Mechanistic studies of the inactivation of crotonase by (methylenecyclopropyl)formyl-CoA

Full text of DOI:10.1021/ja952621b

J. Am. Chem. Soc. 1996, 118, 275-276
275
Mechanistic Studies of the Inactivation of Crotonase
by (Methylenecyclopropyl)formyl-CoA
Ding Li, Zhihong Guo, and Hung-wen Liu*
Department of Chemistry, UniVersity of Minnesota
Minneapolis, Minnesota 55455
ReceiVed August 4, 1995
Figure 1.
Enoyl-CoA hydratase (crotonase, EC 4.2.1.17) catalyzes the
reversible hydration of a ∆2,3-unsaturated enoyl-CoA substrate
to the corresponding 3-hydroxyacyl-CoA product.1 This reac-
tion is the second step in the â-oxidation pathway of fatty acid
metabolism and is also an important step in the catabolism of
branched-chain amino acids. Crotonases of different origins
are known2 and a wide variety of enoyl-CoAs of varied chain
length with or without substituents at the R- or â-C are
accommodated as substrates.3 Early studies revealed that bond
formation/cleavage at R- and â-positions during the hydration/
dehydration occurs in a concerted manner4 and that the overall
stereochemistry is syn.5 The driving force of this catalysis has
been attributed to the capability of crotonase to polarize the
π-electrons of R,â-unsaturated double bond of the substrate in
its active site, hence enhancing the electrophilicity of the â-C.6
While the physiological significance of crotonase is well
established and the mechanism of its catalysis has been well
characterized, interestingly, very few inhibitors are known for
this class of enzymes.3,7,8
In our quest for means to regulate fatty acid metabolism, we
have noted that methylenecyclopropylglycine (1, MCPG),
isolated from the kernels of litchi fruits, had been shown to
cause hypoglycemia in mice and fasted rats.8,9 It was suggested
that 1 is first converted in ViVo into a toxic metabolite,
(methylenecyclopropyl)formyl-CoA (2, MCPF-CoA),9 which
then interrupts â-oxidation. Early experiments found that 1 is
most inhibitory to 3-oxoacyl-CoA and acetoacetyl-CoA thiolases
and has little effect on enoyl-CoA hydratase in fasted rats.9
However, when 2 was used directly in the incubation with
purified enzymes, it exhibited strong inhibition of crotonase from
pig kidney10 but less notably so for the bovine liver enzyme.10b
The distinct effects of 1 and its metabolites on the activities of
different enoyl-CoA hydratases is puzzling, and although this
inhibition clearly offers an alternate mechanism in controlling
the fatty acid metabolism, little is known about its molecular
activity.
Figure 2.
isolated from pig kidney and bovine liver.12 Interestingly, in
contrast to an early report,10b 2 was found to be more inhibitory
toward the bovine liver enzyme, with a KI of 53 µM and a kinact
of 3.0 × 10-3 min-1 13
.
Since the enzyme activity was not
recovered after extensive dialysis, the inhibition is clearly
irreversible.14 It should also be noted that the inhibition by 2
is likely active site directed, since its effect was suppressed by
40% in the presence of an equivalent of acetoacetyl-CoA, which
is a known competitive inhibitor of crotonase.3a As delineated
in Figure 2, the inactivation may be attributed to the electrophilic
nature of the cyclopropane moiety, which, by virtue of its
electron-withdrawing substituent, is readily susceptible to nu-
cleophilic attack (route A).15 While the initial attack, in
principle, could occur at two distinct sites, both routes would
lead to identical adduct(s). The covalent derivatization could
also be achieved by direct attack of the thioester carbonyl,
followed by releasing of CoA to form an acyl-enzyme adduct
(route B). However, the tendency of 2 to release its structural
strain by ring cleavage, generating a reactive intermediate (3)
capable of trapping an enzyme nucleophile, may also account
To gain insight into the toxicity of MCPF-CoA on crotonase,
we have chemically prepared 2 in racemic form (Figure 1).11
The crude product was purified and tested with crotonase
(1) There are three types of enoyl-CoA hydratase in higher animals, two
in mitochondria as short-chain enoyl-CoA hydratase (crotonase) and
trifunctional protein (Uchida, Y.; Izai, K.; Orii, T.; Hashimoto, T. J. Biol.
