Deuterium Isotope Effects during Carbon-Hydrogen Bond
Cleavage by Trimethylamine Dehydrogenase
IMPLICATIONS FOR MECHANISM AND VIBRATIONALLY ASSISTED HYDROGEN
TUNNELING IN WILD-TYPE AND MUTANT ENZYMES*
Jaswir
Basran
,
Michael J.
Sutcliffe§, and
Nigel S.
Scrutton
¶
From the Departments of
Biochemistry and
§ Chemistry, University of Leicester, University Road,
Leicester LE1 7RH, United Kingdom
Received for publication, February 7, 2001, and in revised form, March 29, 2001
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ABSTRACT |
His-172 and Tyr-169 are components of
a triad in the active site of trimethylamine dehydrogenase (TMADH)
comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies
with trimethylamine as substrate have indicated that mutation of
His-172 to Gln reduces the limiting rate constant for flavin reduction
~10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton,
N. S. (1999) Biochem. J. 341, 307-314). A kinetic
isotope effect (KIE = kH/kD)
accompanies flavin reduction by H172Q TMADH, the magnitude of which
varies significantly with solution pH. With trimethylamine, flavin
reduction by H172Q TMADH is controlled by a single macroscopic ionization (pKa = 6.8 ± 0.1). This ionization
is perturbed (pKa = 7.4 ± 0.1) in reactions
with perdeuterated trimethylamine and is responsible for the apparent
variation in the KIE with solution pH. At pH 9.5, where the functional
group controlling flavin reduction is fully ionized, the KIE is
independent of temperature in the range 277-297 K, consistent with
vibrationally assisted hydrogen tunneling during breakage of the
substrate C-H bond. Y169F TMADH is ~4-fold more compromised than
H172Q TMADH for hydrogen transfer, which occurs non-classically.
Studies with Y169F TMADH suggest partial thermal excitation of
substrate prior to hydrogen tunneling by a vibrationally assisted
mechanism. Our studies illustrate the varied effects of compromising
mutations on tunneling regimes in enzyme molecules.
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INTRODUCTION |
Quantum tunneling effects in enzymatic hydrogen transfer reactions
are being observed in a growing number of enzyme systems (1-13). Some
have been modeled using the classical formulations of transition state
theory (14), incorporating a tunneling correction that accounts for
tunneling below the saddle point of the potential energy surface (15).
Recently, however, experimental observations have suggested that
tunneling in some enzymes is vibrationally assisted (6, 7, 12, 13, 16,
17), and the potential energy barrier to the reaction should thus be
seen as fluctuating rather than static. The fluctuating potential
energy surface derives from the thermal vibrations of the protein,
which drive the hydrogen tunneling reaction (for recent reviews, see
Refs. 18-21). Theoretical treatments of vibrationally assisted
tunneling reactions have been presented, with the dynamic component
reflecting substrate vibrations, protein vibrations, or both. Antoniou
and Schwartz (22) and Borgis and Hynes (23) have provided theoretical
descriptions of hydrogen tunneling facilitated by vibrations in the
substrate. Bruno and Bialek (24) proposed that hydrogen tunneling is
coupled to protein vibrations, but their treatment is specific for
hydrogen transfer from the substrate vibrational ground state.
Kuznetsov and Ulstrup (25) have provided a more general
treatment of enzymatic hydrogen tunneling involving coupling between
the tunneling modes and the environment. In the context of
vibrationally assisted tunneling mechanisms, major experimental
challenges are now presented. These include correlation of (i) protein
flexibility with the degree of tunneling (26), (ii) barrier shapes and
tunneling regimes with compromised rates of transfer with "slow"
substrates and in mutant enzymes (12), and (iii) experimental data with computational studies of the quantum dynamics of hydrogen transfer (27).
