The effect of active-site isoleucine to alanine mutation on the DHFR catalyzed hydride-transfer

Article abstract of DOI:10.1039/c0cc02988b

Comparison of the nature of hydride transfer in wild-type and active site mutant (I14A) of dihydrofolate reductase suggests that the size of this side chain at position 14 modulates H-tunneling.

Full text of DOI:10.1039/c0cc02988b

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The effect of active-site isoleucine to alanine mutation on the DHFR
catalyzed hydride-transferw
a
ab
Vanja Stojkovic, Laura L. Perissinotti, Jeeyeon Lee,c Stephen J. Benkovicc and
´
Amnon Kohen*a
Received 2nd August 2010, Accepted 28th September 2010
DOI: 10.1039/c0cc02988b
Comparison of the nature of hydride transfer in wild-type and
active site mutant (I14A) of dihydrofolate reductase suggests
that the size of this side chain at position 14 modulates
H-tunneling.
addressed the role of remote residues in modulating the
hydride-tunneling process.4,8,9 The latter studies supported
the hypothesis that a long range dynamically coupled network
acts in the chemical transformation (C–H - C transfer), as
predicted by hybrid quantum/classical molecular dynamics
simulations.11,12
Understanding the nature of the hydrogen-transfer and how
the protein active site structures and dynamicsz contribute
to these reactions is of contemporary interest. Numerous
experimental and theoretical data suggest that quantum
mechanical tunneling of a nucleus has a role in a large number
of enzyme catalyzed hydrogen transfer reactions.1–7 Owing to
the multidimensional and quantum-mechanical nature of a
hydrogen transfer reaction, the rate of the transfer is sensitive
to the protein’s motions that modulate the height and the
width of the reaction’s barrier. Subtle changes in the protein
structure, some of which take place on the time scale of the
hydride-transfer step, might lead to more efficient barrier
penetration and H-tunneling. Here we present kinetic data
and molecular modeling studies suggesting that an active site
mutation modulates the nature of the hydride-transfer in the
enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3), from
Escherichia coli. The intrinsic kinetic isotope effects (KIEs) for
the hydride transfer, and their temperature dependence were
evaluated. Our findings implicate the hydrophobic side chain
of Ile14 residue, positioned behind the hydride-donor (reduced
nicotinamide), in the reorganization of the active site during
the DHFR-catalyzed reaction.
In the present study, Ile14, that holds the nicotinamide ring
of the cofactor (NADPH) close to the hydride acceptor
(H2folate), has been replaced with alanine by site-directed
mutagenesis. The presence of a conserved hydrophobic residue
behind the nicotinamide ring is a common feature of
nicotinamide-dependent enzymes (ref. 13 and 14 and the
PDB) suggesting its critical role in this type of reaction. This
residue holds the nicotinamide ring in close proximity to the
hydride acceptor (e.g. H2folate), and participates in the
organization of the donor and acceptor during the catalyzed
hydride-transfer reaction. NMR studies indicated that
this residue exists as two rotamers in solution, despite
the well-defined orientation in crystal structure (gauche
rotamer).15 The trans rotamer, not observed in either closed
or occluded crystal structure, is significantly populated in
the solution. Modeling studies suggested that in the trans
rotameric state, the side chain of the I14 clashes with the
nicotinamide ring forcing it towards the pterin ring. MD
simulation confirmed that the position of trans population
along the reaction coordinate overlaps with a decrease in the
donor–acceptor distance (DAD).16 Therefore, decrease in the
size of the side chain, like in the I14A DHFR mutant, alters
the DAD and orientation, with minimal effect on the active
site’s electrostatics. By comparing the intrinsic primary (11)
KIEs and their temperature dependencies for the mutant I14A
DHFR with those previously reported for wtDHFR,10 we can
comment on the nature of the hydride transfer and the
contribution of protein dynamics to the enzyme catalyzed
reaction.
DHFR has been a model system for examination of the
physical nature of enzymatic hydride transfers and of the
contribution of protein dynamics to the hydride transfer.4,8,9
DHFR catalyzes the reduction of 7,8-dihydrofolate (H2folate)
to 5,6,7,8-tetrahydrofolate (H4folate), an essential co-factor in
several biosynthetic pathways. Previous measurements of
intrinsic KIEs and their temperature dependences for
wild-type EcDHFR (wtDHFR) inferred quantum mechanical
tunneling of the hydride in the DHFR catalyzed reaction.10
Kinetic studies of several distal mutants of EcDHFR
Competitive H/T and D/T KIEs on the second order rate
constants (kcat/KM) were measured with the I14A-DHFR
mutant using mixtures of [4(R)-3H]-NADPH with [Ad-14C]-
NADPH for 11 H/T, and [4(R)-3H]-NADPH and [Ad-14C,
4(R)-2H]-NADPH for 11 D/T KIEs. The synthesis, purifica-
tion and storage of these labeled cofactors have been presented
elsewhere.17 Observed KIEs were used to calculate intrinsic
KIEs following the Northrop method as described before
(Fig. 1, also see Fig. S1 and Table S1 in the ESIw).4,8–10,18
Table 1 compares the isotope effects on the Arrhenius
activation parameters for the wtDHFR and I14A. The values
for the isotope effects on the Arrhenius prefactors differ
significantly between the two enzymes suggesting noticeable
a Department of Chemistry, The University of Iowa, Iowa City, IA,
USA. E-mail: amnon-kohen@uiowa.edu; Fax: +1 319 335 1270;
Tel: +1 319 335 0234
b
´
´
´
´
´
Departamento de Quımica Inorganica, Analıtica y Quımica Fısica/
INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad
de Buenos Aires, Ciudad Universitaria, Pabello´n II, piso 3,
C1428EHA Buenos Aires, Argentina
c Department of Chemistry, Pennsylvania State University,
University Park, PA, USA
w Electronic supplementary information (ESI) available: The material
and methods used in these studies and more detailed data analysis. See
DOI: 10.1039/c0cc02988b
c
8974 Chem. Commun., 2010, 46, 8974–8976
This journal is The Royal Society of Chemistry 2010

