Formation of an enolate intermediate is required for the reaction catalyzed by 3-hydroxyacyl-CoA dehydrogenase

Article abstract of DOI:10.1016/j.bmcl.2007.03.023

Fluorinated substrate analogs were synthesized and incubated with rat liver 3-hydroxyacyl-CoA dehydrogenase, which reveals that the formation of an enolate intermediate is required for the reaction catalyzed by the enzyme.

Full text of DOI:10.1016/j.bmcl.2007.03.023

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3187–3190
Formation of an enolate intermediate is required for the
reaction catalyzed by 3-hydroxyacyl-CoA dehydrogenase
Xiaojun Liu, Guisheng Deng, Xiusheng Chu, Nan Li, Long Wu and Ding Li*
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China
Received 2 January 2007; revised 6 March 2007; accepted 8 March 2007
Available online 12 March 2007
Abstract—Fluorinated substrate analogs were synthesized and incubated with rat liver 3-hydroxyacyl-CoA dehydrogenase, which
reveals that the formation of an enolate intermediate is required for the reaction catalyzed by the enzyme.
Ó 2007 Elsevier Ltd. All rights reserved.
Numerous diseases have been reported in relation to
fatty acids, such as cardiovascular disease,1 cancer2,
and diabetes.3 The degradation of saturated fatty acids
occurs in a sequence of four reactions referred to as
the b-oxidation cycle.4 Fatty acid oxidation in mito-
chondria is an essential energy generation system for
cells. The partial fatty acid oxidation (pFOX) inhibition
has been reported as a therapy for non-insulin depen-
dent diabetes mellitus (NIDDM) and chronic stable
angina.5 The third step of the mitochondrial b-oxidation
cycle is catalyzed by 3-hydroxyacyl-CoA dehydrogenase
(HAD; EC 1.1.1.35), a membrane-associated long chain
3-hydroxyacyl-CoA dehydrogenase (LCHAD), and
tial enzyme but also for the design of enzyme inhibitors
targeting above-mentioned diseases.
Rat HAD catalyzes the reversible oxidation of the hy-
droxyl group of 3-hydroxyacyl-CoA to a keto group,
concomitant with the reduction of NAD to NADH
(Fig. 1). The dimeric enzyme displays broad substrate
specificity, utilizing substrates with 4–16 carbons in the
acyl chain.9 The enzyme is a ‘B-side’-specific dehydroge-
nase with hydride transfer occurring on the si face of the
nicotinamide ring.10 The crystal structures have been
solved for the apoenzyme, binary complexes of the
enzyme with reduced cofactor or 3-hydroxybutyryl-
CoA substrate, and an abortive ternary complex of the
enzyme with NAD+ and acetoacetyl-CoA.11 Several res-
idues have been confirmed to play essential roles in the
function of the enzyme.12
short
chain
3-hydroxyacyl-CoA
dehydrogenase
(SCHAD).6 SCHAD is also known as type 10 17b-
hydroxysteroid dehydrogenase (HSD10),7 which is a
potential target for intervention of Alzheimer’s disease
(AD), Parkinson’s disease (PD), infantile neurodegener-
ation, and other neural disorders.8 Therefore, mechanis-
tic studies of the reactions catalyzed by the HAD are
important not only for the understanding of this essen-
In our previous study, we have cloned His-tagged rat
mitochondrial HAD, and purified both wild-type and
Ser137Ala variant proteins.12e In this study, we further
constructed Asn208Ala mutant plasmid, and the variant
protein was subsequently obtained through overexpres-
sion in E. coli BL21::DE3 and purification with nickel
metal affinity column chromatography. These wild-type
and variant proteins were used in this study for investi-
gating enzymatic reaction mechanism.
Abbreviations: AD, Alzheimer’s disease; HAD, 3-hydroxyacyl-CoA
dehydrogenase; HSD10, type 10 17b-hydroxysteroid dehydrogenase;
LCHAD, long chain 3-hydroxyacyl-CoA dehydrogenase; NIDDM,
non-insulin dependent diabetes mellitus; PD, Parkinson’s disease;
pFOX inhibition, partial fatty acid oxidation inhibition; SCHAD,
short chain 3-hydroxyacyl-CoA dehydrogenase; UV/vis, ultraviolet–
visible spectroscopy.
It has been noted that a charge transfer complex is
formed when incubating HAD with acetoacetyl-CoA
in a reverse reaction mixture, which shows a broad peak
centered in the 410–420 nm range in UV/vis spec-
trum.11a,12a The enolate species of acetoacetyl-CoA
serves as electron-donor, and NAD+ is suggested as
Keywords: 3-Hydroxyacyl-CoA dehydrogenase; Enolate; Fatty acid
oxidation; Chronic stable angina; Diabetes; Alzheimer’s disease.
*
Corresponding author. Tel.: +852 2788 7824; fax: +852 2788 7406;
e-mail: bhdingli@cityu.edu.hk
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.023