In this work, we have extended our studies of hydrogen tunneling to the
iron-sulfur flavoenzyme trimethylamine dehydrogenase (TMADH1; EC 1.5.99.7), which
catalyzes the oxidative demethylation of trimethylamine to
dimethylamine and formaldehyde (28): (CH3)3N + H2O
(CH3)2NH + CH2O + 2H+ + 2e
. The reaction is initiated by the
cleavage of a C-H bond in one of the substrate methyl groups, and the
reducing equivalents are transferred to a 6-S-cysteinyl-FMN
in the enzyme active site (29). In stopped-flow studies, a large
primary kinetic isotope effect (KIE) accompanies flavin reduction in
native TMADH (30). Following bond cleavage, electrons are transferred
subsequently from the dihydroflavin in two single-electron transfer
events to electron-transferring flavoprotein via the [4Fe-4S] center
of TMADH (31-36). The reductive half-reaction of TMADH is resolved
into three kinetic phases (37, 38): a fast phase representing
two-electron reduction of the flavin, an intermediate phase that
reports on intramolecular electron transfer from dihydroflavin to the
[4Fe-4S] center to generate flavin semiquinone and reduced
iron-sulfur center, and a slow phase that involves formation of a
spin-interacting state of the enzyme and product release (32-34, 38).
Flavin reduction in native TMADH is fast, prohibiting detailed studies
of flavin reduction at elevated temperatures (38). In this study, we
present the first analysis of vibrationally assisted hydrogen tunneling
in two mutant forms of TMADH that have compromised rates of flavin reduction and are thus accessible to study by stopped-flow methods.
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EXPERIMENTAL PROCEDURES |
Trimethyl-d9-amine HCl (99.7%
deuterium; chemical purity > 99% as determined by high
performance liquid chromatography, NMR, and gas chromatography)
was from CK Gas Products Ltd. Titration curves for protiated and
deuterated trimethylamine HCl were generated using 20 mM
solutions (5 ml) of each tertiary amine at 19.5 °C. The pH of the
solution was measured using a Mettler Toledo Inlab 410 electrode after
sequential addition of sodium hydroxide from a concentrated stock (100 mM). All chemicals were of analytical grade where possible.
Wild-type TMADH was purified from Methylophilus methylotrophus (sp. W3A1) as
described by Steenkamp and Mallinson (28), but with the modifications
of Wilson et al. (39). The isolation, expression, and
purification of H172Q and Y169F TMADH have been described previously
(30, 40).
Stopped-flow experiments with TMADH were performed using an Applied
Photophysics SX.18MV stopped-flow spectrophotometer as described
elsewhere (30, 38, 40). For single-wavelength studies, data collected
at 443 nm were analyzed using nonlinear least-squares regression
analysis on an Acorn RISC PC using Spectrakinetics software (Applied
Photophysics). Experiments were performed by mixing TMADH contained in
buffer of the desired pH with an equal volume of substrate contained in
the same buffer at the desired concentration. The concentration of
substrate was always at least 10-fold greater than that of TMADH,
thereby ensuring pseudo first-order reaction conditions. Transients at
443 nm were analyzed as monophasic decreases in absorption, consistent
with previous studies (38). For each substrate concentration, at least
five replica measurements were collected and averaged. The error for
individual rates measured by fitting to a single transient was <0.5%
in all cases, and the error for the rate fitted to averaged transients
was <0.4%. Substrate-reduced TMADH is quite stable to reoxidation by
molecular oxygen (half-life of ~50 min (39)), negating the need to
use anaerobic conditions in the kinetic experiments.
Reaction scheme modeling for flavin reduction by trimethylamine has
been described in detail elsewhere (38). Flavin reduction involves the
reversible formation of a Michaelis complex, followed by the C-H bond
cleavage/flavin reduction step (Equation 1).
