Isopentenyl diphosphate isomerase. Mechanism-based inhibition by diene analogues of isopentenyl diphosphate and dimethylallyl diphosphate

Article abstract of DOI:10.1021/ja056187h

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization cat

Full text of DOI:10.1021/ja056187h

Published on Web 11/15/2005
Isopentenyl Diphosphate Isomerase. Mechanism-Based
Inhibition by Diene Analogues of Isopentenyl Diphosphate
and Dimethylallyl Diphosphate
‡
§
,‡
Zheng Wu, Johan Wouters, and C. Dale Poulter*
Contribution from the Department of Chemistry, UniVersity of Utah, Salt Lake City, UT 84112,
Laboratoire de Chimie Biologique Structurale, 61 Rue de Bruxelles, Namur, Belgium, and
Institut de Recherches Wiame, Campus Ceria, Bruxelles, Belgium
Received September 8, 2005; E-mail: poulter@chemistry.utah.edu
Abstract: Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate
(IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the
isoprenoid biosynthetic pathway. The isomerization catalyzed by type I IDI involves protonation of the
carbon-carbon double bond in IPP or DMAPP to form a tertiary carbocation, followed by deprotonation.
Diene analogues for DMAPP (E-2-OPP and Z-2-OPP) and IPP (4-OPP) were synthesized and found to be
potent active-site-directed irreversible inhibitors of the enzyme. X-ray analysis of the E‚I complex between
Escherichia coli IDI and 4-OPP reveals the presence of two isomers that differ in the stereochemistry of
the newly formed C3-C4 double bond in the hydrocarbon chain of the inhibitor. In both adducts C5 of the
inhibitor is joined to the sulfur of C67. In these structures the methyl group formed upon protonation of the
diene moiety in 4-OPP is located near E116, implicating that residue in the protonation step.
The isomerization of isopentenyl diphosphate (IPP) to dim-
ethylallyl diphosphate (DMAPP) is a crucial activation step in
isoprenoid biosynthesis, during which IPP is converted to its
highly electrophilic isomer. These two molecules are the
building blocks used to construct over 35 000 isoprenoid
compounds found in nature. In Archaea, Eukaryota, and some
Bacteria, IPP is synthesized from acetyl-CoA by the mevalonate
Scheme 1. Protonation-Deprotonation Mechanism for
Isomerization of IPP and DMAPP
Scheme 1). During the reaction, the pro-R hydrogen (blue) is
removed from C(2) of IPP, and a proton from solvent (red) is
added to the re-face at C4 of the double bond to generate the
(MVA) pathway and is the exclusive product from the phos-
phorylation and decarboxylation of the key intermediate me-
valonic acid.1 IPP is required for biosynthesis of DMAPP in
,2
E-methyl group at C(3).
3
the MVA pathway, and IPP isomerase is essential. In most
Type II IPP isomerase is found in Archaea and in Bacteria.10
bacteria and plant chloroplasts, IPP and DMAPP are synthesized
from pyruvate and D-glyceraldehyde phosphate by the methyl-
Typically, a given bacterium has only one form of IPP
isomerase, but, in at least one instance, the type I and type II
4
erythritol phosphate (MEP) pathway. The final step produces
11,12
enzymes coexist.
Type II IPP isomerase requires reduced
both compounds during the reduction of hydroxydimethylallyl
diphosphate. IPP isomerase activity is often found in organisms
that utilize the MEP pathway, but the enzyme is not essential.
flavin and divalent metal cofactors for activity. The stereo-
chemistry of the reaction is similar to that catalyzed by the type
13,14
I enzyme,
although the facial selectivity for proton addition
5,6
Two convergently evolved IPP isomerases are known. The
at C(4) has not yet been determined.
7
type I IPP isomerase was discovered in the late 1950s and is
(
5) Anderson, M. S.; Muehlbacher, M.; Street, I. P.; Proffitt, J.; Poulter, C. D.
found in Eukaryota and Bacteria. The enzyme requires a divalent
metal and catalyzes the antarafacial isomerization of IPP and
J. Biol. Chem. 1989, 264, 19169-19175.
(6) Kandea, K.; Kuzuyama, T.; Takagi, M.; Hayakawa, Y.; Seto, H. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 932-937.
8
9
DMAPP by a protonation-deprotonation mechanism (see
(
(
(
7) Agranoff, B. W.; Eggerer, H.; Henning, U.; Lynen, F. J. Am. Chem. Soc.
1
959, 81, 1254-55.
8) Clifford, K.; Cornforth, J. W.; Mallaby, R.; Philips, R. T. Chem. Commun.
971, 1599-1600.
‡
University of Utah.
Laboratoire de Chimie Biologique Structurale and Institut de Recherches
1
§
9) Muehlbacher, M.; Poulter, C. D. Biochemistry 1988, 27, 7315-7328.
Wiame.
