Transition state analysis of model and enzymatic prenylation reactions

Article abstract of DOI:10.1021/ja069119j

To obtain a transition state (TS) structure for an enzyme-catalyzed prenylation reaction, S_N1 and S_N2 model substitution reactions with dimethylallyl chloride were first studied. ¹³C Kinetic isotope effects (KIEs) for the model reactions were measured by a natural abundance NMR method and used to validate the computational methods that would be used in the subsequent determination of the enzymatic TS structure. Using a primary ¹³C KIE and a secondary ²H KIE measured via mass spectrometry, a TS structure for the enzyme-catalyzed reaction was computed; a density functional level of electronic structure theory using the mPW1N functional in combination with the 6-31+G(d) basis set was employed for those calculations. That structure has a C-O bond length of 1.69 A and a C-S bond length of 3.70 A. While the former bond length is similar to that for a nonenzymatic S_N2 reaction, the latter is considerably (0.90 A) longer, indicating that the enzyme effects catalysis via an "exploded" TS structure. Copyright

Full text of DOI:10.1021/ja069119j

Published on Web 04/17/2007
Transition State Analysis of Model and Enzymatic Prenylation Reactions
Stepan Lenevich, Juhua Xu, Ayako Hosokawa, Christopher J. Cramer, and Mark D. Distefano*
Department of Chemistry, UniVersity of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455
Received December 19, 2006; E-mail: distefan@chem.umn.edu
Protein prenylation involves the attachment of C15 (farnesyl) or
C
20 (geranylgeranyl) groups to proteins and is catalyzed by a class
1
of enzymes known as prenyltransferases. The observation that
inhibition of Ras farnesylation arrests the growth of tumor cells
has been the motivating factor in developing inhibitors of prenyl-
transferases that can serve as anticancer drugs; currently, several
2
candidates are in Phase 3 clinical trials. Mechanistic analysis of
enzymatic reactions can provide insights that are potentially useful
in drug design. Since enzymes must bind to the transition state
(TS) with greater affinity than the ground state, molecules that
mimic the structure of the TS will have the highest possible affinity
for the enzyme. Enzyme inhibitors based on such principles can
3
manifest extraordinary affinity and selectivity; accordingly, we are
Figure 1. Model reactions and derivatization chemistry for SN1 and SN2
prenylation reactions.
interested in determining the TS structure for the reaction catalyzed
by protein farnesyltransferase. Moreover, the detailed knowledge
gained in these experiments should increase our understanding of
how enzymes activate isoprenoid diphosphates for subsequent
reaction. Such knowledge could be particularly useful for manipu-
lating the reactivity of prenyltransferases and the closely related
terpene cyclases for biotechnology purposes.
N N
The reactants and TS structures for the S 1 and S 2 model reactions
were determined at the density functional level of electronic
7
structure theory with the 6-31+G(d) basis set. Analytic vibrational
frequencies were computed for each stationary point, and KIEs were
8
predicted from the usual transition state theory approach. The
At present, the most reliable method to determine TS structure
is through the use of computational methods in conjunction with
experimentally measured kinetic isotope effect (KIE) measure-
13
calculated primary C KIEs are 1.040 for the S
N
2 model reaction
N
and 1.001 for the S 1 model reaction, which are within experimental
error of the measured values, suggesting that the theoretical model
should be useful for related reactions including those involving
isoprenoid diphosphates.
