Hydrogen tunnelling in enzyme-catalysed H-transfer reactions: flavoprotein and quinoprotein systems.

Abstract

It is now widely accepted that enzyme-catalysed C-H bond breakage occurs by quantum mechanical tunnelling. This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling correction) has been driven over the recent years by experimental studies of the temperature dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and aromatic amine dehydrogenase, and the flavoenzymes such as morphinone reductase and pentaerythritol tetranitrate reductase, which produced observations that are also inconsistent with the simple Bell-correction model of tunnelling. However, these data-especially, the strong temperature dependence of reaction rates and the variable temperature dependence of KIEs-are consistent with other tunnelling models (termed full tunnelling models), in which protein and/or substrate fluctuations generate a configuration compatible with tunnelling. These models accommodate substrate/protein (environment) fluctuations required to attain a configuration with degenerate nuclear quantum states and, when necessary, motion required to increase the probability of tunnelling in these states. Furthermore, tunnelling mechanisms in enzymes are supported by atomistic computational studies performed within the framework of modern TST, which incorporates quantum nuclear effects.

Full text

Page 1

, published 29 August 2006
, doi: 10.1098/rstb.2006.1878
361
2006 Phil. Trans. R. Soc. B
Jaswir Basran, Kara E Ranaghan, Adrian J Mulholland, David Leys and Nigel S Scrutton
Michael J Sutcliffe, Laura Masgrau, Anna Roujeinikova, Linus O Johannissen, Parvinder Hothi,
flavoprotein and quinoprotein systems
Hydrogen tunnelling in enzyme-catalysed H-transfer reactions:


References
http://rstb.royalsocietypublishing.org/content/361/1472/1375.full.html#related-urls

Article cited in:

http://rstb.royalsocietypublishing.org/content/361/1472/1375.full.html#ref-list-1

This article cites 89 articles, 9 of which can be accessed free
Email alerting service

here
right-hand corner of the article or click
Receive free email alerts when new articles cite this article - sign up in the box at the top

http://rstb.royalsocietypublishing.org/subscriptions
go to: Phil. Trans. R. Soc. B To subscribe to
on March 26, 2013rstb.royalsocietypublishing.orgDownloaded from

Page 2

Hydrogen tunnelling in enzyme-catalysed
H-transfer reactions: flavoprotein and
quinoprotein systems
Michael J. Sutcliffe1,2,*, Laura Masgrau1,2, Anna Roujeinikova1,3,
Linus O. Johannissen1,2, Parvinder Hothi1,3, Jaswir Basran4,
Kara E. Ranaghan5, Adrian J. Mulholland5,
David Leys1,3and Nigel S. Scrutton1,3
1Manchester Interdisciplinary Biocentre,2School of Chemical Engineering and Analytical Science, and
3Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7ND, UK
4Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, UK
5School of Chemistry, University of Bristol, Cantocks Close, Bristol BS8 1TS, UK
It is now widely accepted that enzyme-catalysed C–H bond breakage occurs by quantum mechanical
tunnelling. This paradigm shift in the conceptual framework for these reactions away from semi-
classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling
correction) has been driven over the recent years by experimental studies of the temperature
dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the
tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and
aromatic amine dehydrogenase, and the flavoenzymes such as morphinone reductase and
pentaerythritol tetranitrate reductase, which produced observations that are also inconsistent with
the simple Bell-correction model of tunnelling. However, these data—especially, the strong
temperature dependence of reaction rates and the variable temperature dependence of KIEs—are
consistent with other tunnelling models (termed full tunnelling models), in which protein and/or
substrate fluctuations generate a configuration compatible with tunnelling. These models
accommodate substrate/protein (environment) fluctuations required to attain a configuration with
degenerate nuclear quantum states and, when necessary, motion required to increase the probability
of tunnelling in these states. Furthermore, tunnelling mechanisms in enzymes are supported by
atomistic computational studies performed within the framework of modern TST, which
incorporates quantum nuclear effects.
Keywords: H-tunnelling; transition state theory; stopped-flow kinetics; kinetic isotope effect;
computational simulation
1. INTRODUCTION
Enzymes are extremely efficient catalysts that can
achieve rate enhancements up to 1021over the
uncatalysed reaction rate (Lad et al. 2003). Our quest
to understand the physical basis of this catalytic
power—pivotal to our understanding of biological
reactions and exploitation of enzymes in chemical,
biomedical and biotechnological processes—is challen-
ging, and has involved sustained and intensive research
efforts for over 100 years (for reviews, see Cannon &
Benkovic 1998; Cleland et al. 1998; Neet 1998;
Warshel 1998; Benkovic & Hammes-Schiffer 2003).
However, our understanding of how enzymes achieve
phenomenal rate enhancements is far from complete.
Recent years have seen new and important activity in
this area, and these studies include roles of protein
‘motion’ (Cameron & Benkovic 1997; Rajagopalan
et al. 2002; Benkovic & Hammes-Schiffer 2003), active
site pre-organization (for reviews, see Cannon et al.
1996; Warshel 1998) and, in particular, the role of
quantum mechanical tunnelling in enzymic hydrogen
transfer (for reviews, see Antoniou et al. 2002; Knapp &
Klinman 2002; Sutcliffe & Scrutton 2002; Liang &
Klinman 2004). Understanding the factors that drive
this H-tunnelling reaction, i.e. transfer occurs through
the energy barrier separating reactant from product, is
key to understand a large number of reactions in
biology; C–H bond cleavage occurs in approximately
50% of all biological reactions, and all these reactions
are likely to involve tunnelling to some degree. Such
non-classical behaviour is expected to transfer a light
particle, such as the H-nucleus, over short distances
since the de Broglie wavelength is 0.63 A˚for protium
and 0.45 A˚for deuterium (assuming an energy of
20 kJ molK1).
Quantum tunnelling studies of H-transfer focused
initially on deviations from values predicted by
Phil. Trans. R. Soc. B (2006) 361, 1375–1386
doi:10.1098/rstb.2006.1878
Published online 12 July 2006
One contribution of 16 to a Discussion Meeting Issue ‘Quantum
catalysis in enzymes—beyond the transition state theory paradigm’.
*Author for correspondence (michael.sutcliffe@manchester.ac.uk).
1375
q 2006 The Royal Society
on March 26, 2013rstb.royalsocietypublishing.orgDownloaded from

Page 3

semi-classical models (in which zero-point energies
(ZPEs), but not tunnelling, have been taken into
account), namely kinetic isotope effects (KIEs),
Swain–Schaad relationships (Kohen & Jensen 2002)
(expððlnðkH=kTÞÞ= ðlnðkD=kTÞÞÞO3:26, where kH, kDand
kTare the rates of transfer of protium, deuterium and
tritium, respectively) or Arrhenius pre-factor ratios
(much greater than unity for a reaction proceeding
purely by tunnelling and less than unity for moderate
tunnelling). Early examples in which H-tunnelling was
inferred from measurements of KIEs include the
quinoprotein bovine serum amine oxidase (Grant &
Klinman 1989), the Zn2C-dependent yeast alcohol
dehydrogenase (Cha et al. 1989) and horse liver alcohol
dehydrogenase (Bahnson et al. 1993), and the flavin-
dependent monoamine oxidase (Jonsson et al. 1994).
These studies were shown to be consistent with the
so-called Bell tunnel correction model of semi-classical
transfer, which invokes tunnelling just below the
classical transition state (TS; Bell 1980). This correc-
tion model accommodates minor corrections to the
rate of a reaction and predicts inflated KIEs and
Arrhenius pre-factor ratios to be less than unity (the
so-called Kreevoy criteria for tunnelling; Kim &
Kreevoy 1992) when KIEs are calculated as a function
of temperature. When new enzyme-catalysed reactions
were studied, a range of primary and secondary KIE
values, Arrhenius pre-factor ratios and temperature
dependences of rates and KIEs were observed. Studies
from our own group (Basran et al. 1999, 2001a,b,
2003; Kohen et al. 1999; Harris et al. 2000), Klinman
(Knapp et al. 2002) and more recently from Kohen
(Sikorski et al. 2004) and Alleman (Maglia & Allemann
2003) have indicated that the simple Bell-correction
model cannot adequately account for observed KIEs in
a number of enzyme systems. This has led to the
so-called full tunnelling models (see for example,
Kuznetsov & Ulstrup 1999; Knapp & Klinman 2002;
Knapp et al. 2002), akin to the established models for
electron transfer, which are consistent with the strong
temperature dependence of reaction rates, the variable
temperature dependence of KIEs and the observed
range of the Arrhenius pre-factor ratio. Given that
protein motion is thought to be an important factor in
driving quantum tunnelling in enzymic H-transfer
reactions, computational methodology for identifying
the residues important in creating reaction-promoting
vibrations in enzymes has also been developed
(Agarwal et al. 2002b; Mincer & Schwartz 2003).
Computational simulations of the reaction (i.e. the
breaking and making of bonds) are performed within
the framework of modern transition state theory
(TST), i.e. including quantum nuclear effects such as
H-tunnelling and, in the more complete applications,
including sampling over protein configurations. The
potential energy is described either by a hybrid
quantum mechanics/molecular mechanics (QM/MM)
potential (for a review, see Friesner & Guallar 2005;
Mulholland 2005) or the empirical valence bond
approach (Warshel 1991; Hammes-Schiffer 2004). In
the former method, which we used in the study of
methylamine dehydrogenase (MADH), atoms involved
in the reaction are treated quantum mechanically and
the rest of the system is treated classically using
molecular mechanics. Contributions of hydrogen
tunnelling to the rate constant can then be calculated
by different approaches (Garcia-Viloca et al. 2004;
Hammes-Schiffer 2004; Olsson et al. 2004). Given the
larger number of degrees of freedom involved, it is still
very challenging for these atomistic methods to address
the temperature dependence of the kinetics, especially
of the KIEs. Thus, most of the studies have been
limited to a single temperature. The results of the first
temperature dependence studies, which have been
presented recently (Hatcher et al. 2004; Olsson et al.
2004; Pu et al. 2005), are encouraging.
Our studies, in which we have investigated H-tun-
nelling in a number of quinoprotein and flavoprotein
enzymes, have provided evidence—when interpreted
within the framework of phenomenological Marcus-
like tunnelling models—consistent with H-transfer by
quantum tunnelling from the vibrational ground state
of the reactive C–H bond of the substrate. This involves
either H-tunnelling in which the KIE is temperature-
independent—we interpret this to correspond to the
absence of gated motion (i.e. no ‘compression’ of the
transfer distance by substrate and/or protein fluctu-
ations) or H-transfer in which the KIE is temperature-
dependent—we interpret this to correspond to the
involvement of gated motion. Our work (Basran et al.
2001a) has also highlighted the importance of energy
barrier shape in determining the rates of H-transfer,
and the concomitant values of KIEs, obtained in
experimental studies.
2. STOPPED-FLOW METHODS TO ACCESS THE
H-TUNNELLING STEPS IN QUINOPROTEINS AND
FLAVOENZYMES
The quinoprotein and flavoprotein enzymes are ideally
suited to studies of H-transfer during substrate
oxidation using stopped-flow methods. Analysis using
the steady-state approach is often compromised by the
inability to focus on a single chemical step, owing to the
existence of multiple barriers for binding, product
release and a number of chemical steps, each of which
may contribute to the overall catalytic rate. Using the
stopped-flow method, the chemical step can often be
isolated and the kinetics of C–H bond breakage
determined without complications arising from other
events in the catalytic sequence. With flavoprotein and
quinoprotein enzymes, the reactions catalysed are
conveniently divided into reductive and oxidative
half-reactions. Enzyme reduction occurs through the
breakage of substrate or coenzyme C–H bonds. The
kinetics of bond breakage is conveniently followed by
absorbance spectrophotometry, since the reaction is
concomitant with the reduction of the redox centre.
Thus, the alternative redox states of flavins and
quinoprotein centres provide a readily available spec-
troscopic probe for following the kinetics of C–H bond
breakage. The oxidative half-reaction usually involves
long-range electron transfer to acceptor proteins (e.g.
cytochromes, copper proteins or other flavoproteins),
but in the case of flavoproteins it can also involve
H-transfer to a second substrate. The absorbance
changes associated with the oxidation of the flavin
again provide a readily available signal for monitoring
1376M. J. Sutcliffe and others
Enzyme-catalysed H-tunnelling
Phil. Trans. R. Soc. B (2006)
on March 26, 2013 rstb.royalsocietypublishing.orgDownloaded from