(10) Rohdich, F.; Bacher, A.; Eisenreich, W. Bioorg. Chem. 2004, 32, 292-
(
(
(
(
1) Poulter, C. D.; Rilling, H. Biosynthesis of Isoprenoid Compounds; Wiley:
308.
New York, 1981.
(11) Kuzuyama, T.; Seto, H. Nat. Prod. Rep. 2003, 20, 171-183.
(12) Laupitz, R.; Hecht, S.; Amslinger, S.; Zepeck, F.; Kaiser, J.; Richter, G.;
Schramek, N.; Steinbacher, S.; Huber, R.; Arigoni, D.; Bacher, A.;
Eisenreich, W.; Rohdich, R. Eur. J. Biochem. 2004, 271, 2658-2669.
(13) Laupitz, R.; Grawert, T.; Rieder, C.; Zepeck, F.; Bacher, A.; Arigoni, D.;
Rohdich. F.; Eisenreich, W. Chem. BiodiVersity 2004, 1, 1367-1376.
(14) Barkley, S. J.; Desai, S. V.; Poulter, C. D. Org. Lett. 2004, 6, 5019-5021.
2) Koyama, T.; Ogura, K. ComprehensiVe Natural Product Chemistry;
Pergmon: Oxford, 1999.
3) Mayer, M. P.; Hahn, F. M.; Stillman, D. J.; Poulter, C. D. Yeast 1992, 8,
7
43-748.
4) Rodr ´ı guez-Concepci o´ n, M.; Boronat, A. Plant Physiol. 2002, 130, 1079-
089.
1
10.1021/ja056187h CCC: $30.25 © 2005 American Chemical Society
J. AM. CHEM. SOC. 2005, 127, 17433-17438
9
17433

A R T I C L E S
Wu et al.
Several lines of evidence support the protonation-deproto-
structure of E. coli IPP isomerase covalently modified by the
IPP analogue that provides insights about the protonation step.
nation mechanism shown in Scheme 1 for type I IPP isomerase.
15
These include proton exchange experiments, substituent effects
Experimental Section
9
,16
on the rate of the reaction,
tight binding of transition-state
9
(E)-3-Methyl-2,4-pentadien-1-ol (E-2-OH). A solution of 1.0 g
(10.4 mmol) of (E)-3-methyl-2-penten-4-yn-1-ol (E-1-OH) in 10.0 mL
of anhydrous ethyl alcohol was reduced using 0.148 g of Lindlar
catalyst. The catalyst was removed by filtration, solvent was removed
by rotary evaporation, and the resulting yellow oil was purified by flash
chromatography on silica gel (3/2 hexane/diethyl ether) followed by
analogues, and irreversible inhibition by mechanism-based
inhibitors. Covalent modification of active-site residues by the
9
irreversible inhibitors and site-directed mutagenesis of the
modified amino acids implicate two amino acids, C67 and E116
1
7
(
Escherichia coli numbering), in the proton-transfer steps.
X-ray analysis of E. coli IPP isomerase shows that these amino
acids are located on opposite sides of the active site.1 E116 is
also part of a hexacoordinate binding site for a divalent metal.
In the structure of the enzyme containing 2-(N,N-dimethylami-
no)ethyl diphosphate, a transition-state analogue for the putative
tertiary cationic intermediate where C(3) is replaced by a
positively charged N-H ammonium moiety, one of the car-
boxylate oxygens in the E116 side chain is coordinated to the
22
distillation at 50-55 °C (Kugelrohr, 3 mmHg) (lit. bp 74 °C at 14
8
mmHg) to give 0.801 g (77%) of a colorless oil:22,23 1H NMR (C
D )
6 6
δ 0.37 (1H, s (broad)), 1.53 (3H, s), 4.40 (2H, dd, JH,H ) 6.6 Hz), 4.96
(1H, d, JH,H ) 10.5 Hz), 5.09 (1H, dd, JH,H ) 17.4 Hz), 5.52 (1H, d,
1
3
J
(
H,H ) 6.6 Hz), 6.33 40 (1H, dd, JH,H ) 17.4, 10.8 Hz); C NMR
CDCl ) δ 12.2, 59.4, 112.6, 132.4, 135.8, 141.7.
E)-5-Bromo-3-methylpenta-1,3-diene (E-2-Br). To a solution of
5 mg (0.42 mmol) of E-2-OH in CH Cl were added 182 mg (0.55
and 144 mg (0.55 mmol) of triphenylphosphine. After
h at room temperature, pentane was added, the resulting suspension
3
(
4
2
2
mmol) of CBr
3
4
+
H-N unit and the other to the divalent metal. In another
structure, where the enzyme was inactivated with the epoxide
derivative of IPP, the oxirane ring had been opened to give a
primary alcohol at C(4) with concomitant formation of a
thioether bond between C(3) of the inhibitor and the sulfhydryl
moiety of C67.17 The X-ray structures of the enzyme‚inhibitor
complexes also contained a second divalent metal, which was
coordinated to nonbridging oxygens at P(1) and P(2) of the
diphosphate moiety, the side-chain carboxylate of E87, and the
amide carbonyl oxygen in C67.
