Type-2 isopentenyl diphosphate isomerase: Evidence for a stepwise mechanism

Article abstract of DOI:10.1021/ja208331q

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These two molecules are the building blocks for construction of isoprenoid carbon skeletons in nature. Two structurally unrelated forms of IDI are known. A variety of studies support a proton addition/proton elimination mechanism for both enzymes. During studies with Thermus thermophilus IDI-2, we discovered that the olefinic hydrogens of a vinyl thiomethyl analogue of isopentenyl diphosphate exchanged with solvent when the enzyme was incubated with D ₂O without concomitant isomerization of the double bond. These results suggest that the enzyme-catalyzed isomerization reaction is not concerted.

Full text of DOI:10.1021/ja208331q

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Type-2 Isopentenyl Diphosphate Isomerase: Evidence for a
Stepwise Mechanism
Nicole A. Heaps‡ and C. Dale Poulter*
Department of Chemistry, University of Utah, 315 South 1400 East RM 2020, Salt Lake City, Utah 84112, United States
S Supporting Information
b
Scheme 1. Stepwise Mechanism for Isomerization of IPP to
DMAPP
ABSTRACT: Isopentenyl diphosphate isomerase (IDI)
catalyzes the interconversion of isopentenyl diphosphate
(IPP) and dimethylallyl diphosphate (DMAPP). These two
molecules are the building blocks for construction of
isoprenoid carbon skeletons in nature. Two structurally
unrelated forms of IDI are known. A variety of studies
support a proton addition/proton elimination mechanism
for both enzymes. During studies with Thermus thermophilus
IDI-2, we discovered that the olefinic hydrogens of a vinyl
thiomethyl analogue of isopentenyl diphosphate exchanged
with solvent when the enzyme was incubated with D2O
without concomitant isomerization of the double bond.
These results suggest that the enzyme-catalyzed isomeriza-
tion reaction is not concerted.
1-OPP was prepared as shown in Scheme 2. Propargyl alcohol2
was deprotonated with n-butyl lithium, followed by addition of
methylthiocyanate to give thiomethyl alkyne 3.14 Treatment with
trifluoroacetic acid gave thioester 4-OH.15 The hydroxyl group
was protected as a t-butyldimethylsilyl ether, and 4-OTBDMS
was converted to vinyl thioether 1-OTBDMS upon treatment
with Petasis reagent.16 The protecting group was removed with
tetra-n-butylammonium fluoride and alcohol 1-OH was phos-
phorylated by treatment with tosyl chloride to give 1-OTs
followed by tris-(tetra-n-butylammonium) hydrogen pyrophosphate.17
1-OTs was unstable and was converted to 1-OPP immediately
after purification by chromatography.
sopentenyl diphosphate isomerase (IDI) catalyzes the inter-
conversion of isopentenyl diphosphate (IPP) and dimethylal-
I
lyl diphosphate (DMAPP). This is a required reaction in the
mevalonate pathway for isoprenoid biosynthesis in Eukaryotes and
Archaea and, while not required, can balance the relative concentra-
tions of IPP and DMAPP in Bacteria and plant chloroplasts, which
synthesize isoprenoid compounds by the methylerythritol phos-
phate pathway. Two independently evolved forms of IDI are known.
IDI-1, discovered in the 1950s, is a zinc metalloprotein found in
Eukaryotes and some Bacteria.1,2 IDI-2, discovered in 2001, is a
flavoprotein found in Archaea and other Bacteria.3 A few bacterial
species contain both forms of IDI and others apparently lack both.