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(Eq. 1)
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The observed rate of flavin reduction
(vobs) is dependent on substrate concentration,
as described by the general hyperbolic expression in Equation 2
(41),
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(Eq. 2)
|
where K is a constant and equal to
(k2 + k3)/k1. As discussed
elsewhere (38), in reactions of TMADH with trimethylamine, the reaction
is modeled such that k2
k3, and K thus approximates to the
enzyme-substrate dissociation constant, Kd. For multiple-wavelength stopped-flow studies, the reaction was monitored using an Applied Photophysics photodiode array detector and operated using XSCAN software. Analysis of photodiode array data was carried out
using PROKIN software (Applied Photophysics). TMADH is stable over the
temperature range used in the stopped-flow studies. This is evident
since the total absorption change for 6-S-cysteinyl-FMN reduction at all temperatures remains constant and is identical to that
observed in spectrophotometric titrations of the enzyme with its
substrate. Enzyme and substrate were equilibrated for 10 min in the
stopped-flow apparatus at the appropriate temperature prior to mixing
and the acquisition of stopped-flow data. The optimal time for
equilibration was determined empirically. Temperature control was
achieved using a thermostatic circulating water bath, and the
temperature was monitored directly in the stopped-flow apparatus using
a semiconductor sensor (Model LM35CZ, National Semiconductor). In
studies of the temperature dependence of bond cleavage, all substrates
were used at saturating concentrations (see "Results and
Discussion"). Studies of the concentration dependence of bond
cleavage at 5 and 35 °C indicated that the value of K in
Equation 2 was not substantially perturbed upon changing temperature. These control experiments thus ensured that substrate was saturating at
all the temperatures investigated in the temperature dependence studies.
Studies of the pH dependence of flavin reduction were conducted at
5 °C. The buffers used were 100 mM potassium phosphate (pH 6.0-7.5), 100 mM sodium pyrophosphate (pH 8.0-8.5),
100 mM sodium borate (pH 9.0-10.0), and 100 mM
glycine/NaCl (pH 10.5 and 11.0). Previous studies have established that
increased ionic strength upon addition of substrate (trimethylammonium
chloride) over the range employed in this study does not affect the
rate of flavin reduction (30). pH profiles for the kinetic parameters k3 and K were constructed, and the
data were fitted to Equations 3 and 4, respectively, to obtain the
relevant pKa values,
|
(Eq. 3)
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(Eq. 4)
|
where EH and E are the catalytic
activities of the protonated and unprotonated forms of the ionization
group, respectively; and Tmax is the theoretical
maximal value of k3/K.
 |
RESULTS AND DISCUSSION |
pH Dependence of Flavin Reduction with Trimethylamine and
Perdeuterated Trimethylamine--
Previously, we analyzed the pH
dependence of flavin reduction by trimethylamine with H172Q TMADH at
5 °C (30). This study revealed the presence of a single kinetically
influential ionization (pKa = 6.8 ± 0.1) in
the enzyme-substrate complex. Similar studies with native TMADH are
compromised owing to the very fast flavin reduction rates (>1200
s
1 (38)). We have, however, analyzed the pH
dependence of the native enzyme using perdeuterated trimethylamine (30)
since the primary KIE (~7) upon C-H bond cleavage/flavin reduction
allowed us to analyze the full kinetic transient on the stopped-flow
time scale. In native TMADH, there are two kinetically influential ionizations in the enzyme-substrate complex, and their origin is of
interest from a mechanistic viewpoint. The upper ionization is
attributed to the side chain of His-172 (since it is lost in H172Q
TMADH (30)); the lower ionization is as yet unassigned, but it most
likely represents the deprotonation of the trimethylammonium cation
((CH3)3NH+) to form trimethylamine
base. Deprotonation of the substrate cation is consistent with a
mechanism of flavin reduction in which the substrate nitrogen lone pair
undergoes nucleophilic addition at the flavin C-4a atom (Fig. 2) (42).
This mechanism is analogous to that proposed recently for monoamine
oxidase A on the basis of structure-activity relationships with
para-substituted benzylamine analogs (43). All ionizable
groups in the active site of TMADH (His-172 (30), Tyr-169 (40),
Tyr-174,2 and
Tyr-60 2) have been mutated without loss of this
ionization, thus supporting assignment to the deprotonation of
substrate itself in the enzyme-substrate complex.
In this study, we have performed an analysis of the pH dependence of
flavin reduction in H172Q TMADH using perdeuterated substrate (Fig.
