Báo cáo Y học: A new conceptual framework for enzyme catalysis Hydrogen tunneling coupled to enzyme dynamics in flavoprotein and quinoprotein enzymes docx

MINIREVIEW
A new conceptual framework for enzyme catalysis
Hydrogen tunneling coupled to enzyme dynamics in flavoprotein and quinoprotein
enzymes
Michael J. Sutcliffe
1,2
and Nigel S. Scrutton
1
Departments of
1
Biochemistry and
2
Chemistry, University of Leicester, UK
Recent years have witnessed high levels of activity in iden-
tifying enzyme systems that catalyse H-transfer by quantum
tunneling. Rather than being restricted to a small number of
specific enzymes as perceived initially, it has now become an
accepted mechanism for H-transfer in a growing number of
enzymes. Furthermore, H-tunneling is driven by the
thermally induced dynamics of the enzyme. In some of those
enzymes that break stable C–H bonds the reaction proceeds
purely by quantum tunneling, without the need to partially
ascend the barrier. Enzymes studied that fall into this cate-
gory include the flavoprotein and quinoprotein amine
dehydrogenases, which have proved to be excellent model
systems. These enzymes have enabled us to study the rela-
tionship between barrier shape and reaction kinetics. This
has involved studies with ÔslowÕ and ÔfastÕ substrates and
enzymes impaired by mutagenesis. A number of key ques-
tions now remain, including the nature of the coupling
between protein dynamics and quantum tunneling. The

wide-ranging implications of quantum tunneling introduce a
paradigm shift in the conceptual framework for enzyme
catalysis, inhibition and design.
Keywords: H-tunneling; transition state theory; protein
dynamics; flavoprotein; quinoprotein; kinetic isotope
effect; computational simulation; quantum mechanics;
stopped-flow kinetics; molecular mechanics.
INTRODUCTION
The text-book description of catalysis states that enzymes
reduce the energy required to surmount the barrier between
reactants and products, which leads to enhanced rates. This
classical over-the-barrier treatment, known as transition
state theory (TST), has been used to depict enzyme-
catalysed reactions over the last 50 years [3]. Indications
that TST cannot be applied indiscriminately came in the late
1980s and the 1990s. In these instances [4–11], however, the
experimental observations could be modelled satisfactorily
by using a modified form of TST which incorporates an
additional component, a quantum tunneling correction
factor [1]; this permits tunneling below the saddle-point of
the potential energy surface (i.e. in these instances, the
saddle point of the potential energy surface is never
reached). The first indication that TST (with tunneling
correction) may not model faithfully all enzyme catalysed
systems came in 1996 [12]; these data show large deviations
from classical TST behaviour. The acid test of the generic
applicability of TST to enzyme catalysed reactions came in
1999 from experimental studies on enzymes catalysing C–H
bond breakage. Our own studies with methylamine dehy-
drogenase [13], and almost simultaneously that of Klinman

and coworkers independently with thermophilic alcohol
dehydrogenase [14], identified that, rather than ascending
the classical energy barrier prior to tunneling, the reaction
proceeds solely by quantum tunneling. Furthermore, this
work illustrated that quantum tunneling is driven by
thermal vibrations of the enzyme-substrate complex, which
serve to increase the tunneling probability (by reducing the
width and/or height of the barrier) sufficiently for tunneling
to occur (Fig. 1). Thus, at the dawn of the 21st century some
enzymes were shown to gain their catalytic power from
quantum mechanics—arguably the key scientific develop-
ment of the 20th century; indeed the first suggestion that
quantum mechanical tunneling may be a significant factor
in chemical reactions involving the transfer of hydrogen was
made by Hund some 70 years ago [15].
Although to some individuals biological systems and
quantum mechanics seem poles apart, with hindsight the
Correspondence to M. J. Sutcliffe or N. S. Scrutton,
Department of Biochemistry, University of Leicester,
University Road, Leicester LE1 7RH, UK.
Fax: + 44 116252 3369, Tel.: + 44 116223 1337,
E-mail: or
Abbreviations:TST,transitionstatetheory;TTQ,tryptophantrypto-
phylquinone; MADH, methylamine dehydrogenase; AADH, aroma-
tic amine dehydrogenase; TMADH, trimethylamine dehydrogenase;
TSOX, heterotetrameric sarcosine dehydrogenase; KIE, kinetic iso-
tope effect; QM/MM, quantum mechanical/molecular mechanical.
Definitions: Strictly, the term ÔsemiclassicalÕ [1] rather than ÔclassicalÕ is
used to indicate the difference in zero point vibrational energies of C–H
and C–D bonds in studies using the kinetic isotope effect as a probe of