Studies with IDI-1 and IDI-2 provide strong evidence for a
protonationÀdeprotonation mechanism for the isomerization of
IPP to DMAPP, where a proton from solvent is added to C4 of
IPP and the pro-R hydrogen is removed from C2.4À7 Epoxide
and diene analogues of IPP and DMAPP are irreversible inhibitors of
IDI-1 and IDI-2, which are activated by protonation and form
covalent adducts with nucleophiles in the active site.6,8À12 Further-
more, IPP and DMAPP analogues that do not support formation of
a carbocationic intermediate are poor substrates for the enzymes.6,13
The data are consistent with a stepwise reaction that proceeds
through a tertiary carbocationic intermediate (Scheme 1) or a
concerted process that proceeds through a late transition state with
substantial carbocationic character. During studies with IPP an-
alogues where the methyl group was replaced by a variety of functional
groups with different electronic properties, we discovered that
Thermus thermophilus IDI-2 catalyzed exchange of the vinyl hydro-
gens in a vinyl thioether analogue (1-OPP) without concomitant
isomerization. These experiments and their implications with regard
to the mechanism of the isomerization reaction are now discussed.
Scheme 2. Synthesis of IPP Thiomethyl Analogue 1-OPP
A 0.7 mL solution of 25 μM T. thermophilus IDI-2 in D2O,
phosphate buffer, pH 7.0, was prepared in an NMR tube.
Incubations at 37 °C were initiated by addition of 1-OPP
1
(12 mM final concentration), and H spectra were collected at
5 min intervals for up to 18 h. Samples were assayed for IDI
activity before and after the incubation to verify that the enzyme
was active at the end of the experiments. The results are shown in
Figure 1. At t = 0, peaks for 1-OPP are seen at 2.21 (methyl group),
2.57 (C2 methylene), 4.05 (C1 methylene), 4.8 (C4 H), and 5.20
(C4 H). Additional peaks are seen at 3.45, 3.55, and 3.68 ppm for
Received: September 3, 2011
Published: November 02, 2011
r
2011 American Chemical Society
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In separate experiments, 1-OPP was incubated with geranyl
diphosphate (GPP) or DMAPP and avian farnesyl diphosphate
synthase at 4 or 30 °C, under conditions normally used for the
chain elongation reaction. Analysis of the mixture from 1-OPP
and GPP by negative ion electrospray HPLC/MS did not show
peaks with retention times in the neighborhood of farnesyl
diphosphate with a mass expected for the thiomethyl farnesyl
analogue from the coupling of 1-OPP and GPP. However,
analysis of the incubation mixture from 1-OPP and DMAPP
by negative ion electrospray HPLC/MS gave a peak at m/z 445
characteristic of a dithiomethyl farnesyl diphosphate analogue.
Repeated attempts to detect the 1-OPP-GPP adduct were
unsuccessful. In a separate experiment, the mixture from a
4 °C incubation of 1-OPP and GPP was immediately treated
with alkaline phosphatase to convert the putative diphosphate
product to the corresponding alcohol. The mixture was
extracted with methyl t-butyl ether, and analysis of the extract
by HPLC/MS (positive ion electrospray) gave a peak at m/z
277.0 with a mass corresponding to the Na+ adduct of the
alcohol. These results indicate that 1-OPP is a substrate for chain
elongation when incubated with DMAPP or GPP, and gives the
expected farnesyl analogues. However, it is apparent that allylic
3-alkyl-3-thiothiomethyl diphosphates are unstable to our stan-
dard incubation conditions and the amount of products isolated
from the reactions was low.
The reactivity of IPP analogues in the isomerization and chain
elongation reactions depends on the ability of the substituent at
C3 to stabilize positive charge. If the methyl group in IPP is
replaced by CF3, the reactivity of the substrate decreases by more
than 106-fold and by more than 102-fold when replaced by
chlorine.13,18,19 These trends for IDI-2 are consistent with the
+
σp values for CF3 (0.612) and chlorine (0.114) relative to
methyl (À0.311) and trends seen for IDI-1.20 The thiomethyl
group in 1-OPP (σp+ = À0.604) stabilizes positive charge at C3
more effectively than a methyl group. On the basis of the rate of
disappearance of the vinyl peak during the first 20 min of the
exchange
incubation, we estimate that kcat
∼ 0.2 sÀ1. Since the
deprotonation step should selectively remove protium rather
Figure 1. NMR spectra for incubations of 1-OPP and IDI-2 in D2O.
than deuterium from C4 in the early stages of the exchange
reaction because of a primary deuterium isotope effect, kcat
exchange
should reflect the rate of protonation of the double bond in IPP.