1 and Table
I). The plot of
k3 versus solution pH retains the
features we reported previously for H172Q TMADH in reactions with
trimethylamine as substrate (shown also in Fig. 1A), but the
macroscopic pKa describing the kinetically
influential ionization in the enzyme-substrate complex is shifted from
6.8 ± 0.1 (trimethylamine) to 7.4 ± 0.1 (perdeuterated
trimethylamine). The isotope dependence of this pKa
value is of interest. Although, in the literature, this
pKa value has not been attributed formally to a
group in the enzyme-substrate complex, as discussed above, the balance
of evidence suggests that it represents the deprotonation of the
substrate molecule itself
((CH3)3NH+
(CH3)3N) on moving from low to high pH. It is
anticipated that perdeuteration of the substrate will affect this
ionization (and see below). (i) the shorter C-D bond results in a
larger charge density, and thus, it is electron-supplying
(i.e. stabilizing the N-H bond) relative to C-H; and (ii)
the perdeuterated substrate has a greater reduced mass for the
(CD3)3N-H stretching vibration and therefore
lies lower in the asymmetric potential energy well. Thus, the
(CH3)3N-H bond dissociates more readily than
the (CD3)3N-H bond, accounting for the
elevated macroscopic pKa value seen with
perdeuterated substrate in our kinetic studies.

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Fig. 1.
pH dependence of flavin reduction in H172Q
TMADH with protiated and perdeuterated trimethylamine. Individual
parameters determined from curve fitting using Equation 2 to plots of
observed rate (vobs) against substrate
concentration are shown in Table I. A, plot of limiting
flavin reduction rate constant (k3) as a
function of solution pH. , protiated trimethylamine
(pKa = 6.8 ± 0.1); , perdeuterated
trimethylamine (pKa = 7.4 ± 0.1).
B, plot of k3/K as a
function of solution pH. , protiated trimethylamine
(pKa1 = 10.2 ± 0.2 and
pKa2 = 10.0 ± 0.2); ,
perdeuterated trimethylamine (pKa1 = 11.3 ± 0.2 and pKa2 = 9.7 ± 0.2). C, histogram illustrating the observed KIE as a
function of solution pH. In A and B, errors
associated with the pKa values are those determined
from curve fitting.
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Table I
Limiting rate constants for flavin reduction and enzyme-substrate
dissociation constants for the reaction of H172Q TMADH with
trimethylamine and perdeuterated trimethylamine
Experimental errors are those from curve fitting to Equation 2.
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The KIE for C-H bond breakage in H172Q TMADH across the pH range is
illustrated in Fig. 1C. Inflated KIE values (>7) are seen in the low pH range. These result from the
isotope-dependent, kinetically influential ionization in
the enzyme-substrate complex (Fig. 1A). Scheme
1 summarizes the prototropic control of
flavin reduction in the enzyme-substrate complex. In Scheme 1, it is assumed that the rate of breakdown of the ES complex to
EP is slow relative to the dissociation steps, so the
dissociation steps remain in thermodynamic equilibrium. Clearly, as a
result of the elevated pKa value seen with
perdeuterated substrate, there is a greater concentration of the
ESH+ (unreactive) complex (i.e. the
lower branch of Scheme 1). The effect of this partitioning between
ES and ESH+ forms of the
enzyme-substrate complex is that the observed KIE is inflated over the
intrinsic value that would be realized if the concentration of the
ES species were equivalent (at a given pH value) for both
perdeuterated and protiated substrate. Only at pH values of 9.5 and
above (where the group identified in the plot of
k3 versus pH is fully ionized and
where the rate of flavin reduction is maximal) is the intrinsic isotope
effect realized, owing to the enzyme being in the ES form
for both protiated and perdeuterated substrate. In this regime, the KIE
approaches a constant value of ~4.5 (Fig. 1C and Table
I).