Page 4

H-transfer to the oxidizing substrate. The ability to
interrogate each half-reaction by stopped-flow methods
simplifies the kinetic analysis substantially and this
makes these enzymes attractive targets in the studies of
H-transfer employing KIEs as probes of enzymic
H-tunnelling. Here, we present our work on the
tryptophan tryptophylquinone (TTQ)-dependent qui-
noproteinamine oxidases such as MADH and aromatic
amine dehydrogenase (AADH), and the flavoenzymes
such as morphinone reductase (MR) and pentaery-
thritol tetranitrate (PETN) reductase. We have
additionally studied H-tunnelling in the flavoenzymes
such as trimethylamine dehydrogenase (Basran et al.
2001b) and heterotetrameric sarcosine oxidase (Harris
et al. 2000).
3. TEMPERATURE DEPENDENCE OF ISOTOPE
EFFECTS AS PROBES OF H-TUNNELLING
The temperature-dependent behaviour of KIEs (i.e.
temperature-dependent versus temperature-indepen-
dent) is a key experimental result when considering the
nature of tunnelling. To explain the experimental range
of results obtained for enzymic systems, it has been
suggested that protein/substrate motions modulate
H-tunnelling. The realization that thermally induced
vibrations (Antoniou & Schwartz 1998) in the protein
scaffold (i.e. a thermally fluctuating energy surface)
might drive H-tunnelling led to the formulation of the
theoretical model of Kuznetsov & Ulstrup (1999),
analogous to the electron transfer theory (Marcus &
Sutin 1985), and is illustrated in figure 1. This model
has been adopted by Knapp & Klinman (Knapp &
Klinman 2002; Knapp et al. 2002), and is of the form:
?
!ðF:C: TermÞ!ðActive Dynamics TermÞ:
ktunnelZðconst:Þ! exp
K ðDG8ClÞ2
4lRT
???
ð3:1Þ
Here, ktunnelis the tunnelling rate constant; const., an
isotope-independentterm
coupling; the term in square parentheses is an
environmental energy term relating the driving force
of the reaction, DG8, and the reorganizational energy, l
(R is the gas constant and T, the temperature in K);
F.C. Term, the Frank–Condon nuclear overlap along
the hydrogen coordinate (HC), arises from the overlap
between the initial and the final states of the hydrogen’s
nuclear wave function. In the simplest limit, when only
the lowest vibrational level is occupied, the F.C. Term
is temperature-independent. Temperature-dependent
‘gating’ (or ‘active’) dynamics, which can be likened to
‘squeezing’ of the HC (figure 1) potential energy
barrier, can modulate the F.C. Term.
This model predicts that if the gating term
dominates (i.e. energy of gating is lesser than thermal
energy), then the observed KIE can be temperature-
dependent since this leads to different transfer dis-
tances for the heavy and light isotopes (Knapp &
Klinman 2002). In this regime, the ratio of Arrhenius
pre-factors (AH/AD) is predicted to be less than unity.
Alternatively, if the Frank–Condon term dominates
(i.e. energy of gating is greater than thermal energy),
describingelectronic
then the KIE will be temperature-independent. In the
latter scenario, occupation of excited vibrational levels
could result in some temperature dependence (Knapp
et al. 2002). However, the Boltzmann distribution at
298 K suggests that tunnelling should be predomi-
nantly from the vibrational ground state of the nuclear
wave function of hydrogen. In the regime in which the
energy of gating is roughly equal to the thermal energy,
gating would play some role in modulating the
tunnelling probability; the temperature-dependent
KIEs would be observed and the AH/ADvalues would
decrease (compared with the regime where the
Frank–Condon term dominates), and could approach
unity (Knapp & Klinman 2002).
Summarizing, within the framework provided by this
model (Kuznetsov & Ulstrup 1999; Knapp & Klinman
2002;Knappetal.2002),thetemperaturedependenceof
theKIEcanbeinterpretedasfollows:(i)H-tunnelling in
which the KIE is temperature-independent corresponds
to either the absence of, or at least no detectable
contributions from, gated motion (i.e. no significant
motion along HC in figure 1) and (ii) H-tunnelling in
whichtheKIEistemperature-dependentcorrespondsto
theinvolvementofgatedmotion(i.e.motionalongHCin
figure1).Accordingtothis,twotypesofmotionsneedto
be considered in enzymic H-tunnelling: (i) those which
r0
E
q
qR
a
b
qP
HC
rH
q*
qR
q* qP
M
DG0
Figure 1. Representation of the model for the hydrogen
transfer reaction used to interpret the experimental data (see
text and references Benkovic & Hammes-Schiffer (2003),
Kohen & Klinman (1998) and Kohen & Klinman (1999) for
more details). The three axes are: E, energy; q, environmental
coordinate (from which the transferred hydrogen atom is
excluded) and HC, hydrogen coordinate. The four vertical
panels showthepotential energy curveas afunction of HCfor
three values of the environmental coordinate: qRis for the
reactant, q?is for the transition state and qPis for the product.
The grey spheres represent the ground state vibrational wave
function of the hydrogen nucleus. The panel labelled M
shows a Marcus-like view of the free-energy curves as
functions of this environmental coordinate. The motions of
the environment modulate the symmetry of the double well,
thus allowing the system to reach a configuration with
(nearly) degenerate quantum states (qZq?), from which the
hydrogen is able to tunnel (F.C. Term in equation (3.1)). The
difference between panels a and b is a gating motion that
reduces the distance between the two wells along the HC-axis
(rH)awayfromitsequilibriumvalue(r0).Thismotionincreases
the probability of tunnelling at the (nearly) degenerate
configuration q?(active dynamics term in equation (3.1)).
Enzyme-catalysed H-tunnelling
M. J. Sutcliffe and others 1377
Phil. Trans. R. Soc. B (2006)
on March 26, 2013rstb.royalsocietypublishing.orgDownloaded from