quantum tunneling. In this review, we have used the term classical to
indicate over-the-barrier transfer to avoid confusion on the part of a
reader less familiar with the concepts of quantised vibrational energy
states. Quantum tunneling allows the hydrogen to travel through the
barrier. This is made possible by wave–particle duality. A particle
cannot pass through – it must pass over-the-barrier. However, wave–
particle duality also gives the hydrogen wave-like properties, and this
allows it to pass through a region (i.e. the barrier) from which a particle
would be excluded. See reference [2] for a more detailed description of
quantum tunneling.
(Received 7 March 2002, revised 21 May 2002, accepted 6 June 2002)
Eur. J. Biochem. 269, 3096–3102 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03020.x
only major surprise is perhaps that the key role of quantum
tunneling in enzyme catalysed H-transfer reactions is only
now being realized. After all, quantum tunneling is an
attractive means of transferring hydrogen from reactant to
product for those enzyme-catalysed reactions with large
activation barriers, where it is difficult to understand how
the reaction can occur over-the-barrier. Additionally, all
regions of the enzyme (not just the active site) likely
contribute to the vibrations that drive quantum tunneling,
thus providing a possible reason for why enzymes are much
larger than the active site alone. Also, vibrationally assisted
quantum tunneling is, for example, well established as a
means of H-transfer in metals [16] and for enzyme-mediated
electron transfer [17,18] (a proton is much heavier than an
electron, reducing the probability for a proton to tunnel; this
is one reason why enzymatic H-tunneling was not consid-
ered a plausible mechanism until experimental results
proved otherwise). H-transfer in nonbiological systems is

also known to occur by quantum tunneling, but at low
temperatures (cf. enzymatic H-tunneling, which occurs at
room temperature); for example, along hydrogen bonds in
benzoic acid dimers [19] and in the cyclic network of four
hydrogen bonds in calix[4]arene [20].
Earlier reviews on enzyme catalysed tunneling have
focussed on the inadequacies of TST for some enzyme
reactions and the first descriptions of H-tunneling aided by
protein motion along the reaction coordinate [2,21–23].
This review article summarizes our more recent kinetic
work on H-transfer by tunneling in quinoprotein and
flavoprotein enzymes that catalyse the oxidation of a
number of amine substrates, and computational studies of
enzymic H-tunneling in these enzymes.
QUINOPROTEIN AND FLAVOPROTEIN
AMINE DEHYDROGENASES
The quinoprotein and flavoprotein amine dehydrogenases
are ideally suited to studies of H-transfer. The reactions
catalysed are conveniently divided into reductive and
oxidative half-reactions. Enzyme reduction occurs by
breakage of a substrate C–H bond, the kinetics of which
are conveniently followed by absorbance spectrophotome-
try owing to reduction of the redox centre (and concomitant
change in absorbance spectrum) in the enzyme active site.
The oxidative half-reaction usually involves long-range
electron transfer to acceptor proteins (e.g. cytochromes,
copper proteins or other flavoproteins). The ability to
interrogate each half-reaction by stopped-flow methods
simplifies substantially the kinetic analysis. Studies of
steady-state reactions are often compromised by the inab-

ility 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
true kinetics of C–H bond breakage determined without
complications arising from other events in the catalytic
sequence. This feature of redox catalysis by the flavoprotein
and quinoprotein enzymes makes them attractive targets for
studies of H-transfer during substrate oxidation. For this
reason, our work has focused on the tryptophan tryptophyl-
quinone (TTQ)-dependent amine oxidases methylamine
dehydrogenase (MADH) and aromatic amine dehydroge-
nase (AADH), and also the flavoenzymes trimethylamine
dehydrogenase (TMADH) and heterotetrameric sarcosine
Fig. 1. Schematic representation of the three
key steps involved in enzyme catalysed H-tun-
neling. The TTQ-substrate iminoquinone
adduct and the active site aspartate (Asp428)
are represented as sticks. Protein dynamics
(step 1) facilitate transfer of a proton from the
TTQ-substrate iminoquinone adduct to
Asp428 by quantum tunneling (step 2). In step
3, the proton is ÔtrappedÕ on the aspartate
carboxyl group by subsequent protein
vibrations.
Ó FEBS 2002 Hydrogen tunneling in enzymes (Eur. J. Biochem. 269) 3097
dehydrogenase (TSOX). High resolution crystallographic
structures are available for MADH and TMADH, which has
also opened up complementary computational chemistry