Fig. 1B illustrates the plot of
k3/K versus pH for both protiated and
perdeuterated substrate. Again, the general features of the plot are
retained for both substrates, indicating that two ionizations in the
free enzyme and/or free substrate are kinetically influential. The
assignments of the ionizations in the
k3/K plot are unknown. Previously, we
hypothesized that the acid limb of the bell-shaped curve might reflect
ionization of Tyr-60, which contacts the substrate in the
enzyme-substrate complex (38). However, studies with the Y60F mutant
now indicate this is not the case since the bell-shaped dependence of
the k3/K plot was still observed in
this mutant.2 We also conjectured (again without
formal demonstration) that the alkaline limb might represent the
deprotonation of trimethylammonium cation when not complexed to the
enzyme. This suggestion was made since it was initially thought that
the substrate cation was the catalytically active species, whereas
based on the isotope evidence presented above, computational studies
(42), and the mechanism of inactivation of TMADH with phenylhydrazine
(42, 44) and by analogy with the mechanism proposed for monoamine
oxidase A (43), it now seems likely that unprotonated substrate is the active species. The pKa value for deprotonation of
trimethylammonium cation is ~9.8, which is close to the values on
both the acid and alkaline limbs of the curve generated from fitting to
the plot of k3/K versus
pH. It thus seems appropriate to reevaluate the assignments of the two
ionizations in the plot of k3/K
versus pH, especially in the light of the proposed mechanism
in which a complex between trimethylamine base and enzyme is the
catalytically active species (Fig. 2). In
such a scenario, it clearly makes more sense for the enzyme to have
evolved a higher affinity for trimethylamine base rather than its
protonated cation. Improved binding (in terms of Kd)
is observed on moving from the acid to alkaline region of the lower
(acid) limb of the k3/K
versus pH plot, and this ionization therefore most likely
represents deprotonation of the substrate cation. The upper, alkaline
limb remains unassigned, but as discussed above, extensive mutagenesis studies have failed to identify a chemical group in the enzyme active
site responsible for this ionization; a possible candidate is the
N3H position of the flavin isoalloxazine ring
(pKa ~ 10). With H172Q TMADH, the enzyme-substrate
dissociation constants measured across the pH range are modestly
elevated with perdeuterated trimethylamine relative to protiated
substrate (Table I). Again, as discussed above, these small differences
are likely related to the different chemical properties of the
(CD3)3N-H and
(CH3)3N-H bonds, which will affect the
pKa for the ionization of the cationic form of the
substrate (pKa for
(CH3)3N-H = 9.8 and
pKa for (CD3)3N-H = 10.1). This will reduce the concentration of the unprotonated form of
perdeuterated substrate compared with unprotonated protiated substrate
at a given pH value, giving rise to an apparent increase in the
enzyme-substrate dissociation constant (since substrate concentration
is measured as the sum of the cationic and free base forms). The
elevated pKa for deprotonation of perdeuterated
substrate is consistent with the observed shift in the acid limb of the
k3/K versus pH plot relative to the curve for protiated substrate (Fig. 1B).
This strengthens the assertion that the acid limb is attributable to the deprotonation of substrate, which is consistent with the mechanism shown in Fig. 2.

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Fig. 2.
Possible mechanism for the oxidation of
trimethylamine by TMADH. The mechanism is analogous to that
proposed recently for monoamine oxidase A (43) and is consistent with
mutagenesis/kinetic (30, 40, 47) and computational (42) studies of
flavin reduction in TMADH, although unequivocal evidence is lacking.
Other potential mechanisms for flavin reduction in TMADH have been
discussed recently and shown to be inconsistent with the available
experimental evidence (38). Enz, enzyme.
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Vibrationally Assisted Hydrogen Tunneling in TMADH--
Studies of
the temperature dependence of the KIE can be used to indicate whether
hydrogen transfer occurs classically or by quantum tunneling (6). The
temperature dependence of the rates of C-H and C-D bond cleavage is
described by the unimolecular rate (Equation 5),
|
(Eq. 5)
|
where kB and h are the Boltzmann
and Planck constants, respectively. Temperature-dependent
rate data can be plotted conveniently using the following form of the
Eyring equation (Equation 6).