Page 5

facilitate attaining a nuclear configuration compatible
with tunnelling (i.e. a configuration with degenerate
quantum states), termed ‘passive dynamics’ and (ii)
thosewhichenhancetheprobabilityoftunnellingonce(i)
has been attained, termed ‘active dynamics’.
Although these phenomenological models set a
simple conceptual framework to interpret the experi-
mental data, it is not possible to completely decouple
motions, nor their effects on H-tunnelling (Mincer &
Schwartz 2004), into active and passive dynamics.
Moreover, in the cases where the KIE is temperature-
dependent (of which, from our own work, there are
only two examples to date; Basran et al. 2001a, 2003),
the reaction can be taking place partly via the over-the-
barrier route and partly by tunnelling; both these over/
through the barrier explanation and/or the dominance
of active gating would be consistent with a tempera-
ture-dependent KIE. Thus, it is neither possible to map
directly from the kinetic data to a detailed picture of the
concomitant changes (motions) at the atomic level, nor
to the nature of the (free) energy barrier separating the
reactants from the products. It should also to be noted
that the experimentally accessible temperature range is
quite narrow. Thus, it is not always possible to show
unambiguously that a given KIE is truly temperature-
independent. Recent computational studies using
the ensemble-averaged variational transition state
theory with multidimensional tunnelling corrections
(EA-VTST/MT) framework (Pu et al. 2005) illustrate
how, even with different tunnelling behaviour for H and
D, nearly temperature-independent KIEs can be
observed in the temperature range of 5–45 8C.
A key role for dynamics has, however, been
advocated from the theoretical studies and experi-
mental observations, for example, Bruno & Bialek
(Bruno & Bialek 1992), Klinman (Liang et al. 2004),
Benkovic (Falzone et al. 1994; Epstein et al. 1995;
Cameron & Benkovic 1997; Osborne et al. 2001),
Hammes-Schiffer (Billeter et al. 2001; Agarwal et al.
2002a,b; Hammes-Schiffer 2002) and Schwartz
(Antoniou & Schwartz 1998; Antoniou & Schwartz
2001; Caratzoulas & Schwartz 2001; Antoniou et al.
2002; Caratzoulas et al. 2002; Mincer & Schwartz 2003;
Schwartz 2003). Care must be taken when interpreting
these studies, as terms such as ‘dynamics’ and ‘protein
motions’ are used by different authors to refer to very
different events. For example, Schwartz uses the term
‘promoting vibrations’ to refer to vibrations on the sub-
picosecond time-scale (Antoniou & Schwartz 2001)
that are coupled to the reaction coordinate and result in
changes in the (quantum) free-energy barrier.
Hammes-Schiffer uses the term ‘promoting motions’
to refer to motions averaged over Schwartz’s faster
‘promoting vibrations’. These promoting motions
occur on the much longer time-scale of the chemical
reaction being catalysed; these thermally averaged
motions affect the free energy. The term ‘dynamical
motions’ refers to those that influence barrier recross-
ing (Benkovic & Hammes-Schiffer 2003). A similar
definition has been used by Warshel (Villa & Warshel
2001; Warshel & Villa-Freixa 2003). A more general
context of atomic and molecular motion has also been
used by others (for example, by Karplus in Garcia-
Viloca et al. (2004)).
Although protein/substrate motions effect H-
tunnelling, the notion that enzymes have evolved to
optimize H-tunnelling by acquiring strategies to
increase the probability of transfer during evolution
remains controversial (see, for example, a recent
News Feature in Nature; Ball 2004). Warshel claims
(Warshel & Villa-Freixa 2003; Olsson et al. 2004) that
the dynamical effects of a reference reaction in solution
will be of the same magnitude as those in the enzyme
and, thus, they will not contribute to explain the
catalytic power of the enzyme (Villa & Warshel 2001).
In a study employing an adenosylcobalamin-dependent
diol dehydratase model reaction, it is also argued by
Finke & Doll (Doll & Finke 2003; Doll et al. 2003) that
this B12-dependent enzyme (which breaks a cobalt–
carbon bond) exploits the same level of quantum
mechanical tunnelling that is available in the reaction
occurring in the absence of the enzyme (i.e. there is no
‘compressive’ motion that preferentially enhances
H-tunnelling in the enzyme over the reaction in
solvent). Moreover, Siebrand & Smedarchina (2004)
have questioned the statistical significance of data
reported over a relatively narrow temperature range for
reactions of wild-type and mutant lipoxygenase; these
data were originally presented as evidence for gated
motion in this enzyme (Knapp et al. 2002). The same
authors, on theoretical grounds, also argue that flexible
proteins are ‘ill-equipped to cause strong local com-
pression’ (Siebrand & Smedarchina 2004, p. 4186).
One needs to be aware of these issues, but our view is
that it is not logical to generalize them on the basis of a
small number of studies and hence a case-by-case
analysis is appropriate.
4. H-TUNNELLING IN FLAVOENZYMES
MORPHINIONE REDUCTASE AND
PENTAERYTHRITOL TETRANITRATE
REDUCTASE
We have used the formulation of Knapp & Klinman
(described earlier) to interpret the anomalous tempera-
ture dependencies of H-transfer in flavin mononucleo-
tide (FMN)-containing MR and the homologous
PETN reductase. In particular, we have studied the
reactions of (i) PETN reductase with nicotinamide
adenine dinucleotide phosphate (NADPH; Basran
et al. 2003); (ii) MR with nicotinamide adenine
dinucleotide (NADH; Basran et al. 2003); and
(iii) MR in the oxidative half-reaction with 2-cyclohex-
enone (Basran et al. 2003), using stopped-flow and
steady-state kinetic methods with protiated and
deuterated nicotinamide coenzymes.
(a) Reductive half-reaction in morphinone
reductase and PETN reductase
The temperature-dependent behaviour of the primary
KIE for flavin reduction in MR and PETN reductase
by nicotinamide coenzyme indicates that quantum
mechanical tunnelling plays a major role in hydride
transfer. In PETN reductase, the KIE is essentially
temperature-independent in the experimentally acces-
sible range, and this contrasts with the strongly
temperature-dependent reaction rates (table 1). The
data are, therefore, consistent with a tunnelling
1378M. J. Sutcliffe and others
Enzyme-catalysed H-tunnelling
Phil. Trans. R. Soc. B (2006)
on March 26, 2013rstb.royalsocietypublishing.orgDownloaded from

Page 6

mechanism governed by passive dynamics and from the
vibrational ground state of the reactive C–H/D bond.
In MR, however, both the reaction rates, with NADH
and the KIE, are temperature-dependent. Analysis
using the Eyring equation (i.e. a plot of ln(k/T) versus
1/T) suggests that hydride transfer has a major
tunnelling component, which unlike in PETN
reductase would be classed as active dynamics
(table 1). We have suggested that these differences
arise from PETN reductase being relatively more rigid
than MR, which would make gating more dominant in
MR, predicting in turn that the KIE would be more
temperature-dependent in MR than in PETN
reductase. In addition, the active site of PETN
reductase might be more optimally configured for
hydride transfer than that of MR, thus requiring little
(or no) vibrational assistance through gated motion. In
other words, the active site of PETN reductase appears
ideally configured to transfer a hydride ion from
NADPH to FMN, and the nuclear reorganization
associated with H-tunnelling (i.e. passive dynamics) is
the major dynamic component. We have compared the
high-resolution crystal structures of MR (Barna et al.
2002) and PETN reductase (Barna et al. 2001) in an
attempt to provide insight into why gating is potentially
more important in MR. Analysis of the structures of
each enzyme suggests that the key factor could be a
double-stranded anti-parallel b-sheet D, against which
the NAD(P)H coenzyme is thought to bind (Barna
et al. 2002). This region harbours arginine residues,
which are important in the recognition of the
20-phosphate of NADPH (PETN reductase), and a
glutamate residue required to form a H-bond with the
20-OH group of NADH (MR). The position of this
sheet diverges at Leu-133 (PETN reductase)/Val-138
(MR) and converges again at Ile-141 (PETN
reductase)/Gly-146 (MR). In addition, there is an
insertion of a glycine residue (Gly-133) in MR
immediately before the start of b-sheet D. These
differences are consistent with MR being more mobile
at physiological temperatures in this region than PETN
reductase, which in turn might assist in a ‘squeezing’ or
‘compression’ of the HC energy barrier in MR
(figure 1). This suggestion is consistent with the
temperature factors of MR (all Catemperature factors
greater than 40; PDB accession code 1GWJ; Berman
et al. 2000) and PETN reductase (all Catemperature
factors less than 20; PDB accession code 1GVQ) in this
region. The next stage of our work to test this
hypothesis is to obtain structural information for the
coenzyme complexes at high resolution and to perform
a more detailed theoretical analysis involving QM/MM,
variational transitionstate
dimensional tunnelling (VTST/MT) and molecular
dynamics studies.
theory withmulti-
(b) Oxidative half-reaction in morphinone
reductase
The oxidative half-reaction of MR with the substrate
2-cyclohexenone and NADH at saturating concen-
trations is fully rate limiting in steady-state turnover.
This has enabled us to investigate the potential
tunnelling regimes in this part of the reaction cycle.
Reduction of 2-cyclohexenone involves hydride
transfer from reduced flavin (FMNH2) and protona-
tion. Thus, two H-transfer reactions are involved in this
oxidative half-reaction (scheme 1). We have demon-
strated that across the experimentally accessible
temperature range, the KIE for hydride transfer from
reduced flavin to the a/b unsaturated bond of
2-cyclohexenone is temperature-independent, in con-
trast to strongly temperature-dependent reaction rates.
In contrast, a large solvent isotope effect (SIE)
accompanies the oxidative half-reaction, which is also
temperature-independent in the experimentally acces-
sible range. Moreover, double isotope effects indicate
that hydride transfer from the flavin N5 atom to 2-
cyclohexenone and the protonation of 2-cyclohexenone
are coupled. Both the temperature-independent KIE
and SIE suggest that (i) gated motion is not required to
compress the energy barrier and (ii) this reaction
proceeds by ground state quantum tunnelling. Our
work with MR was, therefore, the first to show that
both passive and active dynamics are a feature of
H-tunnelling within the same native enzyme; in the
reductive half-reaction we suggest that barrier com-
pression is required to facilitate hydride transfer from
NADH to FMN, whereas in the oxidative half-reaction
Table 1. Tunnelling regimes and associated parameters in various flavoprotein and quinoprotein enzymes.
enzymesubstrate
AH/AD
DHH
(kJ molK1)
DHD
(kJ molK1)KIEa
passiveb
gatedb
reference
MADHc
MADHc
AADHc
AADHc
AADHc
MRc
MRc
PETNRc
methylamine
ethanolamine
tryptamine
dopamine
benzylamine
2-cyclohexenone
NADH
NADPH
13.3
0.57
44.6G0.5
43.5G0.6
45.0G0.5
51.9G1.1
53.5G1.2
51.6G0.7
67.1G0.9
17.1G0.9
43.5G0.8
36.6G0.9
16.8 TIa
14.7 TDa
54.7 TIa
12.9 TIa
4.8 TIa
3.5 TIa
3.9 TDa
4.1 TIa
#
Basran et al. (1999)
Basran et al. (2001a)
Basran et al. (2001a)
Basran et al. (2001a)
Basran et al. (2001a)
Basran et al. (2003)
Basran et al. (2003)
Basran et al. (2003)
#
#
#
9.4
3.7
3.7
0.126
4.1
50.9G0.7
68.1G1.4
17.6G0.9
35.3G0.5
36.4G0.9
#
#
#
#
aTI, temperature-independent KIE, TD, temperature-dependent KIE; KIE values for enzyme–substrate combinations displaying a
temperature-dependent (TD) KIE (i.e. reactions involving gated motion; Knapp & Klinman 2002; Knapp et al. 2002) are given at 298 K.
bThe terms ‘passive’ (i.e. the KIE is (almost) temperature-independent) and ‘gated’(i.e. the KIEis temperature-dependent) dynamicsare taken
from the work of Knapp & Klinman (Knapp & Klinman 2002; Knapp et al. 2002). See text for a discussion on the current limitations of this, and
other, interpretations of factors affecting the temperature (in)dependence of KIEs.
cEnzyme abbreviations: MADH, methylamine dehydrogenase; AADH, aromatic amine dehydrogenase; MR, morphinone reductase; PETNR,
pentaerythritol tetranitrate reductase.
Enzyme-catalysed H-tunnelling
M. J. Sutcliffe and others1379
Phil. Trans. R. Soc. B (2006)
on March 26, 2013 rstb.royalsocietypublishing.orgDownloaded from