studies of H-transfer in these enzymes.
TTQ-DEPENDENT METHYLAMINE
DEHYDROGENASE AND HETEROTERA-
MERIC SARCOSINE OXIDASE
Our initial studies were focused on TTQ-dependent
MADH. TTQ reduction is concerted with C–H bond
cleavage from an iminoquinone intermediate that forms
rapidly in the reductive half-reaction (Fig. 2). The rate of
reduction of the TTQ cofactor has a large kinetic isotope
effect (KIE ¼ 16.8 ± 0.5 at 298 K), larger than the upper
value expected for reactions described by transition state
theory, and is suggestive of tunneling. Tunneling reactions
are associated with KIE values greater than unity, owing to
the higher probability of proton over deuterium tunneling.
The inflated KIE for MADH prompted us to study the
temperature dependence of this reaction. Reactions that
proceed purely by quantum tunneling are independent of
temperature, and thus the KIE should likewise be inde-
pendent of temperature. Our studies of TTQ reduction in
MADH indicated that the value of the KIE was tempera-
ture independent, but significantly the reaction rate was
strongly dependent on temperature! Our explanation of this
anomalous finding was to couple protein dynamics to the
reaction coordinate (Fig. 1); in other words, temperature
dependent fluctuations of the enzyme-substrate complex are
required to ÔdistortÕ the active site into a geometry that is
compatible with a pure tunneling reaction. These (isotope
independent) fluctuations required to drive the tunneling
reaction give rise to the temperature dependence of the
reaction. The inferences drawn from our experimental data

are congruent with theoretical models of H-tunneling in
enzymes that invoke motion in the protein and/or substrate
as part of the tunneling reaction [24–26]. An earlier study [27]
had observed temperature-independent KIE values ( 2–3)
in steady-state reactions catalysed by serine proteases
performed in deuterated solvent, and these were suggested
to indicate tunneling. Note, however, that the effect of D
2
O
on the reaction dynamics is potentially complicated owing to
the exchange of protons throughout the protein scaffold.
The data were modelled on earlier theoretical treatments of
H-tunneling propounded by Dogonadzhe and coworkers
[27] in which thermal vibrations bring the solvent into a
configuration favourable to tunneling.
The observation of pure tunneling coupled to protein
dynamics represents a major departure from the more
traditional Ôstatic barrierÕ, Ôquantum correctionÕ depictions
of TST that have been used to rationalize H-tunneling
effects in enzymes. Pure tunneling is an attractive means of
promoting a reaction that has a high potential energy
barrier. However, H-tunneling occurs over relatively short
distances (e.g.  0.5 A
˚
). A key feature of the Ôdynamic
barrierÕ model is the role of protein motion in transiently
compressing the width of the potential energy barrier, which
promotes the tunneling reaction. Dynamic fluctuations in
protein structure also prevent transfer from the product to
reactant side of the potential energy surface. Following

tunneling from donor to acceptor atoms, distortion of the
active site geometry away from the optimal configuration
effectively traps the H nucleus on the product side of the
barrier. Pure tunneling (i.e. tunneling without first ascending
the barrier) facilitated by protein dynamics is a radically
different view of enzyme catalysis compared with the
alternative over-the-barrier depictions, but how general is
this phenomenon? Soon after our own findings with
MADH, Klinman and colleagues demonstrated extreme
tunneling coupled to protein motion in a thermophilic
alcohol dehydrogenase [14]. They also made the interesting
finding that the tunneling contribution was less at mesophi-
lic temperatures where the low frequency vibrational modes
of the protein are less excited. Our own work has been
extended in the direction of H-tunneling with other amine
oxidases to demonstrate the general importance of pure
tunneling coupled to enzyme dynamics. We have demon-
strated that the C–H/C–D bond breakage catalysed by
TSOX gives rise to a temperature independent KIE and that
reaction rate is strongly dependent on temperature, consis-
tent with a pure tunneling reaction driven by motion of the
enzyme-substrate complex [28]. TSOX is a flavoprotein, and
our work with this enzyme, together with that of Klinman’s
work with thermophilic alcohol dehydrogenase, was an
early indication that pure tunneling reactions may occur in
different enzyme families. More recent reports have also
made the connection between enzyme dynamics and
tunneling [29] and in at least one case tunneling has been
invoked in the reappraisal of the catalytic mechanism of the
aspartate proteinase family of enzymes [30]. Our own work,