4
ments. While a large body of literature exists for KIE measurements
performed on benzylic systems, reports for related allylic systems
are sparse. Thus, as a prelude to enzymatic measurements, it was
decided to investigate several model reactions, shown in Figure 1,
first. Results from such experiments would provide KIE values for
Protein farnesyltransferase (PFTase) catalyzes the transfer of a
prenyl group from farnesyl diphosphate (FPP, 9) to proteins
including Ras that terminate in CVIA and related sequences. Since
the enzyme recognizes only the four C-terminal residues, tetrapep-
tides are efficient substrates for the enzyme with kinetic constants
N N
limiting associative (S 2) and dissociative (S 1) mechanisms and
allow us to validate the computational methods that would be used
in the subsequent determination of the enzymatic TS. For a model
substrate, dimethylallyl chloride (1) was chosen. Solvolysis of 1
in benzyl alcohol (2) was used as a dissociative model while
displacement with triphenylphosphine (5) was employed as an
similar to full-length protein substrates.9 To measure Vmax/K
M
(V/K) KIEs under steady state conditions, the substrate, FPP, must
manifest a low commitment factor (cf).10 Isotope trapping experi-
ments, using FPP as a substrate, to measure the c suggest significant
f
5
11
associative model. Kinetic analysis of these reactions revealed that
commitment to catalysis. In contrast, similar experiments using
the solvolysis reaction was first order in 1 and zero order in 2,
whereas the other reaction was first order in both 1 and 5. To
measure the 13C KIEs for the reaction, an NMR method was
the related, weaker binding isoprenoid (Figure 2), geranyl diphos-
phate (GPP, 10), gave a low value of c (c ) 0.057, data not shown),
f
f
indicating that KIEs measured using GPP would not be masked by
excessive commitment. On the basis of the work described above
with the model substitution reactions, it was noted that the primary
6
employed based on the work of Singleton and others; that approach
involves the integration of 13C NMR spectra obtained at natural
abundance of reactant obtained prior to reaction and after substantial
conversion. In the work reported here, the model reactions were
performed, monitored by GC, and terminated by vacuum distillation
to recover the remaining starting material which was then deriva-
tized by reaction with dimethylmalonate (7) and the resulting
product (8) purified by flash chromatography. 13C NMR spectra
were obtained and integrated using C-4 as an internal standard.
13
C KIE was a particularly sensitive reporter of the extent of bonding
between the electrophilic carbon center and incoming nucleophile.
Thus it was decided to measure the primary 13C KIE using [1- C]-
GPP as a substrate. The labeled substrate was prepared in four steps
13
13
using commercially available [1- C]-triethylphosphonoacetate. For
13
the KIE measurement, a mixture of [1- C]-GPP and unlabeled GPP
was incubated with the peptide N-dansyl-GCVIA (11) in the
presence of PFTase. Reactions were performed either with limiting
peptide or limiting GPP. After enzymatic incorporation, the
farnesylated peptide product, 12, was purified by reversed-phase
HPLC and analyzed by ESI-MS (ion trap instrument) operating in
the zoom scan mode. The mass spectra of the samples were
13
C KIEs were calculated from the ratios of peak areas determined
from samples of the derivatized starting material (8) before and
after reaction. A significant primary 13C KIE at C-1 was measured
in the S
N
2 model reaction (1.040 ( 0.003), whereas a value near
1 model reaction.
unity (0.997 ( 0.003) was observed in the S
N
5796
9
J. AM. CHEM. SOC. 2007, 129, 5796-5797
10.1021/ja069119j CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Proposed TS structure for PFTase-catalyzed reaction based on
calculations using GPP and ethane thiolate. The second isoprene unit is
omitted for clarity. Colors: S (orange), C (green), and O (red).
In summary, we present here a TS structure for the reaction
catalyzed by PFTase that complements X-ray crystallographic
studies15 and provides a clear structural framework for understand-
ing the results of previous mechanistic investigations of this enzyme,
including stereochemical1 and kinetic analyses.
6,17
14,18
The experi-
ments reported here provide powerful insights into an important
class of biological reactions through a combination of model
chemistry, computation, and kinetic analysis. Finally, it should be
noted that the KIE analysis described here was accomplished
through experiments performed with stable isotopes and did not
require radiolabeled compounds. It is likely that the role of ESI-
MS and NMR in KIE analyses of biological processes will continue
to grow in the future.
Figure 2. Isoprenoid substrates and prenylation reaction catalyzed by
PFTase studied here.
deconvoluted into ratios of labeled and unlabeled species using
spectra obtained from pure labeled and unlabeled products; the V/K
13
KIE using [1- C]-GPP as a substrate calculated from that data was
found to be 1.039 ( 0.003. A similar approach was used to measure
2
2
2
Acknowledgment. This work was supported by the National
Institutes of Health (GM58442) and the National Science Founda-
tion (CHE-0610183).
2 2
the R-secondary H KIE, using [1- H ]-GPP (prepared from [1- H ]-
geraniol)12 as a substrate, which was found to be 1.068 ( 0.003.
To determine the structural significance of these KIE values, a TS
structure for the nonenzymatic reaction between ethane thiolate and
GPP via an associative mechanism was computationally determined;
a density functional level of electronic structure theory using the
mPW1N functional in combination with the 6-31+G(d) basis set
was employed for those calculations. An associative model was
chosen for the starting point for these calculations since a significant
Supporting Information Available: Details for the synthesis of
13
[
f
1- C]-GPP, c determination, KIE experiments, TS calculations, and
data analysis are included. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
primary 13C KIE was observed only in the S
2 model reaction
N
(
(
1) Zhang, F. L.; Casey, P. J. Annu. ReV. Biochem. 1996, 65, 241-269.