Page 7

the active site is configured to catalyse hydride and
proton transfer in a concerted fashion without
vibrational assistance through gated motion.
5. H-TUNNELLING IN QUINOPROTEINS
METHYLAMINE DEHYDROGENASE AND
AROMATIC AMINE DEHYDROGENASE
(a) Experimental studies of substrate oxidation
in tryptophan tryptophylquinone-dependent
amine dehydrogenases
Our work on H-tunnelling in quinoprotein enzymes
has focused on TTQ-dependent MADH (Basran et al.
1999, 2001a) and AADH (Basran et al. 2001a). Earlier
studies on the quinoprotein bovine serum amine
oxidase (a topa quinine (TPQ)-dependent amine
oxidase) also demonstrated deviation from semi-
classical behaviour, thus suggesting a role for H-tun-
nelling (Grant & Klinman 1989). In this case, the data
were interpreted in terms of the tunnelling correction
models of Bell. In MADH, TTQ reduction is
concerted with the breakage of C–H bond from an
iminoquinone intermediate that forms rapidly in the
reductive half-reaction (scheme 1; Chen et al. 1992;
Davidson et al. 1992; Hyun & Davidson 1995b). The
rate of breakage of this bond is accessible using
stopped-flow spectrophotometry. The reduction of
TTQ cofactor is associated with a large KIE
(McWhirter & Klapper 1989; Brooks et al. 1993;
Basran et al. 1999; e.g. KIEZ16.8G0.5 at 298 K;
Basran et al. 1999) when methylamine and deuterated
methylamine are used as substrates. The value of KIE
is, therefore, larger than the higher value expected for
reactions described by semi-classical TST (i.e. includ-
ing ZPE but with no tunnelling correction), and is
clearly suggestive of tunnelling. Our studies on TTQ
reduction in MADH indicated that the value of KIE
was temperature-independent in the experimentally
accessible region, but significantly the reaction rate was
strongly temperature-dependent. These findings are
consistent with the dissipative tunnelling models
(Kuznetsov & Ulstrup 1999; Knapp & Klinman
2002; Knapp et al. 2002). Interpreted in the framework
of these models, the oxidation of methylamine by
MADH is dominated by environmental reorganization
associated with tunnelling, and no further motions
(gated motions) are required to explain the kinetic data.
It is interesting to note that an earlier study (Krishtalik
1986) had observed temperature-independent KIE
values (approx. 2–3) in steady-state reactions catalysed
by serine proteases performed in deuterated solvent,
and these were suggested to indicate tunnelling (note,
however, that the effect of D2O on the reaction
dynamics is potentially complicated owing to the
exchange of protons throughout the protein scaffold).
The data were modelled on earlier theoretical treat-
ments of H-tunnelling propounded by Dogonadzhe
and co-workers in which thermal vibrations bring the
solvent into a configuration favourable to tunnelling.
Moreover, at the time of our own work with MADH,
similar observations were made with the thermophilic
alcohol dehydrogenase of Bacillus stearothermophilus
(Kohen et al. 1999), which also indicates that the Bell-
correction model for H-tunnelling was inappropriate
for these enzyme-catalysed reactions. We extended our
work with MADH to study the tunnelling behaviour
with non-physiological substrates. In our work on the
oxidation of ethanolamine by MADH, we found
evidence for quantum tunnelling (table 1). In this
case, however, the KIE was temperature-dependent,
inconsistent with a reaction dominated by passive
dynamics, but consistent with significant active
dynamics (Basran et al. 2001a).
Our more recent studies have focused on the related
TTQ-dependent enzyme AADH, for which Davidson
and co-workers (Hyun & Davidson 1995a) observed a
large KIE with the aromatic substrate dopamine. Using
similar stopped-flow approaches to those reported for
studies with MADH, we have studied the oxidation of
three different substrates: dopamine, benzylamine and
tryptamine. We have shown that the rate of TTQ
reduction by dopamine in AADH has a large,
temperature-independent KIE (KIEZ12.9G0.2),
consistent with tunnelling involving passive, but not
gated, dynamics. We have also demonstrated that
H-transfer is compromised with benzylamine as
substrate and that the KIE is deflated (4.8G0.2),
although the KIE remains temperature-independent.
As with dopamine, however, the reaction rates are
strongly temperature-dependent. Tryptamine is a ‘fast’
substrate for AADH, thus the rate of TTQ reduction
could only initially be determined at low temperatures
using the stopped-flow method with protiated sub-
strate. It is said that an exceptionally large KIE (54.7G
1.0; single measurement at 4 8C) is observed for the
breakage of substrate C–H/D bond, and the reaction
rate (for C–D bond breakage) is strongly temperature-
dependent. The exceptionally large KIE for this
substrate suggests a major role for H-tunnelling in
substrate oxidation, and by comparison with studies
using dopamine and benzylamine we inferred that only
passive dynamics is involved in H-tunnelling. Our very
recent studies (Masgrau et al. 2006) have utilized a
stopped-flow instrument with a smaller dead time
(0.5 ms), and confirmed that the KIE with tryptamine
N
N
N
N
O
O
R
H
H
H
O
H
A
H
N
N
H2N
O
Asn 189
His 186
Scheme 1. Proposed scheme for the oxidative half-reaction of
morphinone reductase. The identity of the proton donor in
the oxidative half-reaction is not known.
1380M. J. Sutcliffe and others
Enzyme-catalysed H-tunnelling
Phil. Trans. R. Soc. B (2006)
on March 26, 2013 rstb.royalsocietypublishing.orgDownloaded from