was filtered, and solvent was removed at reduced pressure. The residue
was purified by distillation at 40 °C (Kugelrohr, 3 mmHg) to give 49
24 1
mg (62%) of a colorless oil: H NMR (CDCl ) δ 1.85 (3H, d, JH,H )
3
1
.5 Hz), 4.13 (2H, d, JH,H ) 8.4 Hz), 5.12 (1H, d, JH,H ) 10.8 Hz),
5.30 (1H, d, JH,H ) 17.1 Hz), 5.38 (1H, d, JH,H ) 10.8 Hz), 5.78 (1H,
dd, JH,H ) 17.4, 10.8 Hz).
(Z)-3-Methyl-2,4-pentadien-1-ol (Z-2-OH). Following the proce-
dure described for E-2-OH, 2.36 g (24.5 mmol) of (Z)-3-methyl-2-
pentene-4-yn-1-ol (Z-1-OH) was reduced using 0.40 g of Lindlar
catalyst to give 1.67 g (75%) of a colorless oil:2
2,23 1
6 6
H NMR (C D ) δ
Replacement of the active-site cysteine in yeast IPP isomerase
0
.76 (1H, s (broad)), 1.72 (3H, s), 4.21 (2H, t, JH,H ) 6.6 Hz), 5.01
by serine produced a modest 2-fold increase in KM but reduced
(
1H, d, JH,H ) 11.1 Hz), 5.13 (1H, dd, JH,H ) 17.3, 1.0 Hz), 5.57 (1H,
4
17
kcat by ∼10 . The related alanine mutant was inactive.
Likewise, substitution of the active-site glutamate with glutamine
or valine gave inactive proteins. Interestingly, an X-ray structure
of the C67A mutant of E. coli IPP isomerase, which had been
13
d, JH,H ) 6.6 Hz), 6.68 (1H, dd, JH,H ) 17.4, 10.8 Hz); C NMR
(CDCl ) δ 20.1, 58.5, 115.2, 130.6, 134.0, 134.6.
3
(Z)-5-Chloro-3-methylpenta-1,3-diene (Z-2-Cl). To a solution of
0.367 g (2.75 mmol) of NCS in 10 mL of CH
.86.4 mg (3.0 mmol) of dimethyl sulfide. The mixture was allowed
to warm to 0 °C for 5 min and then cooled to -40 °C before 246 mg
2.51 mmol) of Z-2-OH was added. The suspension was allowed to
2 2
Cl at -30 °C was added
1
treated with an epoxy analogue of IPP, showed that the protein
was covalently modified.19 In this case the oxirane ring had
opened to form an ester linkage between C(3) of the inhibitor
and the side-chain carboxylate moiety of E116. Thus, the
(
warm to 0 °C and stirred for 2 h. The resulting solution was washed
with saturated NaCl, solvent was removed at reduced pressure, and
the resulting yellow oil was distilled at 35 °C (Kugelrohr, 4 mmHg) to
“
inactive” protein retained the ability to activate and open the
oxirane ring, suggesting a role for E116 in the protonation step
of the isomerization reaction.
25 1
give 0.175 g (60%) of a colorless oil: H NMR (NMR (CDCl
.72 (3H, s) 4.21 (2H, d, JH,H ) 6.6 Hz), 5.01 (1H, d, JH,H ) 11.1 Hz),
5.31 (1H, dd, JH,H ) 17.3, 1.0 Hz), 5.57 (1H, d, JH,H ) 6.6 Hz), 6.68
1H, dd, JH,H ) 17.4, 10.8 Hz); 13C NMR (CDCl
) δ 20.1, 58.5, 115.2,
30.6, 134.0, 134.6.
-Methylene-4-penten-1-ol (4-OH). Hexane was removed from 8.4
3
) δ
1
Diene analogues of oxidosqualene have been used to intercept
carbocationic intermediates as allylic cations, which react with
active-site nucleophiles and abort the cascade of cyclization
reactions leading to the tetracyclic skeleton polycyclic triter-
(
1
3
3
mL (17 mmol) of a solution of butyllithium (2.0 M in hexane) before
a solution containing 170 mg (16.8 mmol) of diisopropylamine, 1.68
mL of (1.68 mmol) of potassium tert-butoxide (1.0 M in THF), and
2
0,21
penes.
