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.. Earlier reviews on enzyme catal
Trang 1M I N I R E V I E W
A new conceptual framework for enzyme catalysis
Hydrogen tunneling coupled to enzyme dynamics in flavoprotein and quinoprotein enzymes
Michael J Sutcliffe1,2and Nigel S Scrutton1
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 slow and fast 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
I N T R O D U C T I O N
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: sjm@le.ac.uk or nss4@le.ac.uk
Abbreviations: TST, transition state theory; TTQ, tryptophan
trypto-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 semiclassical [1] rather than classical 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)
Trang 2only 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
Q U I N O P R O T E I N A N D F L A V O P R O T E I N
A M I N E D E H Y D R O G E N A S E S
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 trapped on the aspartate
carboxyl group by subsequent protein
vibrations.
Trang 3dehydrogenase (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
T T Q - D E P E N D E N T M E T H Y L A M I N E
D E H Y D R O G E N A S E A N D H E T E R O T E R A
-M E R I C S A R C O S I N E O X I D A S E
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 (KIE¼ 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 distort 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 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 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 static barrier, quantum correction 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 dynamic barrier 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.
Trang 4computational chemistry methods, which are described in
the following section
C O M P U T A T I O N A L S T U D I E S
O F H - T U N N E L I N G I N M E T H Y L A M I N E
D E H Y D R O G E N A S E
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 methylotrophusMADH (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 usingGAUSSIAN94 [33] andAMBER4.1 [34] A
reaction path profile was then generated, usingPOLYRATE
[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
A R O M A T I C A M I N E D E H Y D R O G E N A S E :
S L O W E R S U B S T R A T E S C O M P R O M I S E
R E A C T I O N R A T E S B Y D I F F E R E N T
M E A N S
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 faecalisAADH [39], the fast substrates dopam-ine and tryptamdopam-ine, and the slow substrate benzylamdopam-ine 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 activa-tion 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 ± 1.1 kJÆmol)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
Trang 5considered 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
C O M P R O M I S I N G M U T A T I O N S
A N D E N Z Y M A T I C H - T U N N E L I N G
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 relaxed 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].
Trang 6prior 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 relaxed 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 slow substrate ethanolamine
[39]
F U T U R E P E R S P E C T I V E S
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
A C K N O W L E D G E M E N T S
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.
R E F E R E N C E S
1 Bell, R.P (1980) The application of tunnel corrections in chemical kinetics In The Tunnel Effect in Chemistry pp 51–140 Chapman
& Hall, London.
2 Sutcliffe, M.J & Scrutton, N.S (2000) Enzymology takes a quantum leap forward Phil Trans Roy Soc Series A 358, 367–386.
3 Kraut, J (1988) How do enzymes work? Science 242, 533–540.
4 Cha, Y., Murray, C.J & Klinman, J.P (1989) Hydrogen tun-neling in enzyme reactions Science 243, 1325–1330.
5 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, 6597–6605.
6 Jonsson, T., Edmondson, D.E & Klinman, J.P (1994) Hydrogen tunneling in the flavoenzyme monoamine oxidase B Biochemistry
33, 14871–14878.
7 Kohen, A., Jonsson, T & Klinman, J.P (1997) Effects of protein glycosylation on catalysis: changes in hydrogen tunneling and enthalpy of activation in the glucose oxidase reaction Biochem-istry 36, 2603–2611.
8 Alston, W.C., 2nd, Kanska, M & Murray, C.J (1996) Secondary H/T and D/T isotope effects in enzymatic enolization reactions Coupled motion and tunneling in the triosephosphate isomerase reaction Biochemistry 35, 12873–12881.
9 Karsten, W.E., Hwang, C.C & Cook, P.F (1999) Alpha-sec-ondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of 1-malate by NAD-malic enzyme Biochemistry 38, 4398–4402.
10 Whittaker, M.M., Ballou, D.P & Whittaker, J.W (1998) Kinetic isotope effects as probes of the mechanism of galactose oxidase Biochemistry 37, 8426–8436.