Page 8

does not show a significant temperature dependence in
the experimentally accessible range.
We have used KIEs as a key indicator of H-tun-
nelling, and used the phenomenological Marcus-like
models to interpret the experimental results. However,
as mentioned earlier, it is not possible to map directly
from the KIE to the nature of either (at the atomic
level) the protein/substrate motions or (the shape of) of
the barrier through which tunnelling occurs. It is clear,
however, that for a chemical reaction such as proton
transfer in AADH (Basran et al. 2001a) and MADH
(Faulder et al. 2001), the shape of the barrier is
complex in nature and cannot be adequately re-
presented by the rectangular barrier used to model
electron transfer. To gain insight into the details of the
reaction at the atomic level, computational methods
can be used.
(b) Computational studies of substrate oxidation
in tryptophan tryptophylquinone-dependent
amine dehydrogenases
From the earlier discussion, it is clear that the factors
which give rise to the observed kinetic data for these
systems are far from trivial. To date, several compu-
tational studies elucidating how these enzymes might
work and facilitate tunnelling have been published on
MADH (Faulder et al. 2001; Alhambra et al. 2001,
2002; Tresadern et al. 2002, 2003a,b). Both methyl-
amine and ethanolamine substrates have been studied.
The reaction step modelled corresponds to the C–H
bond breakage involving abstraction by the active site
base (Asp 428) of a proton from iminoquinone
(figure 2). As mentioned earlier, this proton transfer
has been attributed to an elevated experimental KIE,
which was taken as an indicator of H-tunnelling. In all
these cases, the calculation of the rate constants and the
H-tunnelling contributions was performed within the
framework of VTST/MT corrections (Truhlar et al.
1985; Truhlar et al. 2001; Gao & Truhlar 2002) and a
hybrid QM/MM scheme (figure 3) was used to
describe the potential energy surface. Within the QM/
MM approach (Warshel & Levitt 1976; Field et al.
1990 and for a recent review, see Friesner & Guallar
2005; Mulholland 2005), a small region in the active
site containing the atoms involved in the breaking and
forming of bonds is treated by quantum mechanical
methods, whereas the rest of the solvated enzyme is
described by a molecular mechanics force field. There-
fore, this approach allows the atomic-level modelling of
the reaction process in the presence of the enzyme (see
Gao & Truhlar 2002 for a review). The VTST/MTrate
constantforaunimolecularreactioncanbeexpressedas:
kðTÞ ZkðTÞkBT
h
exp
K DGTS;oðTÞ
RT
??
;
ð5:1Þ
where (DGTS,o(T)) is the standard-state molar free
energyof activationat temperature T, with thequantized
vibrational energy included; k(T), the transmission
coefficient, which accounts for quantum nuclear effects
on the reaction coordinate and dynamical recrossing; T,
kB, h and R are the temperature, the Boltzmann, Planck
and the gas constants, respectively.
Our (preliminary) work on MADH-methylamine
(Faulder et al. 2001) used Gaussian 94 (Frisch et al.
1995) and AMBER (Pearlman et al. 1995) for the QM/
MMcalculations,withthesemi-empiricalParameterized
Model 3 (PM3) method (Stewart 1989) and the
link-atom approach (Singh & Kollman 1986; Stewart
1989; Field et al. 1990; Maseras & Morokuma 1995;
for interfacing this QM region with the rest of the
protein modelled by MM). A reactant complex was
found using a single protein configuration, optimizing
the QM region embedded in the frozen MM environ-
ment, and the corresponding saddle point and product
complex were identified. From this saddle point, the
minimum energy path (MEP) was calculated downhill
until the energy approached that of the reactant and the
product complex using POLYRATE v. 7.4 (Steckler
et al. 1997); for the QM region, the first and second
derivatives of the energy were also calculated along this
MEP. The rate constant was then calculated including
a multidimensional treatment of H-tunnelling. The
results (figure 4) suggested that approximately 96% of
the reaction proceeds by tunnelling, with the remaining
approximately 4% proceeding via the classical over-
the-barrier route. It was also shown that tunnelling
needs to be included to obtain a KIE value comparable
(a)
NH
CH
C
CH2
O
NH
HN
CH
C
H2C
NH
O
O
O
(b)
O
6
54
O
O–
H_B
O–
CH3
CH3
CH2
H2O
+ H2O
H2N
O
OH
B–
HNH
O
HO
C
C
HB
B–
k3
CH2
CH2
C
NH
NH
H
C
HN
CH2
C
CC
O
C
C
C
231
C
C
+
+
+
+
..
HNH
Figure 2. (a) The structure of the tryptophan tryptophylqui-
none (TTQ) cofactor. (b) Reaction mechanism of the
reductive half-reaction of MADH. Steps enclosed in the
box represent binding steps. A similar scheme has been
proposed for the reaction of AADH with aromatic primary
amines. The tunnelling step is denoted k3.
Enzyme-catalysed H-tunnelling
M. J. Sutcliffe and others1381
Phil. Trans. R. Soc. B (2006)
on March 26, 2013rstb.royalsocietypublishing.orgDownloaded from

Page 9

to the experimental one, and that a multidimensional
(i.e. more comprehensive) treatment of tunnelling also
improved the result: a value of 6.1 was obtained when
tunnelling was omitted, 9.6 when tunnelling was
included by a simple one-dimensional model (i.e. the
Wigner correction; Wigner 1932; Truhlar et al. 1985)
and 11.1 when the small-curvature tunnelling (Liu
et al. 1993) was used. Thus, our calculated value was in
qualitative agreement with the corresponding experi-
mental value of 16.8G0.5 (all at 298 K; Basran et al.
1999).
In the same year, an independent study on the
MADH-methylamine system was published (Alhambra
et al. 2001; Alhambra et al. 2002). The authors
determined an even larger degree of tunnelling for the
H-transfer step by increasing the rate of reaction
100-fold,i.e.99%oftheH-transferreactionsproceeding
via tunnelling and 1% of the reactions proceeding via
the classical over-the-barrier route (again at 298 K).
Their predicted KIE of 18.3 at 298 K is in good
agreement with the experimental result (Basran et al.
1999) and also larger than our calculated value.
Despite the qualitative agreement between their work
and ours, the schemes followed differ. The main
differences are that they used specific reaction par-
ameters (PM3-SRP; Gonza ´lez-Lafont et al. 1991) to
improve the PM3 potential energy surface, and that
they employed the EA-VTST/MT approach (Truhlar
et al. 2001; Gao & Truhlar 2002). In short, this
approach derives the activation free energy and the
tunnelling transmission coefficient by averaging over
protein configurations instead of using a single MEP.
Despite the differences in the methods used, the
picture emerging from our work and that of others for
this proton transfer is qualitatively the same. First, the
two heavy atoms (C on the iminoquinone and O on the
Asp) approach each other until the distance is reduced
sufficiently for (a significant amount of) tunnelling to
take place through the potential energy surface (note
that the tunnelling probability is related inversely to the
tunnelling distance). Tunnelling is further enhanced by
a corner-cutting mechanism as the C–H (and later the
O–H) stretching modes become coupled to the
reaction coordinate. This, together with a reaction
coordinate dominated by the motion of the light
particle (H-nucleus), results in a shorter effective
tunnelling path, which is responsible for an enhance-
ment of the tunnelling probability. The importance of
this corner-cutting effect in modulating tunnelling has
been recognized for a long time and was used in the
work of Tresadern et al. (2002) to explain the different
tunnelling behaviour among MADH, liver alcohol
dehydrogenase and soybean lipoxygenase. Although
similar potential energy barriers were obtained for
three hydrogen transfer reactions, these three enzymes
exhibit different degrees of tunnelling. The authors
rationalized this in terms of different donor–acceptor
distance behaviour during the reaction, and linked this
to corner-cutting and different curvature of the reaction
paths.
Tresadern et al. (2003a,b) have also studied the
reaction of MADH with ethanolamine, which turned
out to be a much more complex system as a number of
substrate conformers were identified, differing by the
position of the hydroxyl group of ethanolamine. When
the most stable conformer (denoted EA(A) by the
authors; the hydroxyl group hydrogen bonded to a
water molecule, which was in turn hydrogen bonded to
another residue) was studied, the results did not agree
completely with the experimental observations.
Indeed, contrary to the experiment, more tunnelling
was obtained for ethanolamine than for methylamine.
When the other conformer was considered (denoted
EA(B); the hydroxyl group of ethanolamine hydrogen
bonded to the non-reactive oxygen atom of the Asp428;
figure 3), the energy profile and the kinetic results were
significantly different. A higher barrier, a more positive
energy of reaction and (concomitantly) much smaller
5
10
–2–101
s (bohr amu1/2)
Energy (kcal mol–1)
0
–5
Figure 4. Energetics along the reaction path for proton
tunnelling. Potential energy relative to reactant (solid black
line), and minimum energy path (including zero-point
energy; solid grey line) calculated to obtain the KIEs for
proton transfer. The reaction coordinate, s, is the mass-
weighted difference in position from the transition state
(1 bohrZ0.529 A˚), with sZ0 at the top of the barrier and
negative at the reactant side.
N
C
O
NH
H
N
H
N
O
H
C
H2
H
O
O
H
N
H
H
H
Asp-384
Asp-428
TTQ
QM
MM
Figure 3. Schematic of the active site in methylamine
dehydrogenase, illustrating the QM/MM partition used in
the calculations and the mechanism corresponding to the
tunnelling reaction. The QM region is shown on a white
background and partof the surrounding MMregion on agrey
background, with link atoms circled; hydrogen bonds are
depicted by dashed lines.
1382M. J. Sutcliffe and others
Enzyme-catalysed H-tunnelling
Phil. Trans. R. Soc. B (2006)
on March 26, 2013rstb.royalsocietypublishing.orgDownloaded from

