Experimental evidence for hydrogen tunneling when the isotopic arrhenius prefactor (Ah/Ad) is unity

Full text of DOI:10.1021/ja806354w

Published on Web 12/05/2008
Experimental Evidence for Hydrogen Tunneling when the Isotopic Arrhenius
Prefactor (AH/AD) is Unity
Sudhir C. Sharma and Judith P. Klinman*
Departments of Chemistry and Molecular and Cell Biology, UniVersity of California,
Berkeley, California 94720-1460
Received August 11, 2008; E-mail: klinman@berkeley.edu
Scheme 1. Mechanism of H · Abstraction Catalyzed by SLO-1
Elevated values for primary deuterium kinetic isotope effects
(kH/kD),1 deviations of the temperature dependence of kH/kD from
semiclassical predictions1 and the breakdown of multiple isotope
effects from simple reduced mass relationships (Swain-Schaad
relationship)2,3 have been used extensively in the detection of
hydrogen tunneling near room temperature. In particular, the
temperature dependence of kH/kD has emerged as an important
diagnostic of H-tunneling, with isotopic Arrhenius prefactor ratios
(AH/AD) that are distinct from unity providing strong evidence for
tunneling. However, the case is ambiguous when the value of AH/
AD approaches unity (range of 0.8 to 1.4) and is generally interpreted
as a reaction involving no tunneling.1 Herein, we report experi-
mental evidence for an isotopic Arrhenius prefactor ratio of unity
in an enzymatic reaction that undergoes extensive tunneling of both
protium and deuterium. Such an observation has been predicted
within the environmentally coupled tunneling model put forth by
Knapp et al.4
the vicinity of the active site cofactor Fe3+-OH. A previous study
demonstrated that single mutations, Leu546Ala and Leu754Ala, lead
to a moderately temperature-dependent KIE and an elevated Ea in
comparison to WT, whereas the more distal mutation, Ile553Ala,
exhibits an enhanced temperature-dependent KIE with an inverse
Arrhenius prefactor ratio.4 A regular increase in the temperature
dependence of the KIE following a progressive decrease in the
bulkiness of the side chain at residue 553 was subsequently
reported.12
The observation that the weakly temperature-dependent KIE of
WT SLO-1 becomes more temperature-dependent upon the intro-
duction of mutations at or near the active site has been explained
within a Marcus-like, full tunneling model that allows for distance
sampling (gating) between the H-donor and acceptor.4,8
According to a Bell tunneling model,1 all three hydrogen isotopes
can cross a reaction barrier at some point below the classical
transition state. Protium, with the longest de Broglie wavelength,
can cross the barrier at a much lower and wider point than deuterium
or tritium. A major consequence of this tunneling mechanism is
an inflated deuterium kinetic isotope effect (KIE) that exceeds the
semiclassical limit of ∼7. At or near room temperature, where the
majority of biological catalysts operate, this type of tunneling leads
to the trend: Ea(H) < Ea(D) < Ea(T), and the corresponding trend
in Arrhenius prefactors: AH < AD < AT. There are a number of
enzymatic reactions where the temperature dependence of experi-
mental KIEs has been found to produce AH/AD, AH/AT and/or AD/
AT , 1.5
Arrhenius behavior deviations such that Ea(H) - Ea(D) ≈ 0,
leading to temperature-independent KIEs with AH/AD . 1, have
also been observed for enzymes that transfer hydride, hydrogen
atom, and proton.6 These surprising, temperature-independent KIEs
in enzyme-catalyzed reactions, together with the observation of
Swain-Schaad deviations that cannot be accommodated within a
Bell model,7 have led to new theories for H-transfer in condensed
phase that are much closer to Marcus models for electron transfer.4,8
The enzyme soybean lipoxygenase-1 (SLO-1) catalyzes the regio-
and stereospecific conversion of linoleic acid (9,12-(Z,Z)-octade-
canoic acid) (LA) to produce 13(S)-hydroperoxy-9(Z),11(E)-octa-
decadienoic acid [13-(S)HPOD]. The first part of this process is a
proton-coupled electron transfer where the proton is transferred to
the oxygen of the Fe3+-OH and the electron is transferred to the
iron center,9 Scheme 1. Published kinetic data for wild-type (WT)
SLO-1 indicate an extremely large KIE on kcat (kH/kD ) 81) and a
small Arrhenius prefactor and activation energy, together with
experimental KIEs that are largely temperature-independent.10 The
crystal structure of SLO-111 indicates a substrate binding site lined
by hydrophobic side chains, with the residues Leu546 and Leu754 in
r0
-(∆G°+λ)2
-mHωHr2H
ktun ) (const) exp
⁄
exp
⁄
×
{
}
{
}
∫
(4λRT)
2p
r1
exp{-Ex ⁄ kbT} dX (1)
As represented in eq (1), the efficiency of hydrogen transfer is
dependent upon three major factors: (1) A Marcus term that contains
λ, the sum of outer and inner sphere reorganization to the reaction
barrier, and ∆G°, the reaction driving force; this term is temperature
dependent but only weakly isotope independent; (2) the Franck-
Condon overlap, that defines the probability of wave function
overlap between the H-donor and acceptor as a function of the
mass (m), frequency (ω), and distance traveled (r) by the transferred
hydrogen, and is strongly mass dependent; and (3) a donor/acceptor
distance sampling term that reflects the barrier encountered, Ex, in
reducing the distance between the H-donor and acceptor. The third
exponential term connects both temperature and mass dependencies
within a single function. Under this model, a close approach
between the H-donor and acceptor distance, that ensures efficient
wave function overlap for both protium and deuterium transfer,
leads to similar enthalpies of activation for H · and D · transfer.