11 Nesheim, J.C & Lipscomb, J.D (1996) Large kinetic isotope effects in methane oxidation catalyzed by methane mono-oxygenase: evidence for C-H bond cleavage in a reaction cycle intermediate Biochemistry 35, 10240–10247.
12 Jonsson, T., Glickman, M.H., Sun, S & Klinman, J.P (1996) Experimental evidence for extensive tunneling of hydrogen in the lipoxygenase reaction: implications for enzyme catalysis J Am Chem Soc 118, 10319–10320.
13 Basran, J., Sutcliffe, M.J & Scrutton, N.S (1999) Enzymatic H-transfer requires vibration-driven extreme tunneling Bio-chemistry 38, 3218–3222.
14 Kohen, A., Cannio, R., Bartolucci, S & Klinman, J.P (1999) Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase Nature 399, 496–499.
15 Hund, F (1927) Interpretation of molecular spectra III Remarks
on the oscillation and rotation spectra of molecules with more than two nuclei Z Phys 43, 805–826.
16 Stoneham, A (1990) Quantum diffusion in solids J Chem Soc Faraday Trans 86, 1215–1220.
17 DeVault, D (1980) Quantum mechanical tunnelling in biological systems Q Rev Biophys 13, 387–564.
18 Marcus, R.A., & Sutin, N (1985) Electron transfers in chemistry and biology Biochim Biophys Acta 811, 265–316.
19 Neumann, M., Brogham, D.F., McGloin, C.J., Johnson, M.R., Horsewill, A.J & Tromsdorff, H.P (1998) Proton tunneling in benzoic acid crystals at intermediate temperatures: nuclear mag-netic resonance and neutron scattering studies J Chem Phys.
109, 7300–7311.
Fig 4 Our proposed mechanism for the oxidation of trimethylamine by
TMADH, which is based on mechanistic studies [45,46] and by analogy
with the work of Edmondson on monoamine oxidase [47] It is important
to note that steps 1 and 2 of this scheme are likely to be concerted; the
second species shown is therefore not intended to represent a true
enzyme intermediate Enz, enzyme.
Trang 720 Horsewill, A.J., Jones, N.H & Caciuffo, R (2001) Evidence for
coherent proton tunneling in a hydrogen bond network Science
291, 100–103.
21 Scrutton, N.S., Basran, J & Sutcliffe, M.J (1999) New insights
into enzyme catalysis: ground state tunnelling driven by protein
dynamics Eur J Biochem 264, 666–671.
22 Kohen, A & Klinman, J.P (1999) Hydrogen tunneling in
biol-ogy Chem Biol 6, R191–R198.
23 Sutcliffe, M.J & Scrutton, N.S (2000) Enzyme catalysis:
over-the-barrier or through-the over-the-barrier Trends Biochem Sci 25, 405–408.
24 Bruno, W.J & Bialek, W (1992) Vibrationally enhanced
tun-neling as a mechanism for enzymatic hydrogen transfer Biophys.
J 63, 689–699.
25 Antoniou, D & Schwartz, S.D (1997) Large kinetic isotope
effects in enzymatic proton transfer and the role of substrate
oscillations Proc Natl Acad Sci USA 94, 12360–12365.
26 Kuznetsov, A.M & Ulstrup, J (1999) Proton and hydrogen atom
tunnelling in hydrolytic and redox enzyme catalysis Can J.
Chem 77, 1085–1096.
27 Krishtalik, L.I (1986) Charge transfer reactions in electrochemical
and chemical processes pp 244–298 Consultants Bureau, New
York.
28 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
Bio-chemistry 39, 1189–1198.
29 Northrop, D.B & Cho, Y.K (2000) Effect of pressure on
deu-terium isotope effects of yeast alcohol dehydrogenase: evidence
for mechanical models of catalysis Biochemistry 39, 2406–2412.
30 Northrop, D.B (2001) Follow the protons: a low barrier
hydro-gen bond unifies the mechanisms of the aspartic proteases Acc
Chem Res 34, 790–797.
31 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 dehydrogenase J Am Chem Soc 123,
8604–8605.