Chem. 1992, 267, 1034) and one in peroxisomes as bifunctional protein
(Osumi, T.; Hashimoto, T. Arch. Biochem. Biophys. 1980, 203, 372).
(2) Crotonase has been purified from bovine liver,3c rat liver (Furuta,
S.; Miyazawa, S.; Osumi, T.; Hashimoto, T.; Ui, N. J. Biochem. 1980, 88,
1059), pig heart (Fong, J. C.; Schulz, H. Methods Enzymol. 1981, 71, 390),
and pig kidney (Buettner, H. Ph.D. Thesis, University of Konstanz, 1988).
(3) (a) Waterson, R. M.; Hill, R. L. J. Biol. Chem. 1972, 247, 5258. (b)
Steinman, H. M.; Hill, R. L. J. Biol. Chem. 1973, 248, 892. (c) Steinman,
H. M.; Hill, R. L. Methods Enzymol. 1975, 35, 136.
(12) The bovine liver enzyme was purchased from Sigma (St. Louis,
MO). The pig kidney enzyme was purified to near homogeneity by a
sequence similar to that used in the purification of pig heart crotonase.2
The specific activity of the purified enzyme is 293 units/mg (1 unit ) 1
µmol of product formation per minute).
(13) The kinetic parameters of inactivation were derived from plotting
the apparent inactivation rate, obtained by incubating crotonase (9 µM, 6.3
nmol) with 2 of varied concentration (9-600 µM), versus inhibitor
concentration. Unless otherwise specified, 50 mM potassium phosphate
buffer, pH 7.5, was used in all experiments.
(14) Both covalent modification of the enzyme active site and formation
of a very tight-binding complex had been proposed for the irreversible
inactivation of pig kidney crotonase by 2.10b
(15) (a) Suckling, C. J. Angew. Chem., Int. Ed. Engl. 1988, 27, 537. (b)
Liu, H.-w.; Walsh, C. T. In The Chemistry of the Cyclopropyl Group;
Pappoport, Z., Ed.; Wiley-Interscience: New York, 1987; Part 2, p 969.
(4) Bahnson, B. J.; Anderson, V. E. Biochemistry 1991, 30, 5894.
(5) Willadsen, P.; Eggerer, H. Eur. J. Biochem. 1975, 54, 247.
(6) (a) D’Oridine, R. L.; Tonge, P. J.; Carey, P. R.; Anderson, V. E.
Biochemistry 1994, 33, 12635. (b) D’Oridine, R. L.; Bahnson, B. J.; Tonge,
P. J.; Anderson, V. E. Biochemistry 1994, 33, 14733.
(7) He, X.-Y.; Yang, S.-Y.; Schulz, H. Arch. Biochem. Biophys. 1992,
298, 527.
(8) Grey, D. O.; Fowden, L. Biochem. J. 1962, 82, 385.
(9) Melde, K.; Jackson, S.; Bartlett, K.; Sherratt, H. S. A.; Ghisla, S.
Biochem. J. 1991, 274, 395.
(10) (a) Schulz, H.; Fong, J. M. Methods Enzymol. 1981, 72, 604. (b)
Melde, K.; Buettner, H.; Boschert, W.; Wolf, H. P. O.; Ghisla, S. Biochem.
J. 1989, 259, 921.
(11) Lai, M.-t.; Liu, L.-d.; Liu, H.-w. J. Am. Chem. Soc. 1991, 113, 7388.
0002-7863/96/1518-0275$12.00/0 © 1996 American Chemical Society

276 J. Am. Chem. Soc., Vol. 118, No. 1, 1996
Communications to the Editor
Figure 3.
for the eventual toxicity of 1 (route C). Routes B and C are
less likely due to the facts that CoA is not a structural entity
unique for 2 and the cyclopropyl ring hydrogen is not
particularly acidic. Whether the molecular basis of the virulent
consequences of 1 is caused by the nucleophilic trapping via
an intact methylenecyclopropyl moiety or by the ring-opened
form of the inhibitor can be discerned by scrutinizing the
inactivation process using labeled 2.
the target enzyme activates the cyclopropane for nucleophilic
addition by oxidation or protonation of the appended groups
that render them more electron-withdrawing.15,18 However, no
such catalysis-produced activation appears to be necessary for
the inactivation of crotonase by 2, as deduced from this study.
