From the Department of Biochemistry, University of
Leicester, University Road, Leicester LE1 7RH, United Kingdom, the
¶ Department of Chemistry, University of Leicester, University
Road, Leicester LE1 7RH, United Kingdom, and the
§ Department of Medical Biochemistry, Ohio State University,
Columbus, Ohio 43210
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ABSTRACT |
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Tyr-169 in trimethylamine dehydrogenase is one
component of a triad also comprising residues His-172 and Asp-267. Its
role in catalysis and in mediating the magnetic interaction between FMN
cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme.
Tyr-169 is shown to play an important role in catalysis (mutation to
phenylalanine reduces the limiting rate constant for bleaching of the
active site flavin by about 100-fold) but does not serve as a general
base in the course of catalysis. In addition, we are able to resolve
two kinetically influential ionizations involved in both the reaction
of free enzyme with free substrate (as reflected in
klim/Kd), and in the
breakdown of the Eox·S complex (as reflected
in klim). In EPR studies of the Y169F mutant,
it is found that the ability of the Y169F enzyme to form the
spin-interacting state between flavin semiquinone and reduced 4Fe/4S
center characteristic of wild-type enzyme is significantly compromised.
The present results are consistent with Tyr-169 representing the
ionizable group of pKa ~9.5, previously
identified in pH-jump studies of electron transfer, whose deprotonation
must occur for the spin-interacting state to be established.
Trimethylamine dehydrogenase (TMADH, EC
1.5.99.7),1 an iron-sulfur
containing flavoprotein from the bacterium Methylophilus methylotrophus (sp. W3A1), catalyzes the
oxidative demethylation of trimethylamine to dimethylamine and
formaldehyde. The enzyme is a homodimer, and each subunit contains an
unusual covalently linked 6-S-cysteinyl FMN cofactor and a
bacterial ferredoxin-type 4Fe/4S center, as well as 1 equivalent of
tightly bound ADP of unknown function (1-6). The physiological
electron acceptor of TMADH is an electron-transferring flavoprotein, a
62-kDa heterodimer containing 1 equivalent each of FAD (7) and AMP (8).
Electron-transferring flavoprotein is thought to oxidize reduced TMADH
in two successive one-electron steps, cycling between the quinone and
(anionic) semiquinone oxidation states. The availability of a high
resolution structure for TMADH (6) and the cloned and overexpressed
gene for the enzyme (10, 11) has made it possible to examine many aspects of the reaction mechanism by conventional site-directed mutagenesis. These have included studies of the role of (i) the 6-S-cysteinyl FMN in catalysis (11-13), (ii) cation- The reaction of TMADH with trimethylamine exhibits three sequential
kinetic phases (16-18): a fast phase that represents bleaching of the
6-S-cysteinyl FMN, an intermediate phase that reflects intramolecular electron transfer from dihydroflavin to the 4Fe/4S center to generate the flavin semiquinone and reduced 4Fe/4S center, and a slow phase that involves formation of an unusual spin-interacting state of the enzyme in which the unpaired magnetic moments of the
reduced 4Fe/4S center and flavin semiquinone are strongly ferromagnetically coupled (18-21).
In the crystal structure of TMADH, Tyr-169 lies in van der Waals
contact with the pyrimidine ring of the flavin cofactor, and is
hydrogen-bonded to His-172 (which is also in van der Waals contact with
the flavin). In order to ascertain the catalytic significance of
Tyr-169 in TMADH, we have isolated a Y169F mutant enzyme and analyzed
its kinetic behavior. We find that mutation of this residue to
phenylalanine reduces the limiting rate constant for flavin reduction
by a factor of approximately 100, but does not function as an active
site base. The mutation also significantly reduces the ability of the
flavin semiquinone to interact magnetically with the reduced 4Fe/4S
center of the two-electron reduced enzyme. This is due to a substantial
decrease in the equilibrium amount of enzyme possessing flavin
semiquinone and reduced 4Fe/4S center, presumably by perturbing the
semiquinone/hydroquinone half-potential of the active site flavin, and
a decrease in the magnetic interaction between the centers in mutant
enzymes possessing this electron distribution. The result suggests that
Tyr-169 in all likelihood represents the ionizable group of
pKa ~ 9.5, previously identified in pH-jump
studies of electron transfer (22), whose deprotonation must occur for
the spin-interaction state to be established.