However, when the donor/acceptor distance deviates from its
optimal position, gating will impact the rate of D · transfer to a
greater extent due to its smaller wavelength and subsequent poorer
wave function overlap. This situation is especially apparent in the
Ile553Gly mutant of SLO-1 where AH/AD ) 0.027 and the observed
KIE is increased two-fold from that of the WT at 30 °C.12
In the case of WT SLO-1, the magnitude and nearly temperature-
dependent KIEs implicate an optimized active site with a fixed and
weakly modulated donor/acceptor distance that results from a
relatively high gating frequency.4,12 Introduction of active site
9
17632 J. AM. CHEM. SOC. 2008, 130, 17632–17633
10.1021/ja806354w CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Kinetic Parameters for SLO-1 and Mutants in 0.1 M Borate Buffer (pH 9.0)a
b
enzyme form
kcat (s-1
)
KIEc
Ea (H) (kcal/mol)
∆Ead (kcal/mol)
AH (s-1
)
AH/AD
kcat/KM (µM-1 s-1
)
WT-SLOe
546Ae
297 (12)
4.8 (0.6)
280 (10)
81 (5)
93 (9)
93 (4)
2.1 (0.2)
4.1 (0.4)
1.9 (0.2)
3.8 (0.4)
0.9 (0.2)
1.9 (0.6)
4.0 (0.3)
2.8 (0.4)
9 × 103 (2 × 103)
4 × 104 (3 × 104)
7 × 103 (2 × 103)
1.1 × 103 (5 × 102)
18 (5)
4 (4)
0.12 (0.06)
1.05 (0.45)
11 (1)
0.33 (0.1)
12 (1)
553Af
546A/553Ag
2.21 (0.09)
128 (3)
0.11 (0.02)
a Data were collected from 15 to 50 °C. b The rate constants are reported for 30 °C. c KIE ) kcat ) kcat(H)/kcat(D). d This is the isotope effect on Ea,
D
∆Ea ) Ea(D) - ∆Ea(H). e Reference 10a. f Reference 4a. g This work.
involvement of full quantum mechanical hydrogen tunneling for
both protium and deuterium, where the isotopic Arrhenius prefactor
ratio is unity. These results have important consequences for many
reactions where the observation of AH/AD ≈ 1 has been automati-
cally attributed to reaction via a semiclassical transition state. As
an alternative, we posit that, within a given enzyme reaction, the
trends of AH/AD provide a measure of the degree to which the
H-donor/acceptor can achieve an optimal configuration for protium
and deuterium wave function overlap. A range of AH/AD values
may be expected that will fall between . 1 and , 1 depending on
Figure 1. Arrhenius plot of kinetic data for Leu546Ala/Ile553Ala SLO-1 double
mutant: data points for protio-linoleic acid [blue filled diamond ([)] and
dideutero linoleic acid [red open circle (O)]; linear fits to the Arrhenius
equation are shown as solid lines; error bars are obscured by the symbol.
the impact of the environment on the tunneling efficiency. The
behavior reported herein is expected to apply equally well to
reactions characterized by relatively small isotope effects, as seen,
for example in numerous enzyme-catalyzed hydride transfer
reactions.14,6d,15-17
packing defects, via deletion of large hydrophobic side chains, alters
the initial H-donor/acceptor distance as well as the oscillator
frequency for distance modulation, causing enhanced temperature
dependencies for the KIE.12,13 Under this premise, two active site
residues, Leu546 and Ile553, have now been simultaneously mutated
to Ala, and the corresponding hydrogen transfer parameters
investigated.
Acknowledgment. This work was funded in part by grants from
the National Institutes of Health (Grant GM025765) and National
Science Foundation (Grant MCB0446395).
Supporting Information Available: Experimental procedures:
preparation of mutant enzyme, protein isolation and details of enzymatic
assay. This information is available free of charge via the Internet at
http://pubs.acs.org.
Table 1 contains a summary of the kinetic data for the double
mutant Leu546Ala/Ile553Ala, in relation to the respective single
mutants Leu546Ala and Ile553Ala, as well as WT SLO-1. The overall
rate of catalysis (kcat) of the double mutant Leu546Ala/Ile553Ala is
decreased one hundred thirty-fold from that of WT and the single
mutant Ile553Ala, and two-fold from the single mutant Leu546Ala.
The rate of catalysis for the double mutant Leu546Ala/Ile553Ala
between 15 and 50 °C is shown in Figure 1. The temperature
dependencies of kcat for both H · and D · abstraction were fitted to
the empirical Arrhenius equation to yield Ea and the Arrhenius
prefactor, A. This double mutant exhibits a more temperature-
dependent isotope effect than WT or the single mutant Leu546Ala
but less than the single mutant Ile553Ala. A particularly significant
observation with this double mutant is that the magnitude of AH/
AD is reduced to unity (1.05), an observation normally attributed
to semiclassical hydrogen transfer. At the same time the experi-
mental KIEs remain exceedingly large at all temperatures, at values
much larger than permitted within a semiclassical H-transfer
model. These observations show the difficulty of accommodating
the experimental observations for the double mutant Leu546Ala/
Ile553Ala using either semiclassical transition state theory or a Bell
tunneling model.
References
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By contrast, the environmentally coupled tunneling model, eq
1, requires at some point that AH/AD ) 1, as a transition between
a rigid optimized active site (Ea(D) - Ea(H) ≈ 0, AH/AD .1) and
a site that has been compromised to the extent that extensive
distance sampling becomes necessary to achieve a close enough
distance between the H-donor and acceptor for efficient tunneling
to occur (Ea(D) > Ea(H), AH/AD , 1). To our knowledge, the data
presented herein provide the first unambiguous evidence for the
(15) Maglia, G.; Allemann, R. K. J. Am. Chem. Soc. 2003, 125, 13372.
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