However, kcatexchange for 1-OPP does not include a correction for
a solvent deuterium isotope effect or a statistical correction to
account for the requirement that two protonations are required
to fully exchange the vinyl protons. In addition, 1-OPP appears
to bind to IDI-2 in a conformation that slows the deprotonation
at C2 relative to protonation and deprotonation at C4. The result
is a rapid exchange of the vinyl protons with solvent and a much
slower isomerization of 1-OPP to its allylic isomer (Scheme 3).
Thus, 1-OPP is more reactive than IPP, although less so than
glycerol from the enzyme storage buffer. After 2 h, the intensities of
peaks at 2.21, 2.37, and 4.05 have decreased by ∼20%, while the
intensities for those at 4.8 and 5.20 have decreased by >90% of their
original values. No peaks were seen that could be attributed to the
allylic isomer of 1-OPP. After 18 h, the peaks at 2.21, 2.37, and 4.05
had decreased by ∼85%, while the peaks at 4.8 and 5.20 disap-
peared. A portion of the 18 h sample was analyzed by HPLC/MS on
a C18 reversed-phase column. A peak eluting at 3.8 min (1:20
acetonitrile/water), with the same retention time as 1-OPP, gave a
negative ion electrospray mass spectrum with a peak at m/z 278.9,
while a control run with 1-OPP gave a peak at m/z 276.9,
confirming the NMR results that the vinyl hydrogens exchanged
with D2O. In a control experiment 1-OPP was incubated in the
reaction buffer without IDI-2 for 24 h. During this time, there was no
discernible change in the NMR spectrum. The slow disappearance
of 1-OPP in the presence of IDI-2 is consistent with isomerization
of the homoallylic diphosphate to its allylic isomer at a rate that is
substantially slower than the rate for proton exchange, followed by
solvolysis of the more reactive allylic isomer. Thus, IDI-2 catalyzes
the facile exchange of the vinyl hydrogens at C4 of 1-OPP without
formation of detectable amounts of its allylic isomer.
+
predicted by the relative σp values for methyl and thiomethyl
groups.
Scheme 3. Proton Exchange and Isomerization of 1-OPP
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COMMUNICATION
The thiomethyl group also increases the reactivity of the allylic
isomer toward solvolysis, which gives a thiomethyl hemiketal
followed by elimination of methanethiol. We anticipate that
methyl vinyl ketone produced from the thiomethyl DMAPP
analogue would be unstable during the incubation. A related
solvolysis reaction was seen following isomerization of 3-cyclo-
propyl-3-butenyl diphosphate to its allylic isomer, where σp+ for the
cyclopropane ring (À0.601) is similar to the thiomethyl value.8,20
In this case, the solvolysis product from (Z)-3-cyclopropyl-2-butenyl
diphosphate was sufficiently stable to be characterized.
A considerable body of evidence with analogues of IPP indicates
that substantial positive charge develops at C3 of IPP during the
isomerization to DMAPP. However, the previous data do not
distinguish between a stepwise mechanism that proceeds through a
carbocationic intermediate and a concerted process with a late
transition state for their isomerization reactions. The rapid
exchange of the vinyl protons in 1-OPP with solvent without
concomitant rearrangement to its allylic isomer shows that the
analogue is protonated by IDI-2 to give an intermediate carbocation,
which loses a proton to return to 1-OPP without a mandatory
isomerization. 1-OPP is the first example of a substrate for IDI in
which protonation and isomerization are decoupled. The trends
in reactivity for IPP and other C3 analogues and the exchange
results for 1-OPP are consistent with a common protonationÀ
deprotonation mechanism for their isomerization reactions.
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’ ASSOCIATED CONTENT
S
Supporting Information. Procedures for synthesis of
b
1-OPP and NMR spectra; descriptions of product studies and
mass spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
poulter@chemistry.utah.edu
Present Addresses
‡Sapphire Energy, 3115 Merryfield Row, San Diego, California
92121, United States.
’ ACKNOWLEDGMENT
This work was supported by NIH grant GM 25521.
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