Page 10

tunnelling effects were obtained. The authors specu-
lated that as the two conformers were close in energy,
they both could be contributing to the observed kinetic
data, in particular, the temperature dependence of the
KIE. Thus, they suggest that the less stable structure,
EA(B), with a smaller KIE, would become more
important at higher temperatures. The possible contri-
bution to the observed kinetic data of different reactant
conformers is certainly an interesting observation and
an issue to be considered when studying the reactivity
of any system (Zhang et al. 2003).
Summarizing, these computational studies confirm
that over-the-barrier processes are not the only
mechanism employed by MADH, particularly with
methylamine, and H-tunnelling enhances the rate of
transfer significantly. A good agreement between the
calculated and the experimental KIE is obtained when
tunnelling is included. Moreover, a multidimensional
treatment of tunnelling is necessary to reproduce the
high experimental KIE values, as simpler tunnelling
models are insufficient. Thus, in accord with the
experimental observations, these computational
studies suggest that these H-transfer reactions invoke
tunnelling. Indeed, the degree of tunnelling calculated
for MADH-methylamine is larger than that for other
protein systems†, the next largest one being 85% in
xylose isomerase (Alhambra et al. 2000; Garcia-Viloca
et al. 2003).
6. CONCLUSIONS
Quantum tunnelling of hydrogen has emerged over the
recent years as a means by which enzymes catalyse
reactions involving hydrogen transfer. The increasing
body of experimental and computational evidences
suggest that H-tunnelling is likely to be adopted
extensively by enzymes. The temperature-dependent
behaviour of KIEs has revealed that enzymes can
catalyse reactions by ‘pure’ quantum tunnelling. KIEs
give insight at the macroscopic level, but not at the
atomic level. Computational studies are used to give
insight into the atomic details of the mechanisms used
bytheenzymesthatinvokequantumtunnelling.Protein
dynamics is thought to modulate these tunnelling
reactions, and a picture of how enzymes achieve this is
beginning to emerge through, for example, phenomen-
ological Marcus-like models and modern TST (which
incorporates quantum nuclear effects). However, many
details at the atomic level remain to be discovered, and
these will be probed in greater detail as more refined
kinetic (e.g. at cryogenic temperatures) and compu-
tational techniques are developed.
†NOTE ADDED IN PROOF
One very recent computational study of H-tunnelling
in AADH (Masgrau et al. 2006) reveals that with
tryptamine the degree of tunnelling is calculated to
exceed 99.9%.
The authors gratefully acknowledge the financial support
from the Biotechnology and Biological Sciences Research
Council (BBSRC), the Engineering and Physical Sciences
Research Council (EPSRC), the Lister Institute of Preventive
Medicine and The Royal Society.
REFERENCES
Agarwal, P. K., Billeter, S. R. & Hammes-Schiffer, S. 2002a
Nuclear quantum effects and enzyme dynamics in
dihydrofolate reductase catalysis. J. Phys. Chem. B 106,
3283–3293. (doi:10.1021/jp020190v)
Agarwal, P. K., Billeter, S. R., Rajagopalan, P. T., Benkovic,
S. J. & Hammes-Schiffer, S. 2002b Network of coupled
promoting motions in enzyme catalysis. Proc. Natl Acad.
Sci. USA 99, 2794–2799. (doi:10.1073/pnas.052005999)
Alhambra, C., Corchado, J., Sanchez, M., Gao, J. & Truhlar,
D. 2000 Quantum dynamics of hydride transfer in enzyme
catalysis. J. Am. Chem. Soc. 122, 8197–8203. (doi:10.
1021/ja001476l)
Alhambra, C., Sanchez, M. L., Corchado, J., Gao, J. L. &
Truhlar, D. G. 2001 Quantum mechanical tunneling in
methylamine dehydrogenase. Chem. Phys. Lett. 347,
512–518. (doi:10.1016/S0009-2614(01)00921-6)
Alhambra,C.,Sanchez,M.L.,Corchado,J.,Gao,J.&Truhlar,
D. G. 2002 Erratum to: “Quantum mechanical tunneling
in methylamine dehydrogenase”. Chem. Phys. Lett. 355,
388–394. (doi:10.1016/S0009-2614(02)00057-X)
Antoniou, D. & Schwartz, S. D. 1998 Activated chemistry in
the presence ofa strongly
vibration. J. Chem. Phys. 108, 3620–3625. (doi:10.1063/
1.475756)
Antoniou, D. & Schwartz, S. D. 2001 Internal enzyme
motions as a source of catalytic activity: rate-promoting
vibrations and hydrogen tunneling. J. Phys. Chem. B 105,
5553–5558. (doi:10.1021/jp004547b)
Antoniou, D., Caratzoulas, S., Kalyanaraman, C., Mincer,
J. S. & Schwartz, S. D. 2002 Barrier passage and protein
dynamics in enzymatically catalyzed reactions. Eur.
J. Biochem. 269, 3103–3112. (doi:10.1046/j.1432-1033.
2002.03021.x)
Bahnson, B. J., Park, D. H., Kim, K., Plapp, B. V. & Klinman,
J. P. 1993 Unmasking of hydrogen tunneling in the horse
liver alcohol dehydrogenase reaction by site directed
mutagenesis. Biochemistry 32, 5503–5507. (doi:10.1021/
bi00072a003)
Ball, P. 2004 By chance, or by design? Nature 431, 396–397.
(doi:10.1038/431396a)
Barna, T. M., Khan, H., Bruce, N. C., Barsukov, I., Scrutton,
N. S. & Moody, P. C. 2001 Crystal structure of
pentaerythritol tetranitrate reductase: “flipped” binding
geometries for steroid substrates in different redoxstates of
the enzyme. J. Mol. Biol. 310, 433–447. (doi:10.1006/
jmbi.2001.4779)
Barna, T., Messiha, H. L., Petosa, C., Bruce, N. C.,
Scrutton, N. S. & Moody, P. C. 2002 Crystal structure
of bacterial morphinone reductase and properties of the
C191A mutantenzyme.
30 976–30 983. (doi:10.1074/jbc.M202846200)
Basran, J., Sutcliffe, M. J. & Scrutton, N. S. 1999 Enzymatic
H-transfer requires vibration-driven extreme tunneling.
Biochemistry 38, 3218–3222. (doi:10.1021/bi982719d)
Basran, J., Patel, S., Sutcliffe, M. J. & Scrutton, N. S. 2001a
Importance of barrier shape in enzyme-catalyzed reac-
tions—vibrationally assisted hydrogen tunneling in trypto-
phantryptophylquinone-dependentaminedehydrogenases.
J. Biol. Chem. 276, 6234–6242. (doi:10.1074/jbc.M008
141200)
Basran, J., Sutcliffe, M. J. & Scrutton, N. S. 2001b Deuterium
isotope effects during carbon-hydrogen bond cleavage
bytrimethylaminedehydrogenase.
mechanism and vibrationally assisted hydrogen tunneling
in wild-type and mutant enzymes. J. Biol. Chem. 276,
24 581–22 587. (doi:10.1074/jbc.M101178200)
Basran, J., Harris, R. J., Sutcliffe, M. J. & Scrutton, N. S.
2003 H-tunneling in the multiple H-transfers of the
catalytic cycle of morphinone reductase and in the
symmetrically coupled
J. Biol.Chem.
277,
Implicationsfor
Enzyme-catalysed H-tunnelling
M. J. Sutcliffe and others 1383
Phil. Trans. R. Soc. B (2006)
on March 26, 2013rstb.royalsocietypublishing.org Downloaded from

