which is an essential nutrient for heart strength and function.6
The inhibition of one enzyme can cause an increase of the
substrate concentration and a decrease of the product
concentration, which may subsequently generate various side
effects. New inhibitors targeting other enzymes in the
mevalonate pathway need to be developed, which could be
used together with statin in a drug combination strategy7 to
lower the dosage of statin.
The first step of the mevalonate pathway is catalyzed by
cytosolic thiolase, which involves condensation of two acetyl-
CoAs to give the acetoacetyl-CoA product.8 We are interested
in this enzyme because its substrate, acetyl-CoA, is a
universal starting material for various biosynthetic pathways,
and its accumulation may render less side effects for our
bodies. The mitochondrial and peroxisomal thiolases are
involved in fatty acid oxidation, which breaks 3-ketoacyl-
CoA to produce two-carbon short acyl-CoA and acetyl-CoA.
Although several bromine-labeled acyl-CoA analogues have
been reported to be thiolase inhibitors,9 they inactivate all
thiolases without selectivity. In our study of the thiolase, we
found that the enzyme has intrinsic isomerase activity as
shown in Figure 2, which was thoroughly characterized in
Figure 3. Cys92, His352, and Cys382 have been identified as
catalytic residues for the reaction catalyzed by rat liver thiolase.
studied.12 We have previously cloned and purified rat liver
thiolase,13 which has high sequence homology with well-
studied Homo sapiens cytosolic thiolase and Zoogloea
ramigera biosynthetic thiolase and was used in the present
study. We think Cys382 may be the catalytic residue for the
isomerase activity of the thiolase because it is responsible
for R-proton abstraction in the biosynthetic direction. There-
fore, we carried out site-directed mutagenesis and obtained
variant proteins C382S and C382A. Kinetic studies were
carried out for the wild-type and variant enzymes on their
isomerase activities, and the result is shown in Table 1. The
Figure 2. Isomerization reaction catalyzed by thiolase.
the present study. On the basis of this result, we synthesized
2-octynoyl-CoA as an isomerase mechanism-based inhibitor.
It should be noted that 2-alkynoyl-CoA can be detoxified in
mitochondria and peroxisomes in vivo by conversion into
3-ketoacyl-CoA catalyzed by enoyl-CoA hydratase.10 There-
fore, only cytosolic thiolase should be selectively inactivated
by 2-alkynoyl-CoA in vivo.
Table 1. Kinetic Parameters for Isomerase Activity of Rat
Thiolase Wild-Type and Variant Enzymes Using
cis-3-Hexenoyl-CoA as Substratea
kcat (s-1
)
KM (µM)
kcat/KM (s-1/µM-1
)
The crystal structures of the thiolases from various sources
have been solved,11 and three amino acids including two
cysteines and one histidine residue have been suggested as
essential catalytic residues as shown in Figure 3. The
mechanisms of thiolase-catalyzed reactions have been well
WT
0.33 ( 0.01
52 ( 18
ND
ND
6.3 × 10-3
C382S
C382A
C92S
(6.3 ( 0.2) × 10-4
(2.1 ( 0.1) × 10-3
0.28 ( 0.01
ND
ND
4.6 × 10-3
61 ( 19
a
ND: Not determined.
(6) Dhanasekaran, M.; Ren, J. Curr. NeuroVasc. Res. 2005, 2, 447-
459.
k
cat values of C382S and C382A decreased 524 and 157 times
(7) Silverman, R. B. The organic chemistry of drug design and drug
action, 2nd ed.; Elsevier Academic Press: Amsterdam; Boston, 2004.
(8) Haapalainen, A. M.; Merilainen, G.; Wierenga, R. K. Trends Biochem.
Sci. 2006, 31, 64-71.
(9) (a) Olowe, Y.; Schulz, H. J. Biol. Chem. 1982, 257, 5408-5413. (b)
Raaka, B. M.; Lowenstein, J. M. Methods Enzymol. 1981, 72, 559-577.
(10) Thorpe, C. Anal. Biochem. 1986, 155, 391-394.
(11) (a) Sundaramoorthy, R.; Micossi, E.; Alphey, M. S.; Germain, V.;
Bryce, J. H.; Smith, S. M.; Leonard, G. A.; Hunter, W. N. J. Mol. Biol.
2006, 359, 347-357. (b) Kursula, P.; Sikkila, H.; Fukao, T.; Kondo, N.;
Wierenga, R. K. J. Mol. Biol. 2005, 347, 189-201. (c) Modis, Y.; Wierenga,
R. K. J. Mol. Biol. 2000, 297, 1171-1182. (d) Modis, Y.; Wierenga, R. K.
Structure 1999, 7, 1279-1290. (e) Mathieu, M.; Modis, Y.; Zeelen, J. P.;
Engel, C. K.; Abagyan, R. A.; Ahlberg, A.; Rasmussen, B.; Lamzin, V. S.;
Kunau, W. H.; Wierenga, R. K. J. Mol. Biol. 1997, 273, 714-728.
for their isomerase activities compared with that of the wild-
type enzyme, which confirmed that Cys382 is the catalytic
residue for its isomerase activity. In comparison, C92S has
(12) (a) Kursula, P.; Ojala, J.; Lambeir, A. M.; Wierenga, R. K.
Biochemistry 2002, 41, 15543-15556. (b) Williams, S. F.; Palmer, M. A.;
Peoples, O. P.; Walsh, C. T.; Sinskey, A. J.; Masamune, S. J. Biol. Chem.
1992, 267, 16041-16043. (c) Palmer, M. A.; Differding, E.; Gamboni, R.;
Williams, S. F.; Peoples, O. P.; Walsh, C. T.; Sinskey, A. J.; Masamune,
S. J. Biol. Chem. 1991, 266, 8369-8375. (d) Thompson, S.; Mayerl, F.;
Peoples, O. P.; Masamune, S.; Sinskey, A. J.; Walsh, C. T. Biochemistry
1989, 28, 5735-5742.
(13) Zeng, J.; Li, D. Protein Expression Purif. 2004, 35, 320-326.
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