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nuclear wavefunction overlap along the hydrogen coordinate,
and is isotopically sensitive. The temperature dependence in
KIEs results from the gating term. The last two terms are
integrated for all the conformations sampled by the DAD.
Like many highly evolved enzymes under physiological
conditions, the wtDHFR exhibited temperature independent
KIEs (Fig. 1 and Table 1),10 consistent with a well reorganized
donor and acceptor with an average DAD that is adequate for
tunneling. The active site mutant I14A, on the other hand,
exhibits temperature dependent intrinsic KIEs. This implies
that at the tunneling-ready conformation, the average DAD is
too long for efficient tunneling at low temperature, but at
higher temperatures more conformations with a shorter DAD
are sampled. This affects D more than H because D can only
tunnel from shorter distances, thus leading to temperature
dependent KIEs.
Fig. 1 Arrhenius plot of intrinsic H/T KIEs for wild-type (red)10 and
mutant (blue) DHFR. The lines represent the nonlinear regression to
an exponential equation. More details are presented in the ESI.w
Table 1 Activation parameters of comparative KIEs
Due to the very different volumes of isoleucine and alanine
(reflecting deletion of ethyl and methyl on the b-carbon of
residue 14), one would expect that in the I14A DHFR mutant
the nicotinamide ring (the donor of the hydride) would be less
sterically restricted to be close to the hydride acceptor. To
better assess the structural aspects of this effect, we modeled
the relative orientation of the donor and acceptor in the
wild-type and mutant starting from the coordinates of the
ternary complex wtDHFR–Folate–NADP+ (PDB entry:
1RX2).27 This ternary complex was chosen because it is the
best structural representation of the reactive complex.27,28 The
ligands were substituted for the reactants (NADPH and
H2folate) and an active site mutation was performed in silico.
A 15 ns molecular dynamics simulation (MD) was performed
using the Amber9 package29 (see ESIw for details). The results
indicate that the mutation leads to an increase in the ground
state DADs as well as a broader distribution of DADs with
wtDHFRa I14A
Semiclassical range
b
AH/AT
DEaTꢀH*/kcal molꢀ1 ꢀ0.1 ꢂ 0.2 0.39 ꢂ 0.06
From ref. 10. Similar trends were observed for H/D and D/T (data
7.0 ꢂ 1.5 4.7 ꢂ 0.5
0.3–1.7
—
a
b
not shown since this is trivial, as they follow the Swain–Schaad
relationship).
differences in the nature of the hydride tunneling. Moreover,
all the isotope effects on the Arrhenius pre-exponential factors
are larger than their semi-classical limits (0.3–1.7).19,20 Similar
findings with the E. coli DHFR have been simulated by high
level QM/MM calculations.21–23 A simple phenomenological
way to rationalize the data is through the Marcus-like models,
also referred to as environmentally coupled tunneling models,
tunneling promoting vibrations, etc. In these models, heavy
atom motion controls the probability of hydrogen transfer.
From this perspective, larger than unity isotope effects on
Arrhenius prefactors and small temperature dependencies in
KIEs indicate a system with a well reorganized donor and
acceptor. A significant temperature dependency in KIEs, on
the other hand, suggests poor reorganization, where the
average DAD is too long for efficient tunneling. In those
cases, thermally activated fluctuation of the DAD allows the
system to sample more conformations to enable tunneling at
higher temperatures. Most Marcus-like models follow the
general expression:2,3,24,25
r1
Z
ꢁ
2
þlÞ =ð4lRTÞ
k ¼ CeꢀðDG
eFðmÞeꢀE =k T dDAD
ð1Þ
b
FðmÞ
r0
Fig. 2 Histogram representation of DAD distribution for wtDHFR
(red) and I14A DHFR (blue). The average structure of reactive
complex, together with residue 14, for each enzyme is presented. The
lines show the fitting of the data to one and two Gaussians, for the
wild type and mutant DHFRs, respectively. For the wtDHFR the C4
(donor) to C6 (acceptor) have an average distance of 3.62 ꢂ 0.16 A.
For I14A DHFR, on the other hand, there are two populations, one
with an average DAD of 3.69 ꢂ 0.17 A, and the other with 4.24 ꢂ 0.41 A.
These distributions are presented in detail in the ESI.w Significantly, I14A
DHFR exhibits larger DAD distribution suggesting the increase in the
flexibility of the nicotinamide ring for the mutant, and smaller population
at short DADs.
In eqn (1), the rate of hydride-transfer is determined by
the isotope independent pre-exponential constant (C), which
represents the fraction of reactive complexes; a Marcus term
(first exponential); a Franck–Condon term (F(m)) and a
‘‘gating term’’ (EF(m)) that describes the temperature and mass
dependence of the DAD fluctuations.2,3,24,25 The Marcus
term is almost isotopically insensitive, and represents the
heavy-atom reorganization that is required to occur before
significant tunneling can take place (i.e., the reorganization
of the donor and acceptor toward a ‘‘tunneling ready’’
conformation).3,26 The Franck–Condon term describes the
c
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Chem. Commun., 2010, 46, 8974–8976 8975