32 Hall, R.J., Hindle, S.A., Burton, N.A & Hillier, I.H.J (2000)
Aspects of hybrid QM/MM calculations: The treatment of
the QM/MM interface region and geometry optimization
with an application to chorismate mutase J Comput Chem 21,
1433–1441.
33 Frisch, M.J., Trucks, G.W., Schlegel, H.B., Gill, P.M.W.,
Johnson, B.G., Robb, M.A., Cheeseman, J.R.A.K.T., Petersson,
G.A., Montgomery, J.A., Raghavachari, K et al (1995)
Gaussian94 Gaussian Inc, Pittsburgh PA.
34 Pearlman, D.A., Case, D.A., Caldwell, J.W., Ross, W.S.,
Cheatham, T.E., Ferguson, D.M., Seibel, G.L., Singh, U.C.,
Weiner, P.K & Kollman, P.A (1995) AMBER University of
California, San Francisco.
35 Corchado, J.C., Chuang, Y.Y., Fast, P.L., Villa, W.P.,
Hu, W.P., Liu, Y.P., Lynch, G.C., Nguyen, K.A., Jackels, C.F.,
Gu, M.Z et al (2000) POLYRATE University of Minnesota, Minneapolis.
36 Alhambra, C., Sanchez, M.L., Corchado, J.C., Gao, J & Truhlar, D.G (2001) Quantum mechanical tunneling in methyl-amine dehydrogenase Chem Phys Lett 347, 512–518.
37 Alhambra, C., Corchado, J., Sanchez, M., Gao, J & Truhlar, D (2000) Quantum dynamics of hydride transfer in enzyme cata-lysis J Am Chem Soc 122, 8197–8203.
38 Hyun, Y.-L & Davidson, V.L (1995) Electron-transfer reactions between aromatic amine dehydrogenase and azurin Biochemistry
34, 12249–12254.
39 Basran, J., Patel, S., Sutcliffe, M.J & Scrutton, N.S (2001) Importance of barrier shape in enzyme-catalyzed reactions Vibrationally assisted hydrogen tunneling in tryptophan trypto-phylquinone-dependent amine dehydrogenases J Biol Chem.
276, 6234–6242.
40 Knapp, M.J., Rickert, K & Klinman, J.P (2002) Temperature-dependent isotope effects in soybean lipoxygenase-1: correlating hydrogen tunneling with protein dynamics J Am Chem Soc.
124, 3865–3874.
41 Jang, M.-H., Basran, J., Scrutton, N.S & Hille, R (1999) The reaction of trimethylamine dehydrogenase with trimethylamine.
J Biol Chem 274, 13147–13154.
42 Trickey, P., Basran, J., Lian, L.-Y., Chen, Z.-W., Barton, J.D., Sutcliffe, M.J., Scrutton, N.S & Mathews, F.S (2000) Struc-tural and biochemical characterization of recombinant wild-type and a C30A mutant of trimethylamine dehydrogenase from Methylophilus methylotrophus (sp W3A1) Biochemistry 39, 7678–7688.
43 Basran, J., Sutcliffe, M.J., Hille, R & Scrutton, N.S (1999) The role of Tyr 169 of trimethylamine dehydrogenase in substrate oxidation and magnetic interaction between FMN cofactor and the 4Fe/4S center J Biol Chem 274, 13155–13161.
44 Basran, J., Sutcliffe, M.J., Hille, R & Scrutton, N.S (1999) Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme– substrate complex Biochem J 341, 307–314.
45 Basran, J., Sutcliffe, M.J & Scrutton, N.S (2001) Optimizing the Michaelis complex of trimethylamine dehydrogenase: identifica-tion of interacidentifica-tions that perturb the ionizaidentifica-tion of substrate and facilitate catalysis with trimethylamine base J Biol Chem 276, 42887–42892.
46 Basran, J., Sutcliffe, M.J & Scrutton, N.S (2001) Deuterium isotope effects during C-H bond cleavage by trimethylamine dehydrogenase: implications for mechanism and vibrationally assisted H-tunneling in wild-type and mutant enzymes J Biol Chem 276, 24581–24587.
47 Miller, J.R & Edmondson, D.E (1999) Structure–activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A Biochemistry 38, 13670–13683.