Chemicals and Enzymes--
Complex bacteriological media were
from Unipath and all media were prepared as described by Sambrook
et al. (23). Trimethylamine, 2,6-dichlorophenolindophenol,
phenazine methosulfate, tetramethylammonium chloride (TMAC), and all
buffers were from Sigma. Sodium dithionite was obtained from Virginia
Chemicals. Perdeuterated trimethylamine HCl (99.7% D) was from CK Gas
Products Ltd. All other chemicals were of analytical grade where
possible. Wild-type TMADH was purified from M. methylotrophus as described by Steenkamp and Mallinson (1) and
modified by Wilson et al. (24). The concentration of
wild-type enzyme was determined using an extinction coefficient of 27.3 mM
The 4Fe/4S center of TMADH was selectively inactivated by treatment
with ferricenium hexafluorophosphate at pH 10, essentially as described
by Huang et al. (27). The protein (30 µM) was
incubated with ferricenium hexafluorophosphate (3 mM)
contained in 50 mM potassium borate buffer, pH 10, at room
temperature for 6 h. Excess oxidant was removed by size-exclusion
chromatography using Sephadex G-25 equilibrated in 20 mM
potassium phosphate buffer, pH 7.0. The flavin site of TMADH treated in
this way remains reducible by trimethylamine, but the 4Fe/4S enter
becomes redox inert and the enzyme is unable to pass electrons on to
electron-transferring flavoprotein (27).
UV/Visible and EPR Spectroscopy--
UV/visible spectra were
recorded using a Hewlett-Packard 8452A single-beam diode array
spectrophotometer. EPR spectra were obtained using a Brüker ER
300 EPR spectrometer equipped with a ER035M gaussmeter and a
Hewlett-Packard 5352B microwave frequency counter. Instrument settings
were 9.45 GHz microwave frequency, 1.00 mW microwave power, 10 G field
modulation, and 100 kHz modulation amplitude. Temperature was
maintained at 15 K using a Brüker ER 4112HV liquid helium
cryostat with an Oxford Instruments ITC4 temperature controller. EPR
samples were prepared as follows: 300 µl of enzyme solutions at the
desired pH were placed in quartz EPR tubes using a long-needle Hamilton
syringe, then treated with either 20-30 µl of a concentrated
substrate solution prepared in the same buffer (sufficient to give at
least 5-fold stoichiometric excess over the enzyme) or with a
comparable amount of TMAC followed by reduction with sodium dithionite.
For the substrate-reduced samples, a final enzyme concentration of 100 µM was used for the wild-type while 200 µM
was used for Y169F to compensate for the incomplete flavinylation in
this mutant protein to facilitate direct comparisons in the data.
UV/visible spectra of each sample were recorded before and after each
addition using a special spectrophotometer cell holder, which
accommodates EPR tubes. Samples were then thoroughly mixed and slowly
frozen by hand in liquid nitrogen. EPR spectra were recorded at both
half- and high-field (in separate sweeps) for each sample.
Kinetic Measurements--
Steady-state kinetic measurements were
performed with a 1-cm light path in a final volume of 1 ml. The desired
concentrations of trimethylamine, phenazine methosulfate, and
2,6-dichlorophenolindophenol were obtained by making microliter
additions from stock solutions to the assay mixture. Assays were
performed in 100 mM sodium pyrophosphate buffer, pH 8.5. Reaction was initiated by the addition of substrate, and the decrease
in absorbance at 600 nm due to reduction of
2,6-dichlorophenolindophenol (
Rapid kinetic experiments were performed using an Applied Photophysics
SX.17MV stopped-flow spectrophotometer. Time-dependent reduction of TMADH by trimethylamine at pH 6.5 and 7.0 was performed using a photodiode array detector. Spectral deconvolution was perfomed
by global analysis and numerical integration methods using PROKIN
software (Applied Photophysics). For single wavelength studies, data
collected at 443 nm were analyzed using nonlinear least squares
regression on an Archimedes 410-1 microcomputer using Spectrakinetics
software (Applied Photophysics). Experiments were performed by mixing
TMADH in buffer of the desired pH, with an equal volume of
trimethylamine at the desired concentration in the same buffer. The
concentration of substrate was always at least 10-fold greater than
that of TMADH, thereby ensuring pseudo first-order conditions. For each
substrate concentration used, at least four replicate measurements were
collected and averaged. Substrate-reduced TMADH is quite stable to
reoxidation in aerobic environments (half-life about 50 min, Ref. 24),
and consequently these stopped-flow experiments were carried out under aerobic conditions.