Page 11

reductive half-reaction of the homologous pentaerythritol
tetranitrate reductase. J. Biol. Chem. 278, 43 973–43 982.
(doi:10.1074/jbc.M305983200)
Bell, R. P. 1980 The application of tunnel corrections in
chemical kinetics. The tunnel effect in chemistry, p. 140.
London, UK: Chapman and Hall.
Benkovic, S. J. & Hammes-Schiffer, S. 2003 A perspective on
enzyme catalysis. Science 301, 1196–1202. (doi:10.1126/
science.1085515)
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat,
T. N., Weissig, H., Shindyalov, I. N. & Bourne, P. E. 2000
The protein data bank. Nucleic Acids Res. 28, 235–242.
(doi:10.1093/nar/28.1.235)
Billeter, S. R., Webb, S. P., Agarwal, P. K., Iordanov, T. &
Hammes-Schiffer, S. 2001 Hydride transfer in liver
alcohol dehydrogenase: quantum dynamics, kinetic iso-
tope effects, and role of enzyme motion. J. Am. Chem. Soc.
123, 11 262–11 272. (doi:10.1021/ja011384b)
Brooks, H. B., Jones, L. H. & Davidson, V. L. 1993
Deuterium kinetic isotope effect and stopped-flow kinetic
studies of the quinoprotein methylamine dehydrogenase.
Biochemistry 32, 2725. (doi:10.1021/bi00061a034)
Bruno, W. J. & Bialek, W. 1992 Vibrationally enhanced
tunneling as a mechanism for enzymatic hydrogen
transfer. Biophys. J. 63, 689–699.
Cameron, C. E. & Benkovic, S. J. 1997 Evidence for a
functional role of the dynamics of glycine-121 of
Escherichia coli dihydrofolate reductase obtained from
kinetic analysis of a site-directed mutant. Biochemistry
36, 15 792–15 800. (doi:10.1021/bi9716231)
Cannon, W. R. & Benkovic, S. J. 1998 Solvation, reorgan-
ization energy, and biological catalysis. J. Biol. Chem. 273,
26 257–26 260. (doi:10.1074/jbc.273.41.26257)
Cannon, W. R., Singleton, S. F. & Benkovic, S. J. 1996 A
perspective on biological catalysis. Nat. Struct. Biol. 3,
821–833. (doi:10.1038/nsb1096-821)
Caratzoulas, S. & Schwartz, S. D. 2001 A computational
method to discover the existence of promoting vibrations
for chemical reactions in condensed phases. J. Chem. Phys.
114, 2910–2918. (doi:10.1063/1.1342817)
Caratzoulas, S., Mincer, J. S. & Schwartz, S. D. 2002
Identification of a protein-promoting vibration in the
reaction catalyzed by horse liver alcohol dehydrogenase.
J. Am. Chem. Soc. 124, 3270–3276. (doi:10.1021/
ja017146y)
Cha, Y., Murray, C. J. & Klinman, J. P. 1989 Hydrogen
tunneling in enzyme reactions. Science 243, 1325–1330.
Chen, L. Y., Mathews, F. S., Davidson, V. L., Huizinga,
E. G., Vellieux, F. M. D. & Hol, W. G. J. 1992 Three-
dimensional structure of the quinoprotein methylamine
dehydrogenase from Paracoccus denitrificans determined by
molecular replacement at 2.8 A˚resolution. Proteins: Struct.
Funct. Genet. 14, 288–299.
Cleland, W. W., Frey, P. A. & Gerlt, J. A. 1998 The low
barrier hydrogen bond in enzymatic catalysis. J. Biol.
Chem.
273, 25 529–25 532.
25529)
Davidson, V. L., Jones, L. H. & Graichen, M. E. 1992
Reactions of benzylamines with methylamine dehydro-
genase. Evidence for a carbanionic reaction intermediate
and reaction mechanism similar to eukaryotic quinopro-
teins. Biochemistry 31, 3385. (doi:10.1021/bi00128a012)
Doll, K. M. & Finke, R. G. 2003 A compelling experimental
test of the hypothesis that enzymes have evolved to
enhance quantum mechanical tunneling in hydrogen
transfer reactions: the beta-neopentylcobalamin system
combined with prior adocobalamin data. Inorg. Chem. 42,
4849–4856.
(doi:10.1074/jbc.273.40.
Doll, K. M., Bender, B. R. & Finke, R. G. 2003 The first
experimental test of the hypothesis that enzymes have
evolved to enhance hydrogen tunneling. J. Am. Chem. Soc.
125, 10 877–10 884. (doi:10.1021/ja030120h)
Epstein, D. M., Benkovic, S. J. & Wright, P. E. 1995
Dynamics of the dihydrofolate reductase-folate complex:
catalytic sites and regions known to undergo confor-
mational change exhibit diverse dynamical features.
Biochemistry
34,11 037–11 048.
35a009)
Falzone, C. J., Wright, P. E. & Benkovic, S. J. 1994 Dynamics
of a flexible loop in dihydrofolate reductase from
Escherichia coli and its implication for catalysis. Biochem-
istry 33, 439–442. (doi:10.1021/bi00168a007)
Faulder, P. F., Tresadern, G., Chohan, K. K., Scrutton,
N. S., Sutcliffe, M. J., Hillier, I. H. & Burton, N. A. 2001
QM/MM studies show substantial tunneling for the
hydrogen-transfer reaction in methylamine dehydro-
genase. J. Am. Chem. Soc. 123, 8604–8605. (doi:10.
1021/ja016219a)
Field, M. J., Bash, P. A. & Karplus, M. 1990 A combined
quantum-mechanical and molecular mechanical potential
for molecular-dynamics simulations. J. Comput. Chem. 11,
700–733. (doi:10.1002/jcc.540110605)
Friesner, R. A. & Guallar, V. 2005 Ab initio quantum
chemical and mixedquantum
mechanics (QM/MM) methods for studying enzymatic
catalysis. Annu. Rev. Phys. Chem. 56, 389–427. (doi:10.
1146/annurev.physchem.55.091602.094410)
Frisch, M. J. et al. 1995 GAUSSIAN 94. Pittsburgh, PA:
Gaussian Inc.
Gao, J. & Truhlar, D. G. 2002 Quantum mechanical methods
for enzyme kinetics. Annu. Rev. Phys. Chem. 53, 467–505.
(doi:10.1146/annurev.physchem.53.091301.150114)
Garcia-Viloca, M., Alhambra, C., Truhlar, D. G. & Gao, J. L.
2003 Hydride transfer catalyzed by xylose isomerase:
mechanism and quantum effects. J. Comput. Chem. 24,
177–190. (doi:10.1002/jcc.10154)
Garcia-Viloca, M., Gao, J., Karplus, M. & Truhlar, D. G.
2004 How enzymes work: analysis by modern rate theory
and computer simulations. Science 303, 186–195. (doi:10.
1126/science.1088172)
Gonza ´lez-Lafont, A., Truong, T. N. & Truhlar, D. G. 1991
Direct dynamics calculations with neglect of diatomic
differential overlap molecular orbital theory with specific
reaction parameters. J. Phys. Chem. 95, 4618–4627.
(doi:10.1021/j100165a009)
Grant, K. L. & Klinman, J. P. 1989 Evidence that
protium and deuterium undergo significant tunneling
in the reaction catalyzed by bovine serum amine
oxidase.
Biochemistry
28,
bi00442a010)
Hammes-Schiffer, S. 2002 Impact of enzyme motion on
activity. Biochemistry 41, 13 335–13 343. (doi:10.1021/
bi0267137)
Hammes-Schiffer, S. 2004 Quantum-classical simulation
methods for hydrogen transfer in enzymes: a case study
of dihydrofolate reductase. Curr. Opin. Struct. Biol. 14,
192–201. (doi:10.1016/j.sbi.2004.03.008)
Harris, R. J., Meskys, R., Sutcliffe, M. J. & Scrutton, N. S.
2000 Kinetic studies of the mechanism of C–H bond
breakge by the heterotetrameric sarcosine oxidase of
Arthrobacter sp 1-IN. Biochemistry 39, 1189–1198.
(doi:10.1021/bi991941v)
Hatcher, E., Soudackov, A. V. & Hammes-Schiffer, S. 2004
Proton-coupled electron transfer in soybean lipoxygenase.
J. Am. Chem. Soc. 126, 5763–5775. (doi:10.1021/
ja039606o)
(doi:10.1021/bi000
mechanics/molecular
6597–6605.(doi:10.1021/
1384M. J. Sutcliffe and others
Enzyme-catalysed H-tunnelling
Phil. Trans. R. Soc. B (2006)
on March 26, 2013rstb.royalsocietypublishing.orgDownloaded from