2) Doll, R. J.; Kirschmeier, P.; Bishop, W. R. Curr. Opin. Drug DiscoVery
DeV. 2004, 7, 478-486.
described above. For the nonenzymatic reaction between GPP and
ethane thiolate, the primary 13C KIE was calculated to be 1.067.
Interestingly, this value is significantly higher than the experimen-
tally measured value of 1.039 determined for the enzymatic reaction,
indicating that the TS structure for the protein-catalyzed process
must be somewhat different. Iterative cycles of altering the C-O
(
3) Taylor Ringia, E. A.; Tyler, P. C.; Evans, G. B.; Furneaux, R. H.; Murkin,
A. S.; Schramm, V. L. J. Am. Chem. Soc. 2006, 128, 7126-7127.
4) Schramm, V. L. Annu. ReV. Biochem. 1998, 67, 693-720.
(
(5) O’Brien, P. J.; Herschlag, D. Biochemistry 2002, 41, 3207-3225.
(
6) Singleton, D. A.; Thomas, A. A. J. Am. Chem. Soc. 1995, 117, 9357-
9358.
(
leaving group) and C-S (nucleophile) bond lengths and calculation
(
7) Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab Initio
13
of the resulting primary C KIE were used to generate a plot of
the variation of the 13C KIE with C-O and C-S bond lengths; a
similar plot was created for the R-secondary H KIE. Using the
Molecular Orbital Theory, 2nd ed.; Wiley: New York, 1986.
(8) Cramer, C. J. Essentials of Computational Chemistry: Theories and
Models, 2nd ed.; John Wiley & Sons: Chichester, UK, 2004.
2
(
9) Moores, S. L.; Schaber, M. D.; Mosser, S. D.; Rands, E.; O’Hara, M. B.;
Garsky, V. M.; Marshall, M. S.; Pompliano, D. L.; Gibbbs, J. B. J. Biol.
Chem. 1991, 266, 14603-14610.
two KIEs reported here enabled us to computationally identify a
set of C-O (1.69 Å) and C-S (3.70 Å) TS bond lengths that are
consistent with both measurements; a proposed structure for the
TS is shown in Figure 3. Interestingly, the C-O bond lengths in
(
10) Northrop, D. B. Biochemistry 1975, 14, 2644-2651.
(11) Furfine, E. S.; Leban, J. J.; Landavazo, A.; Moomaw, J. F.; Casey, P. J.
Biochemistry 1995, 34, 6857-6862.
(
12) Clausen, V. A.; Edelstein, R. L.; Distefano, M. D. Biochemistry 2001,
0, 3920-3930.
(13) Dolence, J. M.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
N
the model S 2 TS and enzymatic TS are essentially the same.
4
However, the C-S bond lengths are substantially different with
the enzymatic TS (3.70 Å), manifesting less bonding to C-1 of GPP
than is present in the model system (2.80 Å). This suggests lower
5008-5011.
(
(
(
(
(
14) Pais, J. E.; Bowers, K. E.; Fierke, C. A. J. Am. Chem. Soc. 2006, 128,
15086-15087.
15) Reid, T. S.; Terry, K. L.; Casey, P. J.; Beese, L. S. J. Mol. Biol. 2004,
N
electron density at C-1 than is typified by S 2 reactions and provides
343, 417-433.
a rationale for why substrates containing electron-withdrawing
substituents decrease the enzymatic reaction rate.13 It should be
noted that the KIEs reported here for yeast PFTase are similar, but
not identical, to KIEs recently reported by Pais et al. for a
mammalian PFTase.14 These variations may reflect differences in
the TS structures between the yeast and mammalian enzymes that
could be exploited for drug design.
16) Mu, Y. Q.; Omer, C. A.; Gibbs, R. A. J. Am. Chem. Soc. 1996, 118,
117-123.
17) Edelstein, R. L.; Weller, V. A.; Distefano, M. D.; Tung, J. S. J. Org.
Chem. 1998, 63, 5298-5299.
18) Huang, C.-c.; Hightower, K. E.; Fierke, C. A. Biochemistry 2000, 39,
2593-2602.
JA069119J
J. AM. CHEM. SOC.
9
VOL. 129, NO. 18, 2007 5797