The absorbance change at 443 nm for Y169F TMADH at values of pH > 7.0 was essentially monophasic, with a single rate constant obtained
from fits of the data to Equation 1,
The observed rate constants were found to exhibit hyperbolic dependence
on substrate concentration and the reaction sequence was modeled as
shown in the general scheme,
Steady-state Kinetic Analyses--
As reported previously, the
steady-state kinetics of wild-type TMADH exhibit excess substrate
inhibition (18, 30, 31). By contrast, Y169F TMADH exhibits well behaved
steady-state behavior: no evidence for substrate inhibition was seen
even at very high substrate concentrations (up to 45 mM;
Fig. 1).
The kinetic parameters KmTMA and
kcat for the mutant protein are 63 ± 3 µM and 2.6 ± 0.03 s The Reaction of Y169F TMADH with TMA--
Reduction of the flavin
in Y169F TMADH by substrate was examined by stopped-flow spectroscopy
at 443 nm over the pH range 6.0 to 11.0. Unlike wild-type enzyme, in
which flavin reduction is essentially monophasic over this pH range
(18), flavin reduction in Y169F is biphasic below pH 7.0 (Fig.
2). At pH 7.0, it is monophasic at high
substrate concentrations but biphasic at [TMA] < 20 mM, suggesting that ionic strength may influence the kinetic behavior. To
explore this possibility, the reaction was repeated in lower ionic
strength buffer (20 mM phosphate buffer instead of 100 mM, pH 7.0) and biphasic kinetic transients are seen even
at high substrate concentrations. However, the limiting rate constant for flavin reduction (faster phase of the biphasic reaction or the
single kinetic phase in monophasic reaction) is essentially unchanged.
The effect of ionic strength is therefore restricted to controlling the
range of substrate concentration over which biphasic kinetic behavior
is seen (probably by influencing a kinetically relevant ionization; see
below) rather than influencing the limiting rate of flavin reduction.
An analysis of the amplitudes for each of the two phases seen at pH 7.0 and below indicates that the slower kinetic phase becomes increasingly
prominent as pH decreases. The two kinetic phases also become more
clearly resolved, principally due to a decrease in the rate constant
for the slower phase. The pH dependence of the amplitudes for the two
phases indicates the presence of a kinetically influential ionization
of apparent pKa 6.2 ± 0.2 (Fig. 2,
inset). While there will be small differences in ionic
strength across the pH range used for the determination of this value,
these are not expected to compromise the analysis significantly.
Spectral Changes Associated with the Reaction of Y169F TMADH with
Substrate--
The reductive half-reaction of wild-type TMADH with TMA
is triphasic (18). However, at the end of this half-reaction, the distribution of the two electrons derived from substrate in the enzyme
is affected by pH (22, 32): at high pH (pH 7.5 and above), formation of
flavin semiquinone and reduced 4Fe/4S center is favored; at low pH
(e.g. pH 6.5), dihydroflavin and oxidized 4Fe/4S center is
preferred, reducing the overall kinetics to nearly monophasic behavior.
Similar pH effects on electron distribution were also seen for the
Y169F TMADH. To simplify the analysis of the absorbance change
associated with flavin reduction in Y169F TMADH, the experiment was
performed at pH 6.5, thereby effectively eliminating the two slower
phases associated with intramolecular electron transfer observed at
higher pH values.