Page 12

Hyun, Y. L. & Davidson, V. L. 1995a Unusually large isotope
effect for the reaction of aromatic amine dehydrogenase A
common feature of quinoproteins? Biochim. Biophys. Acta
1251, 198–200.
Hyun, Y.-L. & Davidson, V. L. 1995b Electron transfer
reactions between aromatic amine dehydrogenase and
azurin. Biochemistry 34, 12 249–12 254. (doi:10.1021/
bi00038a020)
Jonsson, T., Edmondson, D. E. & Klinman, J. P. 1994
Hydrogen tunneling in the flavoenzyme monoamine
oxidase B. Biochemistry 33, 14 871–14 878. (doi:10.
1021/bi00253a026)
Kim, Y. H. & Kreevoy, M. M. 1992 The experimental
manifestations of corner-cutting tunneling. J. Am. Chem.
Soc. 114, 7116–7123. (doi:10.1021/ja00044a024)
Knapp, M. J. & Klinman, J. P. 2002 Environmentally coupled
hydrogen tunneling. Linking catalysis to dynamics. Eur.
J. Biochem. 269, 3113–3121. (doi:10.1046/j.1432-1033.
2002.03022.x)
Knapp, M. J., Rickert, K. & Klinman, J. P. 2002
Temperature-dependent isotope effects in soybean lipox-
ygenase-1: correlating hydrogen tunneling with protein
dynamics. J. Am. Chem. Soc. 124, 3865–3874. (doi:10.
1021/ja012205t)
Kohen, A. & Jensen, J. H. 2002 Boundary conditions for the
Swain–Schaad relationship as a criterion for hydrogen
tunneling. J. Am. Chem. Soc. 124, 3858–3864. (doi:10.
1021/ja016909e)
Kohen, A. & Klinman, J. P. 1998 Enzyme catalysis: beyond
classical paradigms. Acc. Chem. Res. 31, 397–404. (doi:10.
1021/ar9701225)
Kohen, A. & Klinman, J. P. 1999 Hydrogen tunneling in
biology. Chem. Biol. 6, R191–R198. (doi:10.1016/S1074-
5521(99)80058-1)
Kohen, A., Cannio, R., Bartolucci, S. & Klinman, J. P. 1999
Enzyme dynamics and hydrogen tunnelling in a thermo-
philic alcohol dehydrogenase. Nature 399, 496–499.
(doi:10.1038/20981)
Krishtalik, L. I. 1986 Charge transfer reactions in electrochemical
and chemical processes. New York, NY: Consultants Bureau
pp. 244–298
Kuznetsov, A. M. & Ulstrup, J. 1999 Proton and hydrogen
atom tunnelling in hydrolytic and redox enzyme catalysis.
Can. J. Chem. 77, 1085–1096. (doi:10.1139/cjc-77-5-6-
1085)
Lad, C., Williams, N. H. & Wolfenden, R. 2003 The rate of
hydrolysis of phosphomonoester dianions and the excep-
tional catalytic proficiencies of protein and inositol
phosphatases.
Proc.Natl
5607–5610. (doi:10.1073/pnas.0631607100)
Liang, Z.-X. & Klinman, J. P. 2004 Structural bases of
hydrogen tunneling in enzymes: progress and puzzles.
Curr. Opin. struct. Biol. 14, 648–655.
Liang, Z. X., Lee, T., Resing, K. A., Ahn, N. G. & Klinman,
J. P. 2004 Thermal-activated protein mobility and its
correlation with catalysis in thermophilic alcohol dehydro-
genase. Proc. Natl Acad. Sci. USA 101, 9556–9561.
(doi:10.1073/pnas.0403337101)
Liu, Y. P., Lynch, G. C., Truong, T. N., Lu, D. H., Truhlar,
D. G. & Garrett, B. C. 1993 Molecular modeling of the
kinetic isotope effect for the [1,5] sigmatropic rearrange-
ment of cis-1,3-pentadiene. J. Am. Chem. Soc. 115,
2408–2415. (doi:10.1021/ja00059a041)
Maglia, G. & Allemann, R. K. 2003 Evidence for envir-
onmentally coupled hydrogen tunneling during dihydro-
folate reductase catalysis. J. Am. Chem. Soc. 125,
13 372–13 373. (doi:10.1021/ja035692g)
Marcus, R. A. & Sutin, N. 1985 Electron transfers in
chemistry and biology. Biochim. Biophys. Acta 811,
265–322.
Acad.Sci.USA
100,
Maseras, F. & Morokuma, K. 1995 A new ‘ab initioC
molecular mechanics’ geometry optimization scheme of
equilibrium structures and transition states. J. Comput.
Chem. 16, 1170–1179. (doi:10.1002/jcc.540160911)
Masgrau, L., Roujeinikova, A., Johannissen, L. O., Hothi, P.,
Basran, J., Ranaghan, K. E., Mulholland, A. J., Sutcliffe,
M. J., Scrutton, N. S. & Leys, D. 2006 Atomic description
of an enzyme reaction dominated by proton tunneling.
Science 312, 237–241.
McWhirter, R. B. & Klapper, M. H. 1989 In PQQ and
quinoproteins (ed. J. A. Jongejan & J. A. Duine), p. 259.
Dordrecht,The Netherlands:
Publishers.
Mincer, J. S. & Schwartz, S. D. 2003 A computational
method to identify residues important in creating a protein
promoting vibration in enzymes. J. Phys. Chem. B 107,
366–371. (doi:10.1021/jp027017j)
Mincer, J. S. & Schwartz, S. D. 2004 Rate-promoting
vibrationsandcoupled
reactions in the condensed phase: a model for enzymatic
catalysis. J. Chem. Phys. 120, 7755–7760. (doi:10.1063/
1.1690239)
Mulholland,A.J.2005Modelling
mechanisms, specificity and catalysis. Drug Discov. Today
10, 1393–1402. (doi:10.1016/S1359-6446(05)03611-1)
Neet, K. E. 1998 Enzyme catalytic power minireview series.
J. Biol. Chem. 273, 25 527–25 578. (doi:10.1074/jbc.273.
40.25527)
Olsson, M. H., Siegbahn, P. E. & Warshel, A. 2004
Simulations of the large kinetic isotope effect and the
temperature dependence of the hydrogen atom transfer in
lipoxygenase. J. Am. Chem. Soc. 126, 2820–2828. (doi:10.
1021/ja037233l)
Osborne, M. J., Schnell, J., Benkovic, S. J., Dyson, H. J. &
Wright, P. E. 2001 Backbone dynamics in dihydrofolate
reductase complexes: role of loop flexibility in the catalytic
mechanism. Biochemistry 40, 9846–9859. (doi:10.1021/
bi010621k)
Pearlman, D. A. et al. 1995 AMBER. San Francisco, CA:
University of California.
Pu, J. Z., Ma, S. H., Gao, J. L. & Truhlar, D. G. 2005 Small
temperature dependence of the kinetic isotope effect for
the hydride transfer reaction catalyzed by Escherichia coli
dihydrofolatereductase.
8551–8556. (doi:10.1021/jp051184c)
Rajagopalan, P. T., Lutz, S. & Benkovic, S. J. 2002 Coupling
interactions of distal residues enhance dihydrofolate
reductase catalysis: mutational effects on hydride transfer
rates. Biochemistry 41, 12 618–21 268. (doi:10.1021/
bi026369d)
Schwartz, S. D. 2003 Response to comment on “effect of
active site mutation Phe93/Trp in the horse liver alcohol
dehydrogenase enzyme on catalysis: a molecular dynamics
study”. J. Phys. Chem. B 107, 12 372. (doi:10.1021/
jp035474C)
Siebrand, W. & Smedarchina, Z. 2004 Temperature depen-
dence of kinetic isotope effects for enzymatic carbon–
hydrogen bond cleavage. J. Phys. Chem. 108, 4185–4195.
Sikorski, R. S., Wang, L., Markham, K. A., Rajagopalan,
P. T. R., Benkovic, S. J. & Kohen, A. 2004 Tunneling and
coupled motion in the Escherichia coli dihydrofolate
reductase catalysis. J. Am. Chem. Soc. 126, 4778–4779.
(doi:10.1021/ja031683w)
Singh, U. C. & Kollman, P. A. 1986 A combined ab initio
quantummechanicalandmolecular mechanicalmethodfor
carrying out simulations on complex molecular systems:
applications to the CH3ClCClKexchange reaction and gas
phase protonation of polyethers. J. Comput. Chem. 7,
718–730. (doi:10.1002/jcc.540070604)
Kluwer Academic
hydrogen-electrontransfer
enzymereaction
J.Phys.Chem. B
109,
Enzyme-catalysed H-tunnelling
M. J. Sutcliffe and others 1385
Phil. Trans. R. Soc. B (2006)
on March 26, 2013rstb.royalsocietypublishing.org Downloaded from

Page 13

Steckler, R. et al. 1997 POLYRATE. Minneapolis, MN:
University of Minnesota.
Stewart, J. J. P. 1989 Optimization of parameters for
semiempirical methods I. Method. J. Comput. Phys. 10,
209–220.
Sutcliffe, M. J. & Scrutton, N. S. 2002 A new conceptual
framework for enzyme catalysis. Hydrogen tunnelling
coupled to enzyme dynamics in flavoprotein and quino-
protein enzymes. Eur. J. Biochem. 269, 3096–3102.
(doi:10.1046/j.1432-1033.2002.03020.x)
Tresadern, G., McNamara, J. P., Mohr, M., Wang, H.,
Burton, N. A. & Hillier, I. H. 2002 Calculations of
hydrogen tunnelling and enzyme catalysis: a comparison
of liver alcohol dehydrogenase, methylamine dehydro-
genase and soybean lipoxygenase. Chem. Phys. Lett. 358,
489–494. (doi:10.1016/S0009-2614(02)00654-1)
Tresadern, G., Nunez, S., Faulder, P. F., Wang, H., Hillier,
I. H. & Burton, N. A. 2003a Direct dynamics calculations
of reaction rate and kinetic isotope effects in enzyme
catalysed reactions. Faraday Dis. 122, 223–242. (doi:10.
1039/b201183m)
Tresadern, G., Wang, H., Faulder, P. F., Burton, N. A. &
Hillier, I. H. 2003b Extreme tunnelling in methylamine
dehydrogenase revealed by hybrid QM/MM calculations:
potential energy surface profile for methylamine and
ethanolamine substrates and kinetic isotope effect values.
Mol. Phys. 101, 2775–2784. (doi:10.1080/0026897031
000121271)
Truhlar, D. G., Isaacson, A. D. & Garret, B. C. 1985
Generalized transition state theory. In Theory of chemical
reaction dynamics (ed. M. Baer), pp. 65–136. Boca Raton,
FL: CRC Press.
Truhlar, D. G., Gao, J., Alhambra, C., Garcia-Viloca, M.,
Corchado, J., Sa ´nchez, M. L. & Villa `, J. 2001 The
incorporation of quantum effects in enzyme kinetics
modeling. Acc. Chem. Res. 35, 341–349. (doi:10.1021/
ar0100226)
Villa, J. & Warshel, A. 2001 Energetics and dynamics of
enzymatic reactions. J. Phys. Chem. B 105, 7887–7907.
(doi:10.1021/jp011048h)
Warshel, A. 1991 Computer modeling of chemical reactions in
enzymes and solutions. New York, NY: Wiley.
Warshel, A. 1998 Electrostatic origin of the catalytic power of
enzymes and the role of preorganized active sites. J. Biol.
Chem.273,27 035–27 038.(doi:10.1074/jbc.273.42.27035)
Warshel, A. & Levitt, M. 1976 Theoretical studies of
enzymatic reactions: dielectric electrostatic and steric
stabilization of the carbonium ion in the reaction of
lysozyme. J. Mol. Biol. 103, 227–249. (doi:10.1016/0022-
2836(76)90311-9)
Warshel, A. & Villa-Freixa, J. 2003 Comment on “effect of
active site mutation Phe93/Trp in the horse liver alcohol
dehydrogenase enzyme on catalysis: a molecular dynamics
study”. J. Phys. Chem. B 107, 12 370–12 371. (doi:10.
1021/jp034932b)
Wigner, E. P. 1932 U¨ber das u ¨bershrieten von Potentialsch-
wellen bei Chemischen Reaktionen. Z. Phys. Chem. Abt. B
19, 203–216.
Zhang, Y. K., Kua, J. & McCammon, J. A. 2003 Influence of
structural fluctuation on enzyme reaction energy barriers
in combined quantum mechanical/molecular mechanical
studies. J. Phys. Chem. B 107, 4459–4463. (doi:10.1021/
jp022525e)
1386M. J. Sutcliffe and others
Enzyme-catalysed H-tunnelling
Phil. Trans. R. Soc. B (2006)
on March 26, 2013rstb.royalsocietypublishing.orgDownloaded from