and that of others, on the link between dynamics and
tunneling is inferred from the results of kinetic studies. The
findings are of potential fundamental importance, thus an
independent method of assessing the role of tunneling in
enzymes was sought. Our approach here has been to use
Fig. 2. Reproductive half-reaction of MADH.
(A) A reaction mechanism for the oxidation of
methylamine by MADH. The boxed reaction
step is the step studied computationally. The
base in this reaction corresponds to an aspar-
tate residue (Asp428) in MADH. (B) The
active site of MADH; the QM region is shown
unshaded with link atoms circled.
3098 M. J. Sutcliffe and N. S. Scrutton (Eur. J. Biochem. 269) Ó FEBS 2002
computational chemistry methods, which are described in
the following section.
COMPUTATIONAL STUDIES
OF H-TUNNELING IN METHYLAMINE
DEHYDROGENASE
How can we gain a detailed picture of enzymic H-tunneling
reactions at the atomic level? Computational modelling
methods provide an answer in the form of combined
quantum mechanical/molecular mechanical (QM/MM)
methods. These can simulate the contribution of quantum
tunneling to enzyme-catalysed reactions. In the QM/MM
approach, a small region at the active site is treated quan-
tum mechanically, and is coupled to a simpler molecular
mechanics description of the surrounding protein and
solvent (Fig. 2). This allows the reaction catalysed by the
enzyme to be modelled whilst including the effects of the

protein environment.
H-Tunneling in the oxidative demethylation of methyl-
amine by MADH is a system well suited to study using the
QM/MM approach; a crystal structure of MADH has been
determined, and we have a large body of experimental data,
and the H-tunneling step is rate-limiting. The H-tunneling
step, the step we have studied computationally, involves the
abstraction by the active site base (Asp428) of a proton
(C–H bond breakage) from the iminoquinone (Fig. 2). Our
approach [31] was to determine the potential energy surface
over which the reaction proceeds, and then to calculate the
extent of tunneling by following the reaction over this
surface. Details of the approach are as follows. First, the
structure of the iminoquinone was produced by adding
methylamine to the TTQ in the crystal structure of
Methylophilus methylotrophus MADH (Fig. 2B). The par-
tial charges of the iminoquinone were calculated using
SPARTAN
(Wavefunction Inc., Irvine, CA, USA); the entire
protein (including iminoquinone) was then protonated and
solvated, and energy minimized. Next the QM region was
defined as comprising the sidechain of the active site base
and the sidechain of the catalytically active modified
tryptophan of the TTQ that forms an adduct with
methylamine (Fig. 2B). QM/MM calculations were then
performed to determine the reactant, product and transition
state structures, keeping the link atoms and MM atoms
fixed [32] and using
GAUSSIAN
94 [33] and

AMBER
4.1 [34]. A
reaction path profile was then generated, using
POLYRATE
[35] and the transmission coefficient (extent of tunneling)
calculated.
This computational study suggested that approximately
96% of the reaction proceeds by tunneling through the
barrier, whereas only  4% of the reaction occurs via the
classical over-the-barrier route. Similar results were found in
an independent study [36], where 99% of the reaction was
calculated to proceed by tunneling through the barrier and
1% over-the-barrier. This degree of tunneling with MADH
is significantly larger than that observed in other protein
systems; the next largest is approximately 60% of the
reaction proceeding via tunneling in liver alcohol dehydrog-
enase [37]. Also, a significant tunneling correction is needed
to get closer to the experimental KIE value at 298 K; no
tunneling correction yields a KIE of 6.1, the largest
tunneling correction yields 11.1 and the experimental value
is 16.8 ± 0.5 [13]. Interestingly, in the independent study
mentioned [36], the calculated KIE with tunneling correc-
tion was 18.3, falling to 5.9 when tunneling was omitted.
AROMATIC AMINE DEHYDROGENASE:
SLOWER SUBSTRATES COMPROMISE
REACTION RATES BY DIFFERENT
MEANS
Aromatic amine dehydrogenase (AADH), like MADH, is a
TTQ-dependent amine oxidase. AADH transfers electrons,
derived from the deamination of primary amines (aromatic