3188
X. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3187–3190
OH
O
O
O
of the enolate intermediate. Ser137 may play more
+ NAD+
+ NADH + H+
significant role than Asn208, since Ser137Ala completely
prevented the formation of the enolate intermediate,
resulting in an inactive enzyme for 3-hydroxyoctanoyl-
CoA (1). 2-Fluoro-3-hydroxyoctanoyl-CoA (2) was still
a substrate of HAD wild-type enzyme with significantly
lower Vmax value. This may be because the deprotonation
at carbon-2 is stereospecific and only half of the analog
can be deprotonated at carbon-2 to form the enolate
intermediate. The strong electron-withdrawing property
of fluorine atom might also decrease the p orbital overlap
between carbon-2 and -3. 2-Fluoro-3-hydroxyoctanoyl-
CoA (2) is not a substrate for both variant enzymes,
which is then understandable because the enolate inter-
mediate cannot be stabilized by either Ser137 or Asn208
besides the effect of fluorine substitution at carbon-2 of
the analog. 2,2-Difluoro-3-hydroxyoctanoyl-CoA (3)
was no longer a substrate of the wild-type and variant
enzymes. Since compound 3 cannot form an enolate
intermediate, it supports that the formation of an enolate
intermediate may be a prerequisite for subsequent oxida-
tion reaction.
R
SCoA
R
SCoA
Figure 1. HAD catalyzes the reversible oxidoreduction reaction.
BrCR1R2CO2CH3 / Zn
THF, reflux
OH
O
CHO
O
R1 R2
1. NaOH
2. ClCO2-
OH
O
i-Bu
3. NaSCoA
THF, H2O
SCoA
R1 R2
1: R1=R2=H
2: R1=H; R2=F
3: R1=R2=F
Figure 2. Organic syntheses of HAD substrate and substrate analogs.
the electron acceptor. The resultant negative charge on
O3 of the substrate is stabilized by interactions with
the side-chain amide of Asn208 and the hydroxyl group
of Ser137.
We hypothesized that the formation of an enolate inter-
mediate can facilitate the oxidation reaction through
formation of a more stable conjugated oxidation reac-
tion product, as shown in Figure 3. In order to test this
hypothesis, we synthesized 3-hydroxy-4-octenoyl-CoA
(4), 2-fluoro-3-hydroxy-4-octenoyl-CoA (5), and 2,2-
difluoro-3-hydroxy-4-octenoyl-CoA (6) using the same
synthetic strategy as that for the syntheses of com-
pounds 1–3 (Fig. 4).14 These substrate analogs were
incubated with HAD wild-type and variant enzymes,
and the results are shown in Table 1. Apparently, the
introduction of a double bond at carbon-4 of com-
pounds 4 and 5 increased the reaction rate for the wild
type enzyme. Compounds 4 and 5 became the substrates
Interestingly, we found the charge transfer complex ex-
isted not only in the reverse reaction mixture, but also
in the forward reaction mixture when we incubated
HAD with 3-hydroxyacyl-CoA substrate and NAD+
cofactor. This suggests that the formation of an enolate
intermediate may be a prerequisite for the subsequent
oxidation reaction. In order to further study the mecha-
nism of HAD catalyzed reaction, we synthesized a vari-
ety of 3-hydroxyacyl-CoA substrate analogs with
fluorine substitution at carbon-2. The incubation studies
of HAD with these analogs were carried out, which gave
us interesting results.
3-Hydroxyoctanoyl-CoA (1), 2-fluoro-3-hydroxyocta-
noyl-CoA (2), and 2,2-difluoro-3-hydroxyoctanoyl-CoA
(3) were conveniently synthesized using Reformatsky
reaction followed by hydrolysis and coupling with
coenzyme A,13 as shown in Figure 2. These substrate
and analogs were incubated with HAD wild-type and
variant enzymes, and kinetic studies were carried out.
The results of kinetic studies are shown in Table 1.
3-Hydroxyoctanoyl-CoA (1) was still a substrate of
Asn208Ala variant enzyme with significantly decreased
Vmax value, while it was not a substrate of Ser137Ala
variant enzyme at all. This result indicated that both
Asn208 and Ser137 can stabilize the enolate intermedi-
ate of the forward reaction through interaction with O3
-
-
-
OH
O
OH
O
O
O
R
SCoA
R
SCoA
R
SCoA
H
NAD+
-
-
O
O
O
O
O
O
R
SCoA
R
SCoA
R
SCoA
Figure 3. Proposed mechanism for oxidation reaction catalyzed by
HAD.
Table 1. Incubation of HAD with substrate and analogs
Compound
Wild-type
N208A
S137A
KM
Vmax
KM
Vmax
KM
Vmax
1
2
3
4
5
6
34.6 13.9
69.3 16.1
10.5 1.7
1.04 0.15
46.8 7.4
0.28 0.02
No detectable activity
No detectable activity
No detectable activity
53.2 14.3
No detectable activity
No detectable activity
49.6 16.8
No detectable activity
20.0 0.8
46.8 5.1
21.8 0.3
11.7 0.7
1.08 0.18
0.10 0.01
0.46 0.06
0.17 0.02
44.9 13.1
No detectable activity
55.7 13.1
No detectable activity
No detectable activity

X. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3187–3190
OH
3189
O
BrCR1R2CO2CH3 / Zn
THF, reflux
Acknowledgment
CHO
O
The work described in this paper was supported by
grants from City University of Hong Kong (Strategic
Research Grant, Project No. 7001540 and Strategic Re-
search Grant, Project No. 7001836).
R1 R2
1. NaOH
OH
O
2. ClCO2-i-Bu
3. NaSCoA
THF, H2O
SCoA
R1 R2
4: R1=R2=H
5: R1=H; R2=F
6: R1=R2=F
References and notes
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Figure 4. Organic syntheses of HAD substrate analogs.
of the HAD variant enzymes, which indicates that the
loss of the stabilization of the enolate intermediate by
Ser137 or Asn208 can be partially compensated by the
introduction of a double bond at carbon-4. This result
supports that the formation of a more stable conjugated
oxidation reaction product can facilitate the oxidation
reaction. The result indicates the introduction of a dou-
ble bond at carbon-4 cannot compensate the complete
loss of the enolate intermediate, since compound 6 can-
not form an enolate intermediate and was not a sub-
strate of the enzyme. Further incubation studies
showed that compound 6 was a competitive inhibitor
of the wild-type and variant enzymes, which confirm
that it can bind with the enzyme active site although it
cannot be turned over into the product.
In summary, our results from the incubation of HAD
with the fluorinated substrate analogs strongly supports
that an enolate intermediate is present in the HAD cat-
alyzed forward reaction. The enolate can be effectively
stabilized by residues Ser137 and Asn208. This mecha-
nistic study of the HAD catalyzed reaction suggests that
enolate-like organic analogs may be effective inhibitors
of the HAD.
9. Kobayashi, A.; Jiang, L. L.; Hashimoto, T. J. Biochem.
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W.; Banaszak, L. J. Protein Sci. 1999, 8, 2010.
Since enolate intermediates have been found for the
reactions catalyzed by several other enzymes involved
in fatty acid oxidation such as acyl-CoA dehydrogenase,
enoyl-CoA hydratase, 2,4-dienoyl-CoA reductase,
enoyl-CoA isomerase, and dienoyl-CoA isomerase,15
the analogs of enolate intermediates may become multi-
functional enzyme inhibitors in fatty acid oxidation.
Inactivation of only one specific enzyme involved in
fatty acid oxidation can result in accumulation of the
substrate, which could generate various side effects.
The use of several enzyme inhibitors simultaneously tar-
geting several enzymes could produce side effect due to
drug interactions.16 Therefore, the better solution is to
use multifunctional enzyme inhibitors for partial inhibi-
tion of fatty acid oxidation in treating non-insulin
dependent diabetes mellitus (NIDDM) and chronic sta-
ble angina.
12. (a) Barycki, J. J.; O’Brien, L. K.; Strauss, A. W.;
Banaszak, L. J. J. Biol. Chem. 2001, 276, 36718; (b)
Barycki, J. J.; O’Brien, L. K.; Bratt, J. M.; Zhang, R.;
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istry 1999, 38, 5786; (c) He, X. Y.; Deng, H.; Yang, S. Y.
Biochemistry 1997, 36, 261; (d) He, X. Y.; Yang, S. Y.
Biochemistry 1996, 35, 9625; (e) Liu, X.; Chu, X.; Yu, W.;
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13. (a) The spectra data of 3-hydroxyoctanoic acid (for
coupling with coenzyme A in the synthesis of compound
1) are shown as the following: 1H NMR (300 MHz,
CDCl3, TMS) d 0.89 (t, J = 6.9 Hz, 3H), 1.40–1.59 (m, 6
H), 2.40 (dd, J = 16.5, 8.7 Hz, 1H), 2.51 (dd, J = 16.5,
3.6 Hz, 1H), 3.95–4.03 (m, 1H), 6.27 (br s, 2H); (b) The
spectra data of 3-hydroxy-2-fluorooctanoic acid (for
coupling with coenzyme A in the synthesis of compound
2) are shown as the following: 1H NMR (300 MHz,
CDCl3, TMS) d 0.86 (t, J = 6.3 Hz, 3H), 1.28–1.44 (m, 6
H), 1.53–1.63 (m, 2H), 4.05 (dt, J = 27.6, 6.6 Hz, 1H), 4.85
(d, J = 49.2 Hz, 1H), 6.55 (s, 1H); (c) The spectra data of
3-hydroxy-2,2-difluorooctanoic acid (for coupling with
coenzyme A in the synthesis of compound 3) are shown as
SCHAD is a multifunctional enzyme with its major
function as HAD in fatty acid oxidation, which has been
shown as a drug target for intervention of Alzheimer’s
disease (AD), Parkinson’s disease (PD), infantile neuro-
degeneration, and other neural disorders.7,8 Therefore,
this research may also shed light on the design of
enzyme inhibitors treating these important diseases.
1
the following: H NMR (300 MHz, CDCl3, TMS) d 0.88
(t, J = 6.9 Hz, 3H), 1.24–1.37 (m, 6 H), 1.49–1.70 (m, 2H),
3.99–4.10 (m, 1H), 5.41 (br s, 2H).
14. (a) The spectra data of 3-hydroxy-4-octenoic acid (for
coupling with coenzyme A in the synthesis of compound