Upon completion of the faster phase of flavin reduction the spectrum
resembles that of a mixture of oxidized enzyme and enzyme in the
dihydroflavin form (Fig. 3). Following
completion of the slower phase, the spectrum is that of the
dihydroflavin form. The spectral form seen at the end of the faster
phase rules out a sequential two-step reduction process involving a
flavin semiquinone intermediate since this would give rise to a
characteristic flavin semiquinone
spectrum.2 At 1000 s
after initial mixing of enzyme with substrate, the observed spectrum
indicates that electron transfer to the 4Fe/4S center is indeed far
from complete (data not shown). To further confirm that internal
electron transfer to the 4Fe/4S center is not implicated in the
biphasic behavior seen here, the 4Fe/4S center in the enzyme was
selectively inactivated by ferricenium-PF6 (see
"Experimental Procedures"), which is known to render the 4Fe/4S
center redox inert. The reductive half-reaction of the modified enzyme
was studied at pH 6.5, 7.0, and 10.0. Again, the reaction is biphasic
at pH 6.5 and 7.0 and monophasic at pH 10.0, as seen in the untreated
Y169F TMADH, while ferricenium-PF6 inactivated wild-type enzyme
exhibits monophasic behavior at all pH values examined (data not
shown).
Substrate Concentration Dependence and Kinetic Isotope Effects for
Substrate Oxidation--
The substrate concentration dependence of
flavin reduction with Y169F TMADH has been investigated at pH 7.0 (using 20 mM phosphate buffer so that the biphasic behavior
could be resolved throughout the entire substrate concentration range)
and pH 6.5 (using 100 mM
buffer).3 At pH 7.0, both
phases for flavin reduction in Y169F TMADH exhibit hyperbolic
dependence on [TMA]. The limiting rate constant for the faster phase
(43 s Kinetically Influential Ionizations in Y169F--
The reaction of
Y169F TMADH with trimethylamine has been investigated as a function of
pH at 5 °C. The pH dependence of klim seen
with Y169F enzyme reveals two reasonably well resolved
pKa values (pKa 6.7 ± 0.2 and 9.5 ± 0.3; Fig. 4A),
whereas only one ionization is observed for the wild-type enzyme (18). A plot of klim/Kd
versus pH gives a bell-shaped curve (Fig. 4B), as
seen in wild-type enzyme (18), with two pKa values
of 9.7 ± 0.1 and 11.0 ± 0.1 attributable to the ionization of free enzyme and free substrate (pKa of TMA is
9.81), respectively. As in the case of wild-type enzyme, substrate is found to bind preferentially in the cationic form (14, 18). Comparison
of the pH profiles for Y169F and wild-type enzymes indicates that the
pKa values that control flavin reduction are
slightly perturbed on mutating Tyr-169 to Phe. However, all kinetically
influential ionizations seen in the wild-type enzyme remain in the
Y169F mutant enzyme, indicating that Tyr-169 in the wild-type enzyme
either does not ionize over the pH range investigated, or that its
ionization is not kinetically influential for flavin reduction.
UV/Visible Spectra of Y169F TMADH--
The UV/visible absorption
spectra for the oxidized and substrate-reduced forms of wild-type and
Y169F TMADH, along with the corresponding [oxidized] minus
[substrate-reduced] difference spectra are shown in Fig.
5. Oxidized wild-type protein exhibits an
A444 nm/A382 nm
absorbance ratio of about 1.3, whereas that for the Y169F TMADH gives a
ratio around 1.03 due to incomplete flavinylation when expressed in
Escherichia coli. Interestingly, the absorption change
elicited by reduction with excess substrate for this mutant protein is
different from that seen with wild-type protein under the same
conditions (which we have previously shown to be identical in native
and recombinant wild-type enzyme; Ref. 11).4 In particular, the
difference maximum at 365 nm (reflecting accumulation of the flavin
semiquinone form) is absent in the mutant. The observed spectral change
seen with the Y169F mutant is in fact quite reminiscent of that
generated by reduction of wild-type protein to the two-electron reduced
level using sodium dithionite at pH 8.0, where the enzyme principally
contains flavin semiquinone and reduced 4Fe/4S center but their
magnetic moments do not interact (22). This interpretation is further
supported by the EPR spectroscopic studies discussed below. The
implication is that the distribution of reducing equivalents between
the 4Fe/4S and flavin centers in the two proteins are different; a
larger portion of Y169F TMADH exists as flavin hydroquinone and
oxidized 4Fe/4S center rather than flavin semiquinone and reduced
4Fe/4S center, especially at pH 7.0.