We thought that similar analogues of IPP and
DMAPP might be potent mechanism-based inhibitors of IPP
isomerase by creating an electrophilic center in the substrate
analogues that is susceptible to alkylation. We now report the
synthesis of two diene analogues of DMAPP and a diene
analogue of IPP, their irreversible inactivation of type I IPP
isomerase from Schizosaccharomyces pombe, and an X-ray
1
.50 g (16.8 mmol) of 4-methylenetetrahydropyran (3) in 20 mL of
THF (precooled at -75 °C) was added. After 2 h at -50 °C, the reaction
was quenched with saturated potassium dihydrogen phosphate and
extracted with 30/70 diethyl ether/hexane. Solvent was removed at
reduced pressure (bp 68-71 °C at 22 mmHg) to give 0.75 g (50%) of
a colorless oil:2 H NMR (CDCl
3 1
3
) δ 0.80 (1H, s (broad)), 2.52 (2H, t,
J
H,H ) 6.6 Hz), 3.77 (2H, dt, JH,H ) 6.3 Hz), 509-5.30 (4H, m), 6.40
(1H, ddd, JH,H ) 18.0, 10.5, 0.6 Hz); 13C NMR (CDCl
) δ 34.8, 61.1,
14.0, 117.8, 138.5, 142.8.
(
15) Street, I. P.; Christensen, D. J.; Poulter, C. D. J. Am. Chem. Soc. 1990,
1
12, 8577-8578.
3
(
16) Reardon, J. E.; Abeles, R. H. Biochemistry 1986, 25, 5609-5616.
1
(
17) Street, I. P.; Coffman, H. R.; Baker, J. A.; Poulter, C. D Biochemistry 1994,
3
3, 4212-4217.
(
18) Wouters, J.; Oudjama, Y.; Barkley, S. J.; Tricot, C.; Stalon, V.; Droogmans,
L.; Poulter, C. D. J. Biol. Chem. 2003, 278, 11903-11908.
19) Wouters, J.; Oudjama, Y.; Stalon, V.; Droogmans, L.; Poulter, C. D.
Proteins 2004, 54, 216-221.
(22) Burger, B. V.; Plessis, du E.; Garbers, C. F.; Pachler, K. G. R. J. Chem.
Soc., Perkin Trans. 1 1973, 584-587.
(23) Margot, C.; Rizzolio, M.; Scholosser, M. Tetrahedron 1990, 46, 2411-
2424.
(24) Clark, K. B.; Culshaw, P. N.; Griller, D.; Lossing, F. P.; Martinho Simoes,
J. A.; Walter, J. C. J. Org. Chem. 1991, 56, 5535-5539.
(25) Mayr, H.; Heilman, W. Tetrahedron 1986, 42, 6657-6662.
(
(
20) Xiao, X.-Y.; Prestwich, G. D. J. Am. Chem. Soc. 1991, 113, 9673-9674.
21) Ceruti, M.; Rocco, F.; Viola, F.; Balliano, G.; Milla, P.; Arpicco, S.; Cattel,
L. J. Med. Chem. 1998, 41, 540-554.
(
17434 J. AM. CHEM. SOC.
9
VOL. 127, NO. 49, 2005

Mechanism-Based Inhibition of IPP Isomerase
A R T I C L E S
3
-Methylene-4-pentenyl Tosylate (4-OTs). To a solution of 93 mg
3-Methylene-4-pentenyl Diphosphate (4-OPP). Following the
procedure described for E-2-OPP, 230 mg (1.10 mmol) of 4-OTs was
treated with 2.98 g (3.30 mmol) of tris(tetra-n-butyl) ammonium
(
4
0.50 mmol) of p-toluenesulfonyl chloride and 73 mg (0.60 mmol) of
-(N,N-dimethylamino)pyridine (DMAP) in 4 mL of CH
2 2
Cl was added
4
7 mg (0.48 mmol) of 4-OH. After 2 h, the mixture was poured into
pyrophosphate to give 147 mg (43%) of a white solid: UV (25 mM
1
hexane, and the resulting precipitates were removed by filtration.
Solvent was removed at reduced pressure, and the residue was
chromatographed on silica gel (40/60 diethyl ether/hexane) to give 113
mg (92%) of a clear colorless oil: IR (neat) 3100, 3043, 3015, 2959,
NH
4
HCO
3
) λmax 224 (ꢀ 16 600) nm; H NMR (D
2
O/ND
4
OD, pH )
8.0) δ 2.51 (1H, dd, JH,H ) 18.0, 10.5 Hz), 3.98 (2H, dd, JH,H ) 7.2
13
Hz), 5.03-5.31 (4H, m), 6.37 (1H, dd, JH,H ) 18.0, 10.5 Hz); C NMR
(D O/ND OD, pH ) 8.0) δ 34.5 (d, JC,P ) 5.6 Hz), 67.7 (d, JC,P ) 4.8
Hz), 116.9, 120.5, 141.1, 145.3; 31P NMR (D
O/ND OD, pH ) 8.0) δ
-0.46 (1P, d, JP,P ) 22 Hz), -4.63 (1P, d, JP,P ) 22 Hz); HRMS ((-
)-FAB) calcd for C 256.9980, found 256.9972.