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smaller fraction of short distances (Fig. 2, and Fig. S4 in
ESIw), which is in accordance with the kinetic findings (Fig. 1).
The elevated flexibility in the mutant, together with the longer
average DAD suggest that the side chain of residue I14 in the
wtDHFR compresses the donor toward the acceptor as the
system rearranges toward the tunneling-ready state (Fig. S5 in
ESIw). In support of the less restricted active site, we carefully
examined MD trajectories and found that during the time
scale of the simulation, on average 1–2 water molecules
enter the active site cavity filling the void space between the
nicotinamide ring and Ala14. The MD simulation assists in
rationalizing the kinetic data and further supports the
interpretation of these data by the Marcus-like model. We
trust that the findings presented here will lead to rigorous
QM/MM calculations that will more thoroughly examine the
role of this residue along the reaction coordinate.
Notes and references
z The term dynamics is used here for atomic motion, structural
changes, and alteration of conformational populations.
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The reduction in the size of residue 14 appears to correlate
with reorganization of the reacting complex that is less suitable
for H-tunneling than the wild type. The change in the nature
of the hydride transfer relative to wtDHFR is also reflected in
the reduced rate of hydride transfer. The hydride transfer rate
for the protonated ternary complex of EcDHFR at 25 1C was
estimated to be 450 sꢀ1 (pH = 6.5),30 whereas for I14A
DHFR the rate is decreased B14 fold (khyd = 33 ꢂ 3 sꢀ1
,
pH = 6.8).31 This decrease in rate further supports the
hypothesis that the reorganization to the tunneling-ready
conformation is altered by the alanine mutation in such a
way that H-tunneling is less efficient.
A previous study that suggested that the longer DAD and
distorted geometry diminished tunneling in an enzymatic
system was done on horse liver ADH (HLADH), where a
valine residue, analogous to I14 in DHFR, was mutated into
alanine. That mutation increased the ground state DAD, as
suggested by X-ray crystallography with folate and NADP+
ligands. While no temperature dependent studies were
conducted for this mutant of HLADH, the mutation resulted
in a reduced coupled motion and tunneling, as suggested by
mixed labeling 21 Swain–Schaad exponent (SSE) experiments
at a single temperature.13,14 The current study, on the other
hand, allowed for the measurement of the temperature
dependence of KIEs that likewise resulted from a change in
the orientation between the pterin and nicotinamide rings and
changes in the DAD reorganization step. The current findings
also suggest that the active site becomes more flexible, which is
in accord with a larger temperature dependence of the intrinsic
KIEs with I14A DHFR.
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In conclusion, a mutation that decreased the restriction on
location and motion of the hydride-donor (NADPH) appears
to perturb the dynamic behavior at the ground state. Future
studies will expand the series of mutations, structural studies,
and high level simulations in order to obtain a more rigorous
correlation between the size of the side chain and the nature of
the hydride-transfer. Of particular interest would be analysis
similar to that performed in ref. 21, where the DAD’s
distribution is examined along the reaction coordinate.
This work was supported by NSF CHE-0133117 and BSF-
2007256 (for AK) and 1RO1GM092946 (for SJB). LLP
acknowledges CONICET and Dario A. Estrin.
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C. Sagui, V. Babin and P. A. Kollman, University of California,
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c
8976 Chem. Commun., 2010, 46, 8974–8976
This journal is The Royal Society of Chemistry 2010