Full reduction of wild-type TMADH, which requires 3 reducing
equivalents, is observed when titrated with sodium dithionite, however,
the enzyme takes up only two electrons when reduced with excess
substrate or reduced by sodium dithionite in the presence of TMAC (a
substrate analog and inhibitor of TMADH) (19, 31): binding of the
substrate analog perturbs the reduction potential of the flavin
semiquinone/hydroquinone couple such that full reduction of the enzyme
does not occur. When Y169F TMADH is reduced with sodium dithionite in
the presence of TMAC at pH 7.0, however, the final difference
absorption spectrum resembles that for three-electron reduction of
wild-type enzyme (data not shown). This indicates that full reduction
has occurred, consistent with the EPR studies described below. The
results indicate that the oxidation-reduction properties of the mutant
protein are perturbed and that the ability of the Y169F mutant to form
the spin-interacting state is compromised.
EPR Spectra of Y169F TMADH--
EPR spectra of wild-type and Y169F
TMADH reacted with excess substrate are shown in Fig.
6, A-D. Under these
conditions, the wild-type protein contains flavin semiquinone and a
reduced iron-sulfur center whose magnetic moments interact strongly
with each other and give rise to a spin-interaction state with
characteristic g ~ 2 complex high-field EPR signal and an
intense g ~ 4 half-field signal (Fig. 6, A and
B). For the mutant protein, on the other hand, the signal
centered at g ~ 2 is primarily a combination of the axial signal
of flavin semiquinone and the rhombic signal of reduced 4Fe/4S
center (Fig. 6D). The complex EPR signal associated with the
spin-interacting state seen in the wild-type protein is not observed.
In addition, the intensity of the g ~ 4 signal that is
diagnostic of the spin-interacting state is greatly reduced in the
mutant compared with wild-type protein (Fig. 6C). (The g ~ 4.3 signal seen in the spectrum is due to trace amounts of adventitious iron in the sample.) The experiments have also been performed at pH 10.0 and similar results are observed (data not shown).
This indicates that the mutant protein contains a substantial amount of
flavin semiquinone and reduced iron-sulfur center under the present
experimental conditions, but the magnetic moments of the two unpaired
spins (which given the manner in which the samples were prepared must
exist in the same enzyme molecule) do not interact with each other
strongly as in the wild-type protein. Fig. 6, E and
F, shows the EPR spectra observed when the mutant protein is
reduced by sodium dithionite in the presence of TMAC. The rhombic EPR
signal of the reduced 4Fe/4S center is observed at high-field (Fig.
6F) and no half-field signal associated with the
spin-interaction state is seen (Fig. 6E), which is
consistent with the UV/visible results that the mutant protein is fully
reduced to a three-electron reduction level by sodium dithionite even in the presence of TMAC.
Tyr-169 is one of three amino acids comprising a novel Tyr-His-Asp
triad in the active site of TMADH. Our data for the Y169F mutant
clearly indicate that C-H bond cleavage and FMN reduction occur in the
mutant enzyme, albeit at a limiting rate that is approximately 100-fold
slower than is seen with wild-type enzyme. Tyr-169 is in van der Waals
contact with the flavin isoalloxazine ring, and local adjustments in
active site structure (both physical and electronic) as a result of the
mutation are likely to be responsible, at least in part, for the slower
rates observed in the flavin reduction of Y169F TMADH. The
pH-dependence profiles for Y169F are similar to those for wild-type
enzyme, indicating that Tyr-169 is not the group whose ionization
facilitates substrate oxidation and/or substrate binding in wild-type
enzyme. However, oxidation of substrate is controlled by an additional
ionization with pKa of 6.2 in Y169F TMADH, not
observed in the wild-type enzyme. At pH values below this
pKa, enzyme reduction occurs as two kinetically
resolvable steps, only the faster of which appears to be catalytically
significant. The identity of the amino acid residue responsible for the
additional ionization (pKa value 6.2) that controls
the expression of the biphasic reductive transients in Y169F at low pH
remains to be determined, however, it cannot be His-172 (which H-bonds
to Tyr-169 in wild-type enzyme), since recent studies of a mutant H172Q
TMADH suggest that ionization of this residue occurs around pH
8.5 Similarly, this work
indicates that none of these residues is likely to be involved in
abstraction of a proton from substrate to form a carbanion intermediate
(18). Indeed the base catalysis accounts for only a quite modest
portion of the enzyme-catalyzed rate acceleration for substrate
oxidation (17, 18). The lack of an obvious base to support a carbanion
mechanism provides indirect support for homolytic C-H bond cleavage
analogous to the mechanism that has been proposed for the mechanism of
monoamine oxidase (as discussed in Ref. 18).