Since in the normal catalysis the substrate has been shown to
be polarized by an electrophile proximal to the CdO oxygen
(either via a positive charge or strong H-bonds) and a nucleo-
phile near the â-C in the active site of crotonase,6 such a push-
pull model may also explain the activation of 2 and facilitate
the trapping of an active site nucleophile. The large ring strain
associated with the methylenecyclopropyl system may have also
contributed to the reactivity of 2. Interestingly, while a
glutamate residue (Glu164 of the rat liver enzyme) conserved in
enoyl-CoA hydratases19 has been shown to be the base
responsible for R-proton abstraction in the rat liver crotonase,6b
the nucleophile trapped by MCPF-CoA in the bovine liver
enzyme is unlikely a carboxylate residue, since the inhibitor-
enzyme adduct is stable under denaturing alkaline conditions.
Further characterization of the labeled protein will certainly
provide valuable information about the active site of this
important class of enzyme whose crystal structure is still lacking.
Preparation of the tritium-labeled MCPF-CoA (7) was ef-
fected by the sequence shown in Figure 3. The key steps
involved the exchange of the acetylenic hydrogen of 4 with
tritium by treatment of 4 with base and quenching with tritiated
water, and the cyclization of 5 to 6 by n-butyllithium and
dichloroethane.16 The specific radioactivity of the final product
7 was 0.56 mCi/mmol. The covalent nature of this inactivation
was substantiated by a 1:1 stoichiometry between 7 (10 molar
equiv) and bovine liver crotonase (40 nmol), determined after
extensive dialysis of the inactivated enzyme. The resulting
inactivated enzyme was denatured by boiling, and precipitated
protein was collected and redissolved in Tris-HCl buffer (0.1
M, pH 10) containing 6 M guanidine hydrochloride. Interest-
ingly, >93% of the radioactivity was retained in the denatured
protein after overnight incubation. This result, in conjunction
with the failure to detect released CoA in the overnight
incubation mixture with 5,5′-dithiobis(2-nitrobenzoic acid),
provided strong evidence suggesting that modification of
crotonase via an acyl-enzyme adduct is quite unlikely. More-
over, little tritium release (<1%) was detected when 7 (1 molar
equiv) was incubated with crotonase, followed by quenching
with a 10% charcoal solution.17 On the basis of this observation,
route C may also be excluded as a viable mechanism. Thus,
these results are most consistent with route A, in which 2
inactivates crotonase via the intact methylenecyclopropane ring
trapping of an active site nucleophile.
Acknowledgment. This work was supported in part by the National
Institutes of Health (Grant GM 40541) . H.-w.L. is the recipient of a
National Institutes of Health Research Career Development Award (GM
00559).
JA952621B
(18) (a) Haddow, J.; Suckling, C. J.; Wood, H. C. S. J. Chem. Soc.,
Perkin Trans. 1 1989, 1297. (b) Kraus, G. A.; Kim, H.; Thomas, P. J.;
Metzler, D. E.; Metzler, C. M.; Taylor, J. E. Synth. Commun. 1990, 20,
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Chem. Soc. 1993, 115, 2982. (d) Croteau, R.; Alonso, W. R.; Koepp, A.
E.; Shim, J. H.; Cane, D. E. Arch. Biochem. Biophys. 1993, 307, 397.
(19) (a) Yang, X.-Y. H.; Schulz, H.; Elzinga, M.; Yang, S.-Y. Biochem-
istry 1991, 30, 6788. (b) Hoefler, G.; Forstner, M.; McGuiness, M. C.; Hulla,
W.; Hiden, M.; Krisper, P.; Kenner, L.; Lengauer, C.; Ried, T.; Zechner,
R.; Chen, G. L.; Moser, H. W. Genomics 1994, 19, 60. (c) Kamijo, T.;
Aoyama, T.; Miyazaki, J.; Hashimoto, T. Biochem. Biophys. Res. Commun.
1994, 199, 818. Also see references cited in these papers.
It is worth mentioning that examples of cyclopropane-
containing, mechanism-based inactivators are known in which
(16) Baldwin, J. E.; Parker, D. W. J. Org. Chem. 1987, 52, 1475.
(17) Lai, M.-t.; Li, D.; Oh, E.; Liu, H.-w. J. Am. Chem. Soc. 1993, 115,
1619.