2
4
-1
2917, 1650, 1603, 1358, 1309, 1293, 1230, 1188, 1176, 1096, 970 cm ;
2
4
1
UV (CHCl
δ 2.45 (3H, s), 2.59 (2H, td, JH,H ) 7.2, 0.9 Hz), 4.14 (2H, t, JH,H
.2 Hz), 5.0-5.15 (4H, m), 6.29 (1H, dd, JH,H ) 17.6, 10.5 Hz), 7.35
1H, d, JH,H ) 8.4 Hz), 7.79 (1H, d, JH,H ) 8.4 Hz); 13C NMR (CDCl
δ 21.9, 31.1, 68.9, 114.0, 118.6, 128.0, 129.9, 130.0, 133.2, 138.0,
3
3
) λmax 222 (ꢀ 18 600), 197 (ꢀ 13 800) nm; H NMR (CDCl )
)
6 11 2 7
H P O
2
7
7
(
Assay for IPP Isomerase. Schizosaccharomyces pombe and
Escherichia coli IPP isomerase were obtained as described previously.
The activity of the enzymes was measured by the acid-liability protocol
28
3
)
9
16 3
140.7, 144.9; HRMS (EI at 70 eV) calcd for C13H O S 252.0816, found
in a total volume 200 µL of 50 mM HEPES buffer, pH 7.0, containing
2
52.0797.
2
10 mM MgCl , 200 mM KCl, 0.5 mM DTT, 1 mg/mL BSA, and 400
µM [1- C]IPP (10 µCi/µmol). The mixture was equilibrated at 37 °C
and initiated by addition of 10 µL of a solution of enzyme in assay
buffer. Addition of a His tag to E. coli IPP isomerase did not
6
significantly alter its kinetic properties.
14
General Procedure for Synthesis of Diphosphates. The procedure
2
6
of Davission and co-workers was followed with the exception that
HPLC was used in the final purification step instead of flash
chromatography on cellulose. To a solution of 1.5-3.0 equiv of tris-
(
tetra-n-butylammonium) hydrogen pyrophosphate in CH
added a solution of tosylate, chloride, or bromide (0.25-0.50 mM) in
CH CN. The resulting mixture was allowed to stir for 2-3 h at room
temperature. Solvent was removed at reduced pressure, and the resulting
residue was dissolved in a minimal volume of 25 mM NH HCO (ion-
exchange buffer). The clear solution was passed through DOWEX AG
0W-X8 (100-200 mesh, ammonium form) that had been equilibrated
3
CN was
Time-dependent inactivation of the enzyme was measured as follows.
Assay buffer containing varying concentrations of inhibitor was
equilibrated at 37 °C before a 20-fold higher concentration of enzyme
than required for the standard assay was added to a final volume of
200 µL. Samples (10 µL) were removed at different times and added
3
4
3
14
to assay buffer containing 400 µM [ C]IPP (10 µCi/µmol) to a final
volume of 200 µL.
5
with two column volumes of the ion exchange buffer at a flow rate of
one column volume/15 min. The resulting clear solution was concen-
trated by rotary evaporation and lyophilized. The residue was then
Crystallization of E. coli IPP isomerase. Crystals of recombinant
(C-terminal His-tagged) E. coli IPP isomerase were grown at room
temperature by the hanging drop method as described by Oudjama et
al. Briefly, protein (10 mg/mL) was equilibrated against a reservoir
containing PEG2000 (16%), ammonium sulfate (100 mM), and MnCl2
(10 mM) buffered with Tris/maleate at pH 5.5. Complexes with 4-OPP
were obtained by soaking crystals of the enzyme in a 25 mM solution
of the inhibitor in Tris/maleate (100 mM, pH 5.5), PEG2000 (16%),
28
transferred to a centrifuge tube and dissolved in 100 mM NH
The resulting solution was extracted two or three times with a 1:1
mixture of CH CN and 2-propanol. CH CN and 2-propanol were
4 3
HCO .
3
3
removed at reduced pressure, and the aqueous layer was lyophilized.
The residue was by HPLC, and the purified diphosphate was stored at
-
78 °C.
E)-3-Methyl-2,4-pentadienyl Diphosphate (E-2-OPP). A solution
of 90 mg (0.92 mmol) of E-2-Br in CH CN was treated with 2.74 g
3.00 mmol) of tris(tetra-n-butylammonium) hydrogen pyrophosphate.
ammonium sulfate (100 mM), MnCl (10 mM), and glycerol (25%).
2
(
These conditions were identical to those used to crystallize the wild-
type enzyme. After soaking, a crystal was flash frozen and diffraction
data were collected with a Mar345 imaging plate system from
Marresearch equipped with Osmic optics and running on an FR591
rotating anode generator. Diffraction data were processed with the
MarFLM suite. In the presence of metal, the protein crystallizes, with
two molecules in the asymmetric unit, in orthorhombic space group
P2 2 2 with cell parameters a ) 69.3, b ) 72.6, and and c ) 92.5 Å.