The present work clearly demonstrates that Tyr-169 also plays an
important role in mediating the spin-interaction between the flavin
semiquinone and reduced 4Fe/4S center in TMADH. Previous work has shown
that formation of the spin-interacting state of TMADH is governed by a
basic residue located at or near the active site, with
pKa value around 9.5 (22), and our results implicate
Tyr-169 as this basic residue. Although Tyr-169 lies opposite the
flavin ring from the iron-sulfur in TMADH, the importance of this
residue in forming the spin-interaction between the two centers can be
rationalized in the context of the x-ray crystal structure of TMADH
(6). Tyr-169 is located near the C(2) = O group of the flavin
isoalloxazine ring and its van der Waal's surface is in contact with
that of the flavin ring. When a negative charge is developed on the
hydroxyl group of Tyr-169 side chain, due to electrostatic repulsion
the unpaired electron density on the flavin isoalloxazine ring is
reasonably expected to be forced to redistribute away from this residue
toward the 4Fe/4S center, effectively reducing the spin-spin distance.
This may also induce a larger dipole moment on the flavin isoalloxazine
ring, which could be important in promoting the formation of the
spin-interacting state.
The present work also indicates that the reduction potential of the
semiquinone/hydroquinone flavin couple is perturbed in the Y169F
enzyme, as reflected in the shift in oxidation-reduction equilibrium
inferred from the UV/visible spectra. This is supported by the
steady-state kinetic study demonstrating that substrate inhibition is
absent in the Y169F TMADH, and the spectroscopic studies showing that
full reduction of Y169F is achieved with dithionite even in the
presence of TMAC. As described in Ref. 18, substrate inhibition in
wild-type TMADH is accounted for by perturbation of the
semiquinone/hydroquinone flavin couple upon substrate binding to
partially reduced enzyme. Potentiometric studies on Y169F will soon
help to further illustrate this point.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
bonding in substrate recognition (14), and (iii) residues on the
surface of TMADH involved in electron transfer to electron-transferring flavoprotein (15).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cm
1 at 443 nm in 50 mM potassium phosphate buffer, pH 7.0. Recombinant Y169F
TMADH was generated and isolated as described elsewhere (14) and
expressed using the plasmid pSV2tmdveg (11). With this expression
system, the enzyme as isolated possesses its full complement of 4Fe/4S
center and ADP, but a significant portion of the enzyme lacks the
flavin (25, 26). Using spectrophotometric methods reported elsewhere
(11), the fraction of flavinylated enzyme in the Y169F preparations
used in the present study was estimated as approximately 50%; the
mutant enzyme was found to be stoichiometrically assembled with the
4Fe/4S center and ADP. The concentration of the Y169F mutant of TMADH
was determined using an effective extinction coefficient (20.0 mM
1 cm
1 at 443 nm) for oxidized
enzyme, calculated from the extent of the spectral change elicited by
excess substrate (only the flavinylated enzyme is reducible by
substrate). Enzyme solutions of the desired pH were obtained by adding
microliter volumes of a concentrated enzyme stock to buffer at the
desired pH.
= 21, 900 M
1 cm
1) was measured using a
Hewlett-Packard 8452A diode array spectrophotometer. All data were
collected at 30 °C. Data were fitted to the appropriate rate
equation using the fitting program Grafit (28).
where C is a constant related to the initial
absorbance and b is an offset value to account for a
non-zero baseline. Below pH 7, however, kinetic transients for Y169F
TMADH were biphasic and best fit as the sum of two exponentials using
Equation 2,
(Eq. 1)
where kobs1 and
kobs2 are the observed rate constants for the
faster and slower phases, respectively, and C1
and C2 are related to the initial absorbance;
again, b is an offset.