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(Eq. 6)
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The enthalpy of activation (
H
) is
calculated from the slope of the plot;
S
is calculated by extrapolation to the ordinate axis, and
G
is then calculated directly from
Equation 5. The use of Equation 6 in plotting the temperature
dependence of a unimolecular reaction is preferred over the use of the
classical Arrhenius plot. This arises because the Arrhenius equation is
curved (although it appears linear in the accessible temperature range)
and asymptotically approaches infinity at high temperatures. Use of the
Arrhenius plot has led to the development of criteria to indicate
tunneling based on the values for 
Ea (the
difference in the Arrhenius activation energies for protium
versus deuterium transfer) and the
AH:AD ratio (calculated from the intercepts of
the Arrhenius plot for protium and deuterium substrates) (4). The
corresponding parameters calculated from the slopes and intercepts of
plots using Equation 6 are 
H
and
A'H:A'D (the primes are used to distinguish
this ratio from the AH:AD ratio calculated from
the Arrhenius plot). A'H and A'D are the
intercept values, and the value of the ratio of A'H to
A'D is a measure of the extent of tunneling. For
semiclassical reactions, A'H:A'D is unity; for
reactions that occur by vibrationally assisted tunneling from the
substrate vibrational ground state, the value of the
A'H:A'D ratio is equal to the value of the
intrinsic primary KIE (6).
The temperature dependence of flavin reduction in H172Q TMADH with
protiated and perdeuterated trimethylamine is illustrated in Fig.
3, and parameters are given in Table
II. Reactions were performed using 10 mM substrate, thus ensuring saturation of the enzyme
(KH = 0.19 mM and
KD = 0.44 mM) (Table I), and at pH
9.5 to facilitate complete formation of the catalytic ES
(and not ESH+) complex. A striking feature of
the plot shown in Fig. 3 is the temperature independence of the KIE
(
H
= 0.5 ± 5.2 kJ
mol
1) over the temperature range
investigated. The value of the A'H:A'D ratio
(7.8 ± 1.0) is similar to the KIE (4.6 ± 0.4), which is suggestive of hydrogen and deuterium tunneling from the substrate ground state, but reaction rates are clearly still dependent on temperature (
H
~ 41 kJ
mol
1), consistent with a need to assist the
tunneling reaction through vibrational excitation of the protein. The
behavior is similar to observations we have published previously in our
studies of C-H and C-D bond cleavage in tryptophan tryptophylquinone
(TTQ)-dependent amine dehydrogenases (6, 12) and in
a heterotetrameric sarcosine oxidase (7) and also to that seen by
others in thermophilic alcohol dehydrogenase (16) and acyl-CoA
desaturase (13); these findings have also been rationalized in terms of
vibrationally assisted tunneling of hydrogen and deuterium. The
transfer of the hydrogen and deuterium nuclei (from the substrate
vibrational ground state) by a vibrationally assisted tunneling
mechanism in a compromised mutant (H172Q) of TMADH has clear
implications for hydrogen and deuterium transfer in native enzyme.
Although the temperature dependence of the KIE cannot be measured in
native TMADH at pH 9.5 owing to the very fast limiting rates (>1200
s
1 (38)) of hydrogen transfer, we have been
able to perform a study of the temperature dependence of deuterium
transfer (Fig. 3B). Analysis of these data indicates that
H
D (45.7 ± 0.9 kJ
mol
1) in native TMADH is similar to
H
H (41.2 ± 2.6 kJ
mol
1) and
H
D (41.7 ± 2.6 kJ
mol
1) for H172Q TMADH, indicating a similar
energetic requirement to distort the protein geometry into one that is
compatible with hydrogen and deuterium tunneling. The compromised
reaction rates in the H172Q mutant relative to native enzyme are
therefore likely attributable to a widening of the potential energy
barrier for C-H/C-D bond cleavage in the optimally configured
enzyme-substrate complex. These findings are qualitatively similar to
our recent studies with TTQ-dependent aromatic amine
dehydrogenase with a fast (tryptamine) and slow (dopamine) substrate
(12); in both cases, hydrogen transfer was inferred to be from the
substrate vibrational ground state, and the compromised rates observed
with dopamine were attributed to a broadening of the potential energy barrier in the optimal (thermally activated) geometry of the
enzyme-substrate complex.

