3988 J. Am. Chem. Soc., Vol. 122, No. 17, 2000
Wu et al.
2-Butanone (7.2 g, 0.1 mol) was mixed with 10 mL of 48%
hydrobromic acid in an ice-water bath. Bromine was added (10 mL,
0.2 mol) dropwise, followed by the addition of 30 mL of water. The
organic layer was separated and distilled under reduced pressure. The
fraction boiling between 84 and 85 °C (10 mmHg) was collected. The
yield of 1,3-dibromobutan-2-one was 9.31 g (41%). 1H NMR (300 MHz,
CDCl3) δ 1.8 (d, CH3), 4.1 (d, 1 H, CHH′Br), 4.3 (d, 1 H, CHH′Br),
4.8 (q, 1 H, CHBr).
hydroxyacyl-CoAs in order to facilitate the metabolism of fatty
acids containing cis double bonds. Pathways for the metabolism
of fatty acids containing cis double bonds exist in peroxisomes
and include a dienoyl-CoA reductase and a hydroxyacyl-CoA
epimerase.4-8 The epimerase activity is linked to an enoyl-CoA
hydratase that catalyzes the interconversion of trans-2-enoyl-
CoAs and 3(R)-hydroxyacyl-CoAs.8-10 Although each of these
enzymes is stereospecific, the absolute stereospecificity has not
previously been reported. Here we demonstrate that mitochon-
drial enoyl-CoA hydratase can catalyze the formation of 3(R)-
hydroxybutyryl-CoA from trans-2-crotonyl-CoA.
Using NMR spectroscopy we have observed that the enzyme
catalyzes incorporation of solvent deuterium into the pro-2S
position of 3(S)-hydroxybutyryl-CoA, in addition to the expected
exchange of the pro-2R proton. Exchange of the pro-2S proton
has been shown to occur in concert with the formation of 3(R)-
hydroxybutyryl-CoA. This has allowed us to determine the
absolute stereospecificity for the enzyme-catalyzed reaction.
During the time course of the NMR experiment, the total amount
of crotonyl-CoA decreases, indicating a shift in the equilibrium
to the hydroxylated species. This observation is rationalized by
the knowledge that, once formed, the 3(R)-hydroxybutyryl-CoA
will dehydrate to give cis-2-crotonyl-CoA. While the equilibrium
constant for the hydration of trans-2-crotonyl-CoA to 3(S)-
hydroxybutyryl-CoA is 7.5, the equilibrium constant for the
hydration of cis-2-crotonyl-CoA to 3(R)-hydroxybutyryl-CoA
is estimated to be ∼1000 (vide infra), explaining the overall
decrease in crotonyl-CoA during epimerization. This model has
been supported by studying the enzyme-catalyzed hydration of
cis-2-crotonyl-CoA.
To a molar solution of potassium bicarbonate (50 mL) was added
1,3-dibromobutan-2-one (4.5 g, 0.02 mol) over a 5-minute period. The
mixture was stirred for 2.5 h followed by extraction (2 × 15 mL) with
ether. The water layer was acidified with dilute hydrochloric acid and
again extracted with 6 × 20 mL portions of ether. The organic layer
was dried over MgSO4 overnight followed by concentration in vacuo
(bath temperature around 0 °C) to give 1.1 g (65%) cis-crotonic acid.
1H NMR showed the sample to be >98% cis. This compound was
1
refrigerated until further use. H NMR (300 MHz CDCl3) δ 2.1 (d, d,
3H), 5.8 (d, q, 1 H), 6.4-6.6 (m, 1 H).
cis-Crotonyl-CoA was synthesized from cis-crotonic acid using the
mixed anhydride method. Briefly, cis-crotonic acid (30 mg, 0.35 mmol)
was dissolved in anhydrous ether (4 mL) with triethylamine (44 mg,
0.43 mmol), followed by the addition of ethyl chloroformate (37 mg,
0.35 mmol). The solution was stirred in an ice-water bath for 4 h.
The mixed anhydride was then filtered and added dropwise to a solution
of CoA in Na2CO3 (50 mM, pH 8), ethanol, and ethyl acetate (1:1:1)
with stirring at 0 °C. The reaction progress was monitored by following
the concentration of free thiol in solution using 5,5′-dithiol-bis(2-
nitrobenzoic acid) (DTNB). When no free thiol was detected the
solution was concentrated in vacuo to remove the organic solvent and
purified by HPLC (Shimadzu) using a Vydac C18 250 × 4.60 mm
preparative column. Chromatography was performed using ammonium
acetate (100 mM)/1.75% acetonitrile as buffer A and running a 0 to
30% gradient of 95% acetonitrile/5% H2O (buffer B) over 40 min at a
flow rate of 8 mL/min. Elution was monitored at 260 and 290 nm using
a Shimadzu SPD-10A UV-vis detector, and fractions containing cis-
2-crotonyl-CoA were pooled and lyophilized. The desired product was
Experimental Procedures
Chemicals. Coenzyme A (CoA) lithium salt, 3(S)-hydroxybutyryl-
CoA dehydrogenase, L-lactate dehydrogenase, pyruvate, thrombin, and
NAD+ were purchased from Sigma Chemical Co. Deuterium oxide
(99.9%) was purchased from Cambridge Isotope Labs. His-bind resin
was purchased from Novagen.