(Eq. 2)
Data were then fitted to obtain related Kd
and klim values using
kobs = klim[S]/(Kd + [S])
(29).
(Eq. 3)
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 at pH 8.5 and
30 °C, which are approximately 5-fold higher and 6-fold lower,
respectively, than those determined for the wild-type enzyme (13.7 ± 1.7 µM and 15.6 ± 2.4 s
1,
respectively; Ref. 14). The results suggest that Tyr-169 plays only a
relatively small role in the overall catalytic efficiency of the enzyme
in the steady-state.
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Fig. 1.
Steady-state kinetic analyses of the reaction
of wild-type and Y169F TMADH with trimethylamine. Open
circles, data for wild-type TMADH; closed circles, data
for Y169F TMADH. Data for the wild-type were fitted using the equation
described by Falzon and Davidson (30); Y169F data were fitted using the
standard Michaelis-Menten equation.
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Fig. 2.
Reduction of the 6-S-cysteinyl FMN in Y169F
TMADH at different pH values, as monitored by the decrease in
absorbance at 443 nm. TMADH (4 µM) was mixed with
trimethylamine in 100 mM buffer of the appropriate pH (see
"Experimental Procedures") at 5 °C. Kinetic transient A is
fitted to the expression for a monophasic reaction (Equation 1) and
transients B and C are fitted to a biphasic expression (Equation 2).
Substrate concentrations were: transient A, 5 mM; transient
B, 20 mM; transient C, 100 mM.
Inset, plot of contribution made by the fast phase to the
total amplitude change at 443 nm versus solution pH. Data
were fitted to the equation describing a single ionization
(pKa 6.2 ± 0.2).
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Fig. 3.
Deconvoluted spectra for the Y169F TMADH in
the reaction with trimethylamine. The reaction of Y169F TMADH and
trimethylamine at pH 6.5 was followed by multiple wavelength
stopped-flow spectroscopy. The deconvoluted spectra for the reaction
species are presented: initial spectrum (solid line),
intermediate (i.e. end of first phase) (- -) and end
spectra for Y169F TMADH (···).
1 ± 1.7) was about 21-fold less than that seen with
wild-type enzyme (903 s
1 ± 50) (Table
I). The dissociation constants calculated
for the two phases seen with the mutant protein are 35 ± 3.4 and
38 ± 4 mM, respectively, considerably larger than
that seen with wild-type TMADH (6 mM, Table I). At pH 7.0, both phases were found to be sensitive to a kinetic isotope effect of
approximately 7, as seen with wild-type TMADH, when perdeuterated TMA
was used (Table I) indicating that the observed kinetics involve C-H
bond breakage. At pH 6.5, the faster phase of the reaction exhibits
hyperbolic dependence on [TMA], with klim and
Kd of 9 ± 0.8 s
1 and 55 ± 10 mM, respectively. The slower phase, however, is
essentially independent of [TMA] over the concentration range studied
(except in the very low substrate concentration regime), with an
observed rate constant of 0.03 s
1. There is also an
associated loss of primary kinetic isotope effect on the reaction
(Table I), indicating that cleavage of the C-H bond is no longer
rate-limiting. Given the additional observation that the slow phase of
the reaction seen at pH 7.0 is significantly slower than
kcat, we have not pursued the nature of this
slow phase further in the present work.
Limiting rate and equilibrium constants for wild-type and Y169F TMADH
reductive half-reactions at 25 °C, pH 7.0, and pH 6.5
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Fig. 4.
pH dependence of flavin reduction in Y169F
TMADH. A, plot of limiting electron transfer rate
constant, klim, as a function of pH. Data are
fitted to the expression for a double ionization. Macroscopic
pKa values are 6.7 ± 0.2 and 9.5 ± 0.3. B, plot of klim/Kd
as a function of pH. Data are fitted to the expression for a double
ionization. Macroscopic pKa values are 9.7 ± 0.1 and 11.0 ± 0.1. The buffer range was achieved using
phosphate, pyrophosphate, borate, and glycine buffers.
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Fig. 5.