amines are generally preferred over simple aliphatic amines),
to azurin [38]. As with MADH, the rate-limiting step in the
reductive half-reaction is abstraction by an active site base
of a proton (C–H bond breakage) from an iminoquinone
intermediate (Fig. 2). We used stopped-flow kinetics to
study C–H bond breakage in three different substrates by
Alcaligenes faecalis AADH [39], the fast substrates dopam-
ine and tryptamine, and the slow substrate benzylamine.
Again,aswithMADHandTSOX,anindicationasto whether
H-transfer occurs classically or by quantum tunneling was
gained by investigating the temperature dependence of the
rates of C–H and C–D bond breakage, and analysing this
using an Eyring plot. This indicated that, whilst the rates of
both C–H and C–D bond breakage are temperature
dependent for all three substrates, the KIEs are temperature
independent. Also, for dopamine and benzylamine (a) there
was no significant difference between the apparent Ôactiva-
tionÕ energy (or to be more precise the enthalpy of
activation) for C–H and C–D bond breakage (C–H bond
breakage in tryptamine was too rapid to observe above
277 K), and (b) the ratio of the Arrhenius-like pre-
exponential factors [13] was comparable with the KIE.
This illustrates that protium and deuterium do not ascend
the potential energy barrier and that vibrationally assisted
quantum tunneling is the mechanism for H- and D-transfer
for all three substrates. Additionally, the enthalpy of
activation for benzylamine (67.1 ± 0.9 kJÆmol
)1
)is
 15 kJÆmol

)1
higher than both that for trypta-
mine (53.5 ± 1.2 kJÆmol
)1
) and that for dopamine
(51.9±1.1kJÆmol
)1
), suggesting that more energy is
required to deform the enzyme-substrate iminoquinone
intermediate with tryptamine.
How does barrier shape change with substrate? The
relative rates of C–H and C–D bond breakage in dopamine,
tryptamine and benzylamine, and the relative KIEs, give
important insight into the shape of the potential energy
barrier separating reactants from products. Although a
fluctuating potential energy barrier is consistent with
our experimental observations, the tunneling event can be
visualized as a two-step process (see, for example, [2,13,21]).
(Fig. 1). The first step is dynamic, and is required to activate
the enzyme–substrate complex by thermal vibration. In
essence, this leads to a crossing over of the potential energy
surfaces of the enzyme-substrate and enzyme–product
complexes. Once this crossing point is populated, the
second step (H-transfer by quantum tunneling) can occur.
Thus, although the enzyme is dynamic, the barrier can be
considered rigid for the lifetime of the tunneling event. Our
conceptual framework therefore uses a rigid barrier depic-
tion of H-tunneling.
To understand the effect of barrier shape on tunneling
rates, the factors that enhance tunneling need to be

Ó FEBS 2002 Hydrogen tunneling in enzymes (Eur. J. Biochem. 269) 3099
considered. These are (a) a small particle mass and (b) a
small area under the potential energy barrier. The barrier
also needs to be sufficiently high to favour tunneling rather
than classical over-the-barrier reactions; high, narrow
barriers are particularly favoured for efficient tunneling.
Based on these criteria, we investigated the compatibility of
different barrier shapes with our experimental rates and
KIEs [39]. These data are inconsistent with both a
rectangular energy barrier and a truncated parabolic energy
barrier (Fig. 3), two commonly used, idealized barrier
shapes. However, the data are consistent with more complex
barrier shapes (Fig. 3), the true nature of which remain to be
established.
COMPROMISING MUTATIONS
AND ENZYMATIC H-TUNNELING
Over the years, the transition state theory has been the
foundation for our quantitative understanding of the effects
of compromising mutations on enzyme catalysis. Altered
enzymic rates have often been modelled as changes in the
stabilities of transition states and ground states. But how do
we rationalize the effects of compromising mutations in
those enzymes known to catalyse C–H bond breakage by
pure tunneling? Is reduced catalytic rate solely attributable
to changes in barrier width and height? Clearly, increases in
width and height will lead to lower tunneling probability.
Mutations within the active site will also likely affect the
thermal fluctuations coupled to the reaction coordinate, and
may lead to differences in (a) the energetics of barrier
compression (as seen for benzylamine and AADH; [39])