1
obtained in 25% yield as a white powder. H NMR (500 MHz, D2O)
δ 8.56 (s, 1 H), 8.28 (s, 1 H), 6.27-6.22 (dt, 1H, J ) 11.5, 7.0 Hz),
6.14 (d, 1 H, J ) 11 Hz), 4.85 (t, 1 H), 4.61 (s, 1 H), 4.26 (s, 2 H),
4.04 (s, 1 H), 3.85 (q, 1 H), 3.58 (q, 1 H), 3.47 (t, 2 H), 3.03 (t, 2 H),
2.45 (t, 2 H), 2.04 (d, 3 H, J ) 7.0 Hz), 0.91 (s, 3 H), 0.78 (s, 3 H).
MALDI-MS calculated for [C25H39N7O17P3S]- [M - H]-: 834.5;
found: 833.1.
Preparation of trans-2-Crotonyl-CoA, 3(S)-Hydroxybutyryl-CoA
and 4-Dimethylaminocinnamoyl-CoA. trans-2-Crotonyl-CoA was
synthesized from crotonic acid and coenzyme A using the mixed
anhydride method described previously.3 3(S)-Hydroxybutyryl-CoA was
synthesized enzymatically from trans-2-crotonyl-CoA. In a typical
reaction, 3 mM trans-2-crotonyl-CoA was incubated with 1 µM enoyl-
CoA hydratase for 5 min. This enzyme concentration and incubation
time is sufficient to completely hydrate the trans-2-crotonyl-CoA
without resulting in any formation of 3(R)-hydroxybutyryl-CoA.
Following removal of the enzyme using a Centricon, 3(S)-hydroxybu-
tyryl-CoA was purified by HPLC using conditions identical to those
used for trans-2-crotonyl-CoA. 3(S)-Hydroxybutyryl-CoA eluted with
a retention time of 17.6 min compared to 23.6 min for trans-2-crotonyl-
CoA. 4-Dimethylaminocinnamoyl-CoA was synthesized from 4-dim-
ethylaminocinnamic acid and CoA following activation of the acid using
1,1′-carbonyl diimidazole.3
Subsequent HPLC analysis revealed that the cis-2-crotonyl-CoA was
contaminated by ∼25% of the trans isomer. However, prolonged
incubation in solution at room temperature resulted in no further increase
in the amount of trans and consequently we hypothesized that
isomerization had occurred during the coupling reaction. The kinetics
of the hydration of cis-2-crotonyl-CoA were performed at pH 7.4 in
20 mM phosphate buffer by monitoring the decrease in absorbance at
280 nm as described for the trans isomer.3 Spectrophotometric analysis
of the contaminated cis-2-crotonyl-CoA following addition of 0.28 nM
enzyme revealed a fast decrease in absorbance at 280 nm followed by
a slower phase. After the reaction was followed to completion, the ratio
of the absorbance change in the fast and slow phases was 1:4, in keeping
with the observed ratio of trans to cis isomers in the substrate.
Subsequent modification of the HPLC method enabled us to separate
the cis and trans isomers. Chromatography was performed using 50
mM KH2PO4 as buffer A and running a 0 to 50% gradient of methanol
(buffer B) over 50 min at a flow rate of 8 mL/min. Under these
conditions the retention time for cis- and trans-2-crotonyl-CoA was
38.4 and 38.0 min, respectively. Kinetic analysis of the repurified cis-
2-crotonyl-CoA revealed monophasic rates of reaction comparable to
the slow phase observed previously. The repurified cis-2-crotonyl-CoA
Preparation of cis-Crotonyl-CoA. cis-Crotonic acid (isocrotonic
acid) was prepared following the procedure of Rappe as follows.11
(4) Yang, S. Y.; Cuebas, D.; Schulz, H. J. Biol. Chem. 1986, 261, 12238-
43.
(5) Smeland, T. E.; Li, J. X.; Chu, C. H.; Cuebas, D.; Schulz, H. Biochem.
Biophys. Res. Comm. 1989, 160, 988-92.
(6) Hiltunen, J. K.; Palosaari, P. M.; Kunau, W. H. J. Biol. Chem. 1989,
264, 13536-40.
was used for determination of kcat and Km using ∆ꢀ280 4300 M-1 cm-1
.
(7) Smeland, T. E.; Cuebas, D.; Schulz, H. J. Biol. Chem. 1991, 266,
23904-8.
Preparation of Enoyl-CoA Hydratase. Recombinant wild-type rat
mitochondrial enoyl-CoA hydratase was expressed and purified from
cultures of Escherichia coli as described.3,12 This method involves
(8) Malila, L. H.; Siivari, K. M.; Ma¨kela¨, M. J.; Jalonen, J. E.; Latipa¨a¨,
P. M.; Kunau, W. H.; Hiltunen, J. K. J. Biol. Chem. 1993, 268, 21578-85.
(9) Yang, S. Y.; Elzinga, M. J. Biol. Chem. 1993, 268, 6588-6592.
(10) Qin, Y. M.; Haapalainen, A. M.; Conry, D.; Cuebas, D. A.; Hiltunen,
J. K.; Novikov, D. K. Biochem. J. 1997, 328 (Pt 2), 377-82.
(11) Rappe, C. Org. Synth. 1973, 53, 123-126.
(12) Wu, W. J.; Anderson, V. E.; Raleigh, D. P.; Tonge, P. J.
Biochemistry 1997, 36, 2211-2220.