3
(
The ammonium form of the diphosphate was purified by HPLC on an
Asahipak ODP-90 HPLC column with gradient elution (solvent A )
1
1
00 mM NH
:1 (A/B), 35-45 min, 1:1 (A/B)) to give 120 mg (42%) of a white
HCO ) λmax 228 (ꢀ 14 800), 197 (ꢀ 10 600)
OD, pH ) 8.0) δ 1.60 (3H, s), 4.40 (2H, dd,
H,H ) JH,P ) 6.6 Hz), 4.94 (1H, d, JH,H ) 11.1 Hz), 5.26 (1H, d, JH,H
4
HCO
3
, solvent B ) CH
3
CN; 0-20 min (A) 20-35 min
solid: UV (25 mM NH
nm; H NMR (D
J
4
3
1
1 1
1
2
O/ND
4
A data set was recorded at 2.05 Å resolution. Refinement was performed
with the program SHELXL97 using the structure of unliganded IPP
isomerase (PDB reference code: 1hxz) as starting model.
)
17.7 Hz), 5.53 (1H, t, JH,H ) 6.6 Hz), 6.29 (1H, dd, JH,H ) 17.7,
1
3
1
0.8 Hz); C NMR (D
2
O/ND
4
OD, pH ) 8.0) δ 14.1, 65.3 (d, JC,P
)
O/
3
1
Results
5
.2 Hz), 112.7, 129.9 (d, JC,P ) 7.9 Hz), 140.9, 143.2; P NMR (D
2
ND
22 Hz); HRMS ((-)-FAB) calcd for C
56.9966.
Z)-3-Methyl-2,4-pentadienyl Diphosphate (Z-2-OPP). Following
the procedure described for E-2-OPP, 63 mg (0.54 mmol) of Z-2-Cl
was treated with 0.901 g (1.0 mmol) of tris(tetra-n-butylammonium)
4
OD, pH ) 8.0) δ -1.90 (1P, d, JP,P ) 22 Hz), -4.76 (1P, d, JP,P
Synthesis of Diene Analogues of IPP and DMAPP. The
syntheses of DMAPP analogues E-2-OPP and Z-2-OPP and
IPP analogue 4-OPP are shown in Scheme 2. The E- and
Z-isomers of diene analogues for DMAPP (E-2-OH and Z-2-
OH) were prepared from commercially available isomeric
acetylenic alcohols E-1-OH and Z-1-OH by selective hydro-
genation of the triple bond using Lindlar catalyst. Initially both
alcohols were converted to the corresponding bromides. How-
ever, the yield for the Z-isomer was substantially lower, and
the less reactive chloride was prepared instead. The halides were
phosphorylated by treatment with tris(tetra-n-butylammonium)-
hydrogen pyrophosphate to give E- and Z-2-OPP. The diene
)
2
6 11 2 7
H P O
256.9980, found
(
hydrogen pyrophosphate to give 87 mg (52%) of a white solid: UV
1
(
(
7
5
25 mM NH
O/ND
.0 Hz), 5.13 (1H, d, JH,H ) 11.1 Hz), 5.25 (1H, d, JH,H ) 17.4 Hz),
4
HCO
3
) λmax 229 (ꢀ 8900), 196 (ꢀ 6800) nm; H NMR
D
2
4
OD, pH ) 8.0) δ 1.72 (3H, s), 4.44 (2H, dd, JH,H ) JH,P )
.50 (1H, t, JH,H ) 7.3 Hz), 6.71 (1H, dd, JH,H ) 17.2, 10.9 Hz); 13
C
NMR (D
2
O/ND
4
OD, pH ) 8.0) δ 21.6, 64.2 (d, JC,P ) 5.2 Hz), 119.3,
3
1
1
27.7 2 (d, JC,P ) 7.8 Hz), 135.6, 140.1; P NMR (D
2
O/ND
4
OD, pH
)
8.0) δ -1.83 (1P, d, JP,P ) 21 Hz, P1), -4.83 (1P, d, JP,P ) 21 Hz,
256.9980, found 256.9959.
6 11 2 7
P2); HRMS ((-)-FAB) calcd for C H P O
(
27) Hahn, F. M.; Poulter, C. D. J. Biol. Chem. 1995, 270, 11298-11303.
28) Oudjama, Y.; Derbecq, V.; Sainz, G.; Clantin, B.; Tricot, C.; Stalon, V.;
Villeret, V.; Droogmans, L. Acta Crystallogr. Sect. D, Biol. Crystallogr.
2001, 57, 287-288.
(
(
26) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler, K. E.; Muehlbacher,
M.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768-4779.
J. AM. CHEM. SOC.
9
VOL. 127, NO. 49, 2005 17435

A R T I C L E S
Wu et al.