and/or (b) extent of barrier compression (i.e. is the active site
more ÔrelaxedÕ in a mutant enzyme compared with the native
enzyme?). In the latter scenario, the equilibrium distance
between donor and acceptor atoms is larger and thus a
greater degree of thermal motion is required to form a
geometry consistent with quantum tunneling. This has been
discussed recently in reactions catalysed by lipoxygenase
and mutants thereof [40]. We have attempted to gain an
early insight into the effects of compromising mutations on
catalysis by studying C–H and C–D bond breakage in
TMADH. In the wild-type enzyme, C–H bond cleavage is
fast (> 1200 s
)1
[41]), and a number of transient kinetic,
computational and mutagenesis studies [42–45] have indi-
cated that the mechanism of flavin reduction in TMADH
involves nucleophilic attack of the substrate lone pair on the
C4a atom of the flavin followed by C–H bond breakage
(Fig. 4). Evidence now favours the transfer of hydrogen
from the substrate methyl to the flavin N5. The rate of C–H
bond breakage is lowered > 100-fold in a H172Q mutant
TMADH [44], and by an additional factor of 4 in a Y169F
mutant TMADH [43]. Both His172 and Tyr169 are located
near the substrate-binding site and the flavin isoalloxazine
ring as part of a His-Tyr-Asp triad [42], and their mutation
will likely lead to altered dynamics within the active site. A
KIE accompanies flavin reduction in the wild-type and
mutant enzymes [44,46]. The very fast flavin reduction rates
of wild-type TMADH with protiated substrate has preven-
ted us from performing detailed temperature-dependence

studies of this reaction. However, we have shown with
H172Q TMADH that the KIE is independent of tempera-
ture over the experimental range (277–297 K) and that
reaction rates are strongly dependent on temperature [46].
This suggests, as with MADH [13] and TSOX [28], that the
reaction proceeds by pure tunneling driven by protein
motion. Comparable studies with Y169F TMADH
revealed a small temperature dependence on the KIE. The
data cannot be understood in terms of the TST. We have
previously suggested this might be due to partial thermal
excitation of substrate (i.e. partial ascent of the barrier)
Fig. 3. Schematic illustrating how changing
barrier width affects tunneling through a variety
of potential energy barriers. Arrows indicate
the paths of H and D nuclei; solid lines denote
the classically allowed regions and dashed
lines the classically disallowed (quantum tun-
neling) regions. The top two barriers (the
rectangular barrier and truncated parabolic
barrier) are commonly used idealized barrier
shapes; these do not agree with the experi-
mentally observed trends in rates and KIEs
[39]. The bottom barrier is a possible barrier
shape that is consistent with the experiment-
ally observed trends in both rates and KIEs
[39]. In this barrier, it is the narrowest part of
the barrier, rather than the whole barrier, that
becomes progressively wider, with the concave
shoulder becoming progressively less pro-
nounced. For a full discussion of the effects of

barrier shape on tunneling rate and KIE val-
ues see reference [39].
3100 M. J. Sutcliffe and N. S. Scrutton (Eur. J. Biochem. 269) Ó FEBS 2002
prior to H-tunneling by a vibrationally assisted mechan-
ism, although a Boltzman analysis suggests a very small
population in anything other than the vibrational ground
state. An alternative explanation might be found in
invoking a more ÔrelaxedÕ active site with larger degree of
vibrational motion required to reach a geometry consis-
tent with tunneling. Our observations with Y169F
TMADH also find parallels with our recent data for the
reaction of MADH with the ÔslowÕ substrate ethanolamine
[39].
FUTURE PERSPECTIVES
Recent studies have established the importance of H-
tunneling in enzyme catalysis. In the last three years, pure
tunneling (i.e. without partial barrier ascent) driven by
protein motion has become established as a mechanism for
the enzymic breakage of C–H bonds; this may be a general
strategy for these energetically difficult reactions. Our
current understanding of how protein motion is coupled
to the reaction coordinate is lacking, and unravelling this
represents a major challenge for the future. As this
understanding is gained, H-tunneling can then be used as
a tool for (a) increasing the catalytic efficiency of enzymes in
the biotechnology industry (by enhancing the coupling of
dynamics to the reaction coordinate), and (b) producing
more effective enzyme inhibitors in the pharmaceutical
industry (by dampening those vibrations coupled to the
reaction coordinate). Such advances have broader implica-

tions, as they will also give insight into the role of dynamics
in driving classical over-the-barrier reactions—these issues
also impact on the tuning of enzyme performance under
extreme conditions (e.g. high/low temperature). Thus, the
key challenge for the future is elucidating the inseparable
relationship between protein dynamics and classical/quan-
tum enzyme mechanisms.
ACKNOWLEDGEMENTS
The work described in this article was funded by the UK Biotechnology
and Biological Sciences Research Council, the Wellcome Trust and the
Lister Institute of Preventive Medicine. N. S. S. is a Lister Institute
Research Professor.
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3102 M. J. Sutcliffe and N. S. Scrutton (Eur. J. Biochem. 269) Ó FEBS 2002