3190
X. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3187–3190
4) are shown as the following: 1H NMR (300 MHz,
in the synthesis of compound 6) are shown as the
following: H NMR (300 MHz, CDCl3, TMS) d 0.91 (t,
J = 7.2 Hz, 3H), 1.37–1.49 (m, 2H), 2.06–2.14 (m, 2H),
4.56 (dd, J = 15.6, 7.5 Hz, 1H), 6.90 (dt, J = 15.6, 7.2 Hz,
1H), 6.96 (br s, 2H).
1
CDCl3, TMS) d 0.85 (t, J = 7.5 Hz, 3H), 1.33–1.42 (m,
2H), 1.94–2.01 (m, 2H), 2.54 (m, 2H), 4.34–4.49 (m, 1H),
5.45 (ddt, J = 15.3, 6.6, 1.4 Hz, 1H), 5.69 (dt, J = 15.3,
7.2 Hz, 1H), 6.33 (br s, 2H); (b) The spectra data of 3-
hydroxy-2-fluoro-4-octenoic acid (for coupling with coen-
zyme A in the synthesis of compound 5) are shown as the
15. (a) Babbitt, P. C.; Gerlt, J. A. J. Biol. Chem. 1997, 272,
30591; (b) Zeng, J.; Li, D. Biochemistry 2005, 44, 6715; (c)
Alphey, M. S.; Yu, W.; Byres, E.; Li, D.; Hunter, W. N.
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16. (a) Mross, K.; Steinbild, S.; Baas, F.; Reil, M.; Buss, P.;
Mersmann, S.; Voliotis, D.; Schwartz, B.; Brendel, E. Int.
J. Clin. Pharmacol. Ther. 2003, 41, 618; (b) Perez-Reyes,
M. Life Sci. 1994, 55, 541.
1
following: H NMR (300 MHz, CDCl3, TMS) d 0.87 (t,
J = 7.2 Hz, 3H), 1.35–1.44 (m, 2H), 2.03–2.11 (m, 2H),
4.51–4.59 (m, 1H), 4.83 (dd, J = 18.6, 3.0 Hz, 1H), 5.02 (br
s, 2H), 5.55 (dt, J = 15.3, 6.3 Hz, 1H), 5.84 (dd, J = 15.3,
5.1 Hz, 1H); (c) The spectra data of 3-hydroxy-2,2-
difluoro-4-octenoic acid (for coupling with coenzyme A