Scheme 2. Synthesis of Diphosphates E-2-OPP, Z-2-OPP, and 4-OPP
Table 1. Kinetic Constants for Irreversible Inhibition of S. pombe
IPP Isomerase by E-2-OPP, Z-2-OPP, and 4-OPP
analogue for IPP was synthesized from 4-methylene tetrahy-
dropyran. The pyran ring was opened upon treatment with
lithium diisopropyl amide (LDA)/potassium tert-butoxide. Al-
cohol 4-OH was converted to the corresponding tosylate,
followed by treatment with inorganic pyrophosphate to give
inhibitor
k
I
(s-1)
K
I
(µM)
k
I
/K
I
(s-
1
µ
M-1)
E-2-OPP
Z-2-OPP
17 ( 1.2
186 ( 11
43 ( 5.1
0.91 ( 0.08
84 ( 7
3.2 ( 0.2
19
2.2
13
4-OPP
4
-OPP.
Kinetic Studies. Preliminary experiments indicated that S.
the diphosphate group in 4-OPP is similar in both geometries.
A second metal binds to nonbridging oxygens in the diphosphate
moiety, and the stabilizing roles of Lys21, Arg51, Lys55, Glu83,
and Glu87 are confirmed.
pombe and E. coli IPP isomerase was irreversibly inactivated
upon incubation with the diene analogues of IPP and DMAPP.
The kinetic constants for inactivation of the S. pombe enzyme
were measured by incubation of the protein with varying
concentrations of the inhibitors. Samples were removed over a
period of 30 min and diluted 20-fold into buffer containing
1
4
[
C]IPP, and the activity of the enzyme was measured. In a
control experiment, the enzyme retained >97% of its activity
over a period of 1 h when incubated under similar conditions
without addition of an inhibitor. In each case concentration-
dependent first-order inactivation was observed (Figure 1). The
rate constants for inactivation (kinact) were determined from a
semilogarithmic plot of residual enzyme activity versus time.
The rate constants for inhibition at saturating levels of the
inhibitors (kI) and the inhibitor constants (KI) were determined
from replots of kinact versus the reciprocal of the inhibitor
concentrations. The values for kI and KI are given in Table 1.
All of the diene analogues were potent inhibitors of the enzyme.
E-2-OPP was the most efficient inhibitor as measured by kI/
KI. Although Z-2-OPP was ∼10 times more reactive than E-2-
OPP, its KI was substantially higher. 4-OPP was only slightly
less efficient than E-2-OPP. Both of the DMAPP analogues
were alternate substrates for chain elongation by avian FPP
synthase, with steady-state kinetic constants similar to those for
DMAPP (data not shown); however, neither compound inacti-
vated the enzyme.
Crystallographic Structure of the E. coli 4-OPP‚IPP
Isomerase Complex. The quality of the crystallographic data
and the refinement are summarized in Table 2. Unambiguous
electron density in the initial Fourier difference (Fo - Fc) maps
(contoured at 3σ) confirmed the presence of the inhibitor within
the active site of the protein. Simulated-annealing omit maps
of the refined models confirmed the placement on the inhibitor
with a distinct geometry for both molecules in the asymmetric
unit. Electron density is observed consistent with formation of
a covalent bond between the sulfur of Cys67 in the active site
and C5 in 4-OPP and a new carbon-carbon double bond
between C3 and C4. Interestingly, in one protein molecule the
stereochemistry of the double bond in the covalently attached
diene inhibitor is Z, while in the other it is E. The position of
Figure 1. Plots of ln[% inactivation] versus time for incubation of IPP
isomerase with E-2-OPP, Z-2-OPP, and 4-OPP. Panel A, [E-2-OPP] ) 0
(
O), 0.4 (b), 0.5 (0), 0.7 (9), 1.0 (∆), and 2.5 (2) µM. Panel B, [Z-2-
OPP] ) 0 (O), 15 (b), 20 (0), 30 (9), 40 (∆), and 50 (2) µM. Panel C,
[4-OPP] ) 0 (O), 10 (b), 12 (0), 15 (9), 20 (∆), and 25 (2) µM.
17436 J. AM. CHEM. SOC.
9
VOL. 127, NO. 49, 2005

Mechanism-Based Inhibition of IPP Isomerase
A R T I C L E S
Table 2. Statistics of Data Collection and Structure Refinement
for IPP Isomerase‚4-OPP
DMAPP diene analogues 2-E-OPP, 2-Z-OPP, and 4-OPP were
likely candidates for irreversible inhibition of IPP isomerase
by a related mechanism. In this case, an allylic cation is
generated by protonation of the diene in an active site known
to contain carboxylate and thiol nucleophiles. All three dienes
were potent time-dependent irreversible inhibitors of S. pombe
and E. coli IPP isomerase.
