavities at the B3-LYP ⁄
6-311G(d,p) level of theory [36]. The ꢃcombination of the
G3(MP2) gas-phase free energies [DGðgÞ, 1 atm reference]
with
the
free
energies
of
solvation
[DGꢄs ,
1 molÆL)1
fi 1 molÆL)1(aq)] [21] and a reference state
(g)
correction of RT ln(RT) [1 atm(g) fi 1 molÆL)1(g)] yields
the relative free energies in solution [DGꢄðaqÞ], corresponding
to a standard state of 1 molÆL)1. It is these values that
were used to determine Kconc, which were corrected to K
according to Fig. 1. Gaussian archive entries of the gas-
phase and solution calculations of the 1a, 2a, 1b, 2b, 1c, 2c
and H2O can be found in Table S1.
~
Purification of lactyl-CoA dehydratase from
C. propionicum
The following buffers were used: A, 25 mm Tris ⁄ HCl,
1 mm MgCl2, 1 mm EDTA, and 2 mm dithiothreitol; B,
1.5 m NaCl in A; C, 1.5 m ammonium sulfate in A; and D,
150 mm NaCl in A. Pre-equilibration of the columns
allowed purification in about one working day. Frozen cells
(12 g) were suspended in 20 mL of buffer A and opened by
ultrasonication. The cell-free extract was clarified by ultra-
centrifugation for 45 min at 100 000 g and applied onto a
Source 15Q column (1.6 · 15 cm) equilibrated with buf-
fer A. After washing of the column with 25 mL of buf-
fer A, the proteins were eluted in a linear gradient of
Acknowledgements
We gratefully acknowledge support (of D. M. Smith)
by the Croatian Ministry of Science (project 098-
0982933-2937) and the EC (FP6 contract 043749).
Work in Marburg was supported by the Max-Planck
Society, Deutsche Forschungsgemeinschaft and the
1744
FEBS Journal 277 (2010) 1738–1746 ª 2010 The Authors Journal compilation ª 2010 FEBS

A. Parthasarathy et al.
Equilibrium between hydroxyacyl-CoA and enoyl-CoA
Acidaminococcus fermentans. Eur J Biochem 265, 404–
414.
Fonds der chemischen Industrie. We thank T. Selmer,
Fachhochschule Aachen, Germany, for advice on the
purification of lactyl-CoA dehydratase.
14 Hofmeister AE & Buckel W (1992) (R)-lactyl-CoA de-
hydratase from Clostridium propionicum. Stereochemis-
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15 Gomes B, Fendrich G & Abeles RH (1981) Mechanism
of action of glutaryl-CoA and butyryl-CoA dehydro-
genases. Purification of glutaryl-CoA dehydrogenase.
Biochemistry 20, 1481–1490.
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Supporting information
The following supplementary material is available:
Table S1. Gaussian archive entries of the gas-phase
and solution calculations of the species 1a, 2a, 1b, 2b,
1c, 2c, and H2O.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
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