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Fig. 3.
Temperature dependence and KIE data for H172Q
and native TMADH at pH 9.5. A, temperature dependence
plots for H172Q TMADH with trimethylamine ( ) and perdeuterated
trimethylamine ( ). Inset, plot of ln(KIE)
versus 1/T. Substrate concentration was 10 mM. B, temperature dependence plot for native
TMADH with perdeuterated trimethylamine. Parameters evaluated by
fitting to Equation 6 are given in Table II. Substrate concentration
was 5 mM.
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Table II
Calculated parameters from studies of the temperature dependence of
C-H and C-D bond cleavage in native and mutant TMADH enzymes
Values were determined by fitting Equation 6 to data shown in Figs. 3
and 4. Errors were determined from curve fitting.
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We have also investigated the temperature dependence of the KIE in
Y169F TMADH. This mutant is ~4-fold more compromised in its limiting
rate of flavin reduction relative to the H172Q mutant, and analysis of
the temperature dependence of C-H and C-D bond breakage was performed
to probe the effect of further reduction in flavin reduction rate on
the hydrogen and deuterium tunneling mechanism. Our previous studies
with Y169F TMADH indicated that both ionizations seen with native TMADH
(perdeuterated substrate) in the k3
versus pH plot are retained in the mutant TMADH (40). The
more acidic ionization has a similar pKa value in both enzymes (~6.5), whereas the more alkaline ionization is
perturbed to a higher pH value in Y169F TMADH (pKa = 9.5 (40)) relative to native TMADH (pKa = 8.4 with
perdeuterated substrate (30)). This perturbation has been attributed to
the effects of removing a hydrogen bond interaction from the phenolic hydroxy group of Tyr-169 to the imidazole side chain of His-172 in
Y169F TMADH, resulting in displacement of the pKa for the imidazole side chain to a higher pH value (40). A consequence of the perturbed ionization of His-172 in Y169F TMADH is the need to
perform temperature dependence studies of C-H and C-D bond cleavage
at a higher pH value (pH 11) than that used for corresponding studies
with H172Q TMADH (pH 9.5) to ensure full formation of the catalytically
active enzyme species (ES). Studies with Y169F TMADH were
therefore performed at pH 11 with 10 mM substrate
(Kd = 125 µM (40)). Corresponding
studies with native TMADH (perdeuterated substrate) were also performed
to enable comparison with data collected at pH 9.5 for native and H172Q
TMADH.
Our analysis of Y169F TMADH indicates that, unlike with H172Q TMADH,
the KIE is not independent of temperature
(
H
= 3.02 ± 2.5 kJ
mol
1) (Fig. 4).
Also, the A'H:A'D ratio (2.5 ± 0.2)
calculated from the intercept of the temperature dependence plot is
elevated over that expected for semiclassical transfer
(A'H:A'D = unity). The enthalpic contributions
are similar to those seen with H172Q TMADH at pH 9.5. The data are
consistent with a quantum tunneling mechanism, but differential effects
are seen with the different isotopes. The slightly larger enthalpic
contribution with perdeuterated substrate suggests that this substrate
requires thermal activation to partially ascend the potential energy
barrier (thus minimizing barrier width) prior to tunneling. This
is supported by the finding that transfer rates with Y169F < H172Q < native TMADH, suggesting that the Y169F mutant presents
the widest barrier to hydrogen transfer.

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Fig. 4.
Temperature dependence and KIE data for Y169F
and native TMADH at pH 11.0. A, temperature dependence
plots for Y169F TMADH with trimethylamine ( ) and perdeuterated
trimethylamine ( ). Inset, plot of ln(KIE)
versus 1/T. Substrate concentration was 10 mM. B, temperature dependence plot for native
TMADH with perdeuterated trimethylamine. Parameters evaluated by
fitting to Equation 6 are given in Table II. Substrate concentration
was 5 mM.