Crystal Data
space group
P212121
cell dimensions (Å)
68.633
7
9
2
0.681
0.773
subunits per asymmetric unit
Data Set
The isomeric forms of covalently bound 4-OPP are shown
in Figure 2. The structures mostly differ in the stereochemistry
of the C3-C4 double bond and the conformation of the
hydrocarbon chain of the bound analogue. The most likely
mechanism for inactivation involves protonation of the meth-
ylene group in 4-OPP to give isomeric allylic cations, which
span C3, C4, and C5, followed by alkylation of the thiol group
in C67 (Scheme 4). Since the barrier to rotation about the C3-
C4 partial double bond in the allylic cation should exceed 10
wavelength (Å)
highest resolution (Å)
total reflections
1.54179
2.03
55557
26732
23693
93.3 (93.2)
8.0 (27.5)
6.2
unique reflections
observed reflections (I > 2σ(I))
a
completeness (%)
a
Rmerge (%)
I/σ(I)
Refinement
resolution range (Å)
10.0-2.0
2831
52
85
18.8/22.1
23.9
32
kcal/mol, we conclude that 4-OPP is bound in s-cis and s-trans
conformations in the E‚I complex prior to protonation.
number of protein atoms
number of ligand atoms
number of water molecules
Rcryst (observed/all data) (%)
b
Rfree (%)
rms deviations
bond lengths (Å)
bond angles (Å)
0.007
0.022
Ramachadran Plotc
core region (%)
inner core region (%)
95.8
72.8
a
Statistics for the highest resolution shell (2.08-1.97 Å) are given in
b
parentheses. Free test subset represents 10% of total unique reflections.
As defined by SHELXPRO, Gly and Pro residues excluded.
c
29
Figure 2. Structures of the isomeric IPP isomerase‚4-OPP complexes.
Discussion
Scheme 4. Mechanism for Inhibition of Type I IPP Isomerase by
The substrates for chain elongation,30,31 isomerization, and
cyclization reactions31 in isoprenoid metabolism are processed
by reactions that proceed through discrete carbocationic inter-
mediates. A common feature of these reactions is the addition
9
4-OPP
+
of an electrophile (R3 ) to a carbon-carbon double bond to
generate a tertiary cation, as illustrated in Scheme 3. While the
carbocationic intermediates are sufficiently electrophilic to
alkylate any of the nucleophiles typically found in enzyme active
sites, enzymes that process carbocations typically do not suffer
inactivation during catalysis. However, if the structure of the
intermediate is altered in a manner that changes the location of
the positive charge in the active site, alkylation is commonly
seen. For example, diene analogues of oxidosqualene (R2 )
sCHdCH2), which generate allylic cations during cyclization,
One of the carboxyl oxygens in the side chain of E116 is
coordinated with a tightly bound divalent metal ion in type I
IPP isomerase. The other carboxyl oxygen points toward the
substrate in the binding pocket. The distance between the non-
coordinated oxygen and the newly formed methyl group in the
Z-adduct is 2.80 Å, and thus E116 is the only amino acid that
could protonate the diene to give the structure that was seen.
In the E-adduct, the methyl group is rotated to increase the
distance to 3.98 Å, but again the non-coordinated oxygen of
E116 is the only obvious source for the proton. This oxygen is
also strongly hydrogen-bonded to the positively charged nitrogen
in the tightly bound transition-state analogue 2-(N,N-dimethy-
20
irreversible inactivate oxidosqualene cyclase and squalene-
hopene cyclase21 during turnover. We reasoned that the IPP and
Scheme 3. Electrophilic Additions to Carbon-Carbon Double
Bonds in Substrates and Diene Mechanism-Based Inhibitors
(
29) Sheldrick, G. M.; Schneider, T. R. Methods Enzymol. 1997, 277, 319-
343.
(
30) Erickson, H.; Poulter, C. D. J. Am. Chem. Soc. 2003, 23, 6885-6888.
(
31) Liang, P.-H.; Ko, T.-P.; Wang, A. Eur. J. Biochem. 2002, 269, 3339-
3354.
(32) Allinger, N. L.; Siefert, J. H. J. Am. Chem. Soc. 1975, 97, 752-760.
J. AM. CHEM. SOC. VOL. 127, NO. 49, 2005 17437
9

A R T I C L E S
Wu et al.
lamino)ethyl diphosphate.18 Thus, E116 is a likely active-site
acid in the protonation-deprotonation mechanism for type I
IPP isomerase. Coordination with the divalent metal would
enhance the acidity of the carboxyl group, although the state of
protonation of that residue in the resting enzyme has not been
established. In addition, the tertiary carbocationic intermediate
would be stabilized by the carboxylate moiety following the
protonation step.
In summary, diene analogues of IPP and DMAPP are potent
active-site-directed irreversible inhibitors of type I IPP isomerase.
During the inhibition reaction, the IPP analogue 4-OPP is
protonated at its C3 methylene to generate an allylic cation that
alkylates the thiol group in C67 to form a primary thioether.
An X-ray structure of inactivated E. coli IPP isomerase shows
that the methyl group generated by protonation is located near
E116, thereby implicating that residue in the protonation step
of the isomerization reaction.
Acknowledgment. This project was supported by NIH Grant
GM 25521 (C.D.P.) and an IISN Grant from the FNRS
(Belgium). The authors thank Y. Oudjama and V. Durisotti for
purification of the E. coli enzyme and crystallization assays.
JA056187H
17438 J. AM. CHEM. SOC.
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VOL. 127, NO. 49, 2005