UV/visible spectra of wild-type and Y169F
TMADH. A, spectra of oxidized wild-type (upper
solid line), substrate-reduced wild-type (. . . . . ), oxidized
Y169F (- - - -) and substrate-reduced Y169F TMADH (lower solid
line) in 0.1 M phosphate buffer, pH 7.0. B,
[oxidized] minus [substrate-reduced] difference spectra of
wild-type (solid line) and Y169F TMADH (···) in 0.1 M phosphate buffer, pH 7.0. C, spectra of
oxidized wild-type (upper solid line), substrate-reduced
wild-type (···), oxidized Y169F (- - - -) and
substrate-reduced Y169F TMADH (lower solid line) in 0.1 M borate buffer, pH 10.0. D, [oxidized] minus
[substrate-reduced] difference spectra of wild-type (solid
line) and Y169F TMADH (···) in 0.1 M borate
buffer, pH 10.0.
View larger version (28K):
[in a new window]
Fig. 6.
EPR spectra of wild-type and Y169F TMADH at
pH 7.0. A, half-field EPR spectrum of substrate-reduced
wild-type TMADH. B, high-field EPR spectrum of
substrate-reduced wild-type TMADH. C, half-field EPR
spectrum of substrate-reduced Y169F TMADH. D, high-field EPR
spectrum of substrate-reduced Y169F TMADH. E, half-field EPR
spectrum of Y169F TMADH reduced with sodium dithionite in the presence
of TMAC. F, high-field EPR spectrum of Y169F TMADH reduced
with sodium dithionite in the presence of TMAC.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Mewies for assistance with mutagenesis in the early stages of this work and Craig Hemman for valuable help in the EPR experiments.
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council (to N. S. S.), the Royal Society (to M. J. S. and N. S. S.), and National Science Foundation Grant MCB 9420185 (to R. H.).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.
To whom correspondence should be addressed: Dept. of
Biochemistry, University of Leicester, Adrian Building, University
Road, Leicester LE1 7RH, UK. Tel.: 44-116-223-1337; Fax:
44-116-252-3369; E-mail, nss4{at}le.ac.uk.
2 The transient formation of an anionic semiquinone would normally be observed readily at 365 nm. However, single wavelength studies performed at 365 nm indicated that transient formation of an anionic semiquinone did not occur en route to formation of the dihydroflavin.
3 Given that trimethylamine is predominantly protonated at the pH values employed here and the wide substrate concentration range used in the experiments (0 to 120 mM), control experiments have been performed in the absence and presence of 0.2 M potassium chloride to study the effect of ionic strength on kinetics. Additionally, in separate experiments and at selected pH values (6.5, 7.0, and 7.5), ionic strength was kept constant by balancing the substrate and potassium chloride concentrations over the entire substrate concentration range studied. In all cases, the values for the limiting rate constants for both phases and enzyme-substrate dissociation constants were found to be identical (within experimental error). The data therefore demonstrate that ionic strength influences only the relative spectral change associated with each of the two kinetic phases (presumably by perturbing the apparent pKa of about 6.2) and does not affect the observed rate constants for each phase or the corresponding dissociation constant for the E·S complex.
4 It is to be emphasized that while the incomplete flavinylation of the recombinant Y169F mutant prevents a direct spectral comparison of its absorbance spectrum with that of the fully flavinylated native protein, a direct comparison can be made between the substrate-induced difference spectra seen with the two forms of the enzyme, as only that portion of the mutant protein possessing flavin can become reduced by substrate. All discussion and conclusions here are based on these substrate-induced difference spectra and not on the absolute spectra themselves. Use of substrate as reductant ensures that the entirety of the spectral change seen with the mutant arises from enzyme that possesses the full complement of redox-active cofactors, as the deflavo form of the enzyme cannot be reduced by substrate. In determining the concentration of the Y169F protein, we use an effective extinction coefficient that gives the concentration of the fully functional, flavin-containing portion of the enzyme, not simply the total concentration of polypeptide. In both Figs. 5 and 6, spectra of native and Y169F enzyme are presented that have been normalized on a per-flavin basis so that a direct comparison can be made in extinction coefficient and EPR intensity.
5 J. Basran, M. J. Sutcliffe, R. Hille, and N. S. Scrutton, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: TMADH, trimethylamine dehydrogenase; FMN, flavin mononucleotide; TMAC, tetramethylammonium chloride.
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REFERENCES |
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