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The enthalpic contribution to deuterium transfer (and by inference,
hydrogen transfer) in native TMADH (which is assumed to transfer via a
vibrationally assisted mechanism from the substrate ground state by
inference from the H172Q TMADH data) is similar at pH 11 (48.2 ± 0.7 kJ mol
1) and pH 9.5 (45.7 ± 0.9 kJ
mol
1). It is worth noting that at any single
pH value, the enthalpic contribution for native TMADH is larger than
that for the mutant enzymes by ~3-4 kJ
mol
1 (Table II). The barrier in native TMADH
is therefore more rigid than the corresponding barriers in the mutant
enzymes. This may reflect the removal of the side chain hydrogen bond
between Tyr-169 and His-172 in each mutant enzyme, making the active
site less restrained and thus more readily deformed.
Our analysis of H172Q and Y169F TMADH establishes a link between
protein structure and tunneling characteristics for a vibrationally assisted tunneling mechanism. Klinman and co-workers (45) have also
established a link between the extent of hydrogen tunneling and
mutations in the active site of horse liver alcohol dehydrogenase, and
this is similarly likely to reflect increased barrier widths (as
revealed through crystallographic analysis of mutant enzymes (45, 46))
and altered dynamics in the active site of the mutant enzymes. Our own
studies reported herein and our analysis of the reduction of aromatic
amine dehydrogenase with slow substrates (12) suggest that enzymes can
tolerate increases in barrier size when catalyzing hydrogen and
deuterium transfer by quantum tunneling. Transfer from the substrate
vibrational ground state can occur for small increases in barrier width
(and therefore relatively small reductions in reaction rate). For very
slow substrates or highly compromised mutants, the barrier may be too
wide for transfer from the substrate vibrational ground state level
(particularly for deuterium), and partial barrier ascent may be
required to narrow the transfer distance. Additionally, as seen in
reactions of aromatic amine dehydrogenase with benzylamine (a very slow substrate), the enthalpic contribution for barrier compression may also
be raised, which in turn will also affect the rate of transfer (12).
Our studies therefore illustrate the varied effects of using slow
substrates or engineering compromising mutations into the active site
of those enzymes that catalyze hydrogen transfer by vibrationally
assisted mechanisms.
Concluding Remarks--
Deuterium isotope studies with H172Q TMADH
suggest that oxidation of substrate proceeds from an enzyme-substrate
complex in which trimethylamine base is bound in the active site of the
enzyme. The optimal rate of flavin reduction thus requires enzyme to
preferentially bind trimethylamine, and not the protonated cation.
Kinetic and computational data are consistent with a mechanism
involving nucleophilic attack of the substrate nitrogen lone pair at
the flavin C-4a atom and C-H bond cleavage initiated by the flavin N-5
atom as proposed for monoamine oxidase A. Temperature dependence
studies of the rate of C-H bond cleavage are consistent with a
vibrationally assisted tunneling mechanism from the substrate ground
state in both H172Q and native TMADH, i.e. the C-H and C-D
bond breaking processes occur entirely by tunneling effects.
Compromised rates of C-H bond cleavage in Y169F TMADH are consistent
with a need to thermally activate the substrate to enable hydrogen
tunneling from an excited vibrational state.
 |
FOOTNOTES |
*
This work was supported by grants from the Biotechnology and
Biological Sciences Research Council and the Lister Institute of
Preventive Medicine.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Lister Institute Research Professor. To whom correspondence
should be addressed. Tel.: 44-116-223-1337; Fax: 44-116-252-3369; E-mail: nss4@le.ac.uk.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M101178200
2
J. Basran, M. J. Sutcliffe, and N. S. Scrutton, unpublished data. Results are from analysis of Y60F and Y174F
TMADH. Kinetic studies with Y60F TMADH, trimethylamine, and
perdeuterated trimethylamine indicated that this ionization is
perturbed substantially (pKa ~ 10). This likely
reflects the different electronic environment of the substrate imparted
by Phe-60 (which directly contacts the substrate by forming an
amino-aromatic interaction).
 |
ABBREVIATIONS |
The abbreviations used are:
TMADH, trimethylamine dehydrogenase;
KIE, kinetic isotope effect.
 |
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