From the Department of Medical Biochemistry, The Ohio
State University, Columbus, Ohio 43210 and the § Department
of Biochemistry, University of Leicester, Adrian Building, University
Road, Leicester LE1 7RH, United Kingdom
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ABSTRACT |
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The reductive half-reaction of trimethylamine
dehydrogenase with its physiological substrate trimethylamine has been
examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the
reaction, the flavin reduction/substrate oxidation process. As in
previous work with the slow substrate diethylmethylamine, the reaction
is found to consist of three well resolved kinetic phases. The observed
rate constant for the fast phase exhibits hyperbolic dependence on the
substrate concentration with an extrapolated limiting rate constant
(klim) greater than 1000 s Trimethylamine dehydrogenase (TMADH, EC
1.5.99.7)1 is an iron-sulfur
containing flavoprotein isolated from the bacterium Methylophilus
methylotrophus W3A1 that catalyzes the
oxidative demethylation of trimethylamine to dimethylamine and
formaldehyde (presumably through an imine intermediate that
spontaneously hydrolyzes once dissociated from the
enzyme),
1 at pH
above 8.5, 10 °C. The kinetic parameter
klim/Kd for the fast phase
exhibits a bell-shaped pH dependence, with two pKa
values of 9.3 ± 0.1 and 10.0 ± 0.1 attributed to a basic
residue in the enzyme active site and the ionization of the free
substrate, respectively. The sigmoidal pH profile for
klim gives a single pKa
value of 7.1 ± 0.2. The observed rate constants for both the
intermediate and slow phases are found to decrease as the substrate
concentration is increased. The steady-state kinetic behavior of
trimethylamine dehydrogenase with trimethylamine has also been
examined, and is found to be adequately described without invoking a
second, inhibitory substrate-binding site. The present results
demonstrate that: (a) substrate must be protonated in order
to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site
general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with
native enzyme is considerably faster than observed with enzyme isolated
from Methylophilus methylotrophus that has been
grown up on dimethylamine; and (e) a discrete inhibitory
substrate-binding site is not required to account for excess substrate
inhibition, the kinetic behavior of trimethylamine dehydrogenase can be
readily explained in the context of the known properties of the enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The enzyme is a homodimeric protein having a subunit molecular
mass of 83 kDa, with each subunit containing a covalently linked
6-S-cysteinyl FMN cofactor and a bacterial ferredoxin type 4Fe/4S center; each subunit also possesses 1 equivalent tightly bound
ADP of unknown function (1-7). The physiological electron acceptor for
TMADH is an electron transferring flavoprotein (ETF), an
(Eq. 1)
dimer
of molecular mass 62 kDa. ETF contains 1 equivalent of FAD, which
cycles between oxidized and (anionic) semiquinone oxidation states (8),
and 1 equivalent AMP, whose function remains unclear (9).
Full reduction of TMADH requires three electrons per subunit, two for reduction of the FMN and a third for reduction of the 4Fe/4S center, but only two reducing equivalents are removed from substrate during catalysis. The distribution of reducing equivalents within two-electron reduced enzyme generated by reduction with excess substrate favors the formation of flavin semiquinone and reduced 4Fe/4S center, with the magnetic moments of the two paramagnetic centers interacting strongly to give a spin-interacting state (a triplet state), which is distinguished by a complex EPR signal centered near g ~2 and an unusually intense half-field g ~4 signal (10-16). Reduction of enzyme with dithionite in the presence of the substrate analog and inhibitor tetramethylammonium chloride (TMAC), or by titration with dithionite at high pH also generates this characteristic spin-interacting state.
Previous stopped-flow and freeze-quench EPR kinetic studies have
demonstrated that the reaction of TMADH with trimethylamine consists of
three kinetic phases. The first phase involves a very rapid bleaching
of the enzyme-bound FMN and has been only poorly characterized owing to
its rapid rate (t1/2 2 ms at 500 µM
trimethylamine in 0.1 M pyrophosphate buffer, pH 7.7, 18 °C; Ref. 10). This fast phase is followed by two slower kinetic
phases with spectral changes reflecting intramolecular electron
transfer from reduced flavin to the 4Fe/4S center to give flavin
semiquinone and reduced 4Fe/4S center (10-14, 17). More recently, the
reductive half-reaction has also been investigated using a slow
substrate, diethylmethylamine, and the reaction also exhibits three
kinetic phases (18). On the basis of the kinetic studies with
diethylmethylamine, an overall reductive half-reaction mechanism for
TMADH has been proposed (15, 18): the fast phase represents the two
electron reduction of the flavin cofactor (oxidation of the substrate)
with simultaneous formation of a covalent substrate-flavin intermediate; the intermediate phase reflects intramolecular electron transfer from reduced flavin to the 4Fe/4S center, generating flavin
semiquinone and reduced 4Fe/4S center with intrinsic rapid electron
transfer rate limited by the decay of the covalent adduct (15); and the
slow phase involves dissociation of product and binding of a second
substrate molecule, which perturbs the electron distribution in the
partially reduced enzyme and facilitates formation of the
spin-interacting state. Most recently, the reaction with trimethylamine
has been re-examined with a considerably slower rate constant for the
first phase of the reaction being reported despite the fact that the
experiment was performed at 30 °C (19). In addition, a kinetic
mechanism involving an additional inhibitory substrate-binding site was
proposed, although there is no independent evidence for the existence
of such an additional substrate binding site.
In an effort to clarify the discrepancies concerning the kinetic
behavior of TMADH and to further elucidate its reaction mechanism, a
comprehensive pH dependence study of the enzyme reaction with trimethylamine has been performed, with particular attention paid to
the fast phase of the reaction. By working at 10 °C, we have been
able to characterize the substrate concentration and pH dependence of
the fast phase of the reaction. We find that this phase is indeed very
rapid, with an extrapolated limiting rate constant greater than 1000 s1 at pH 8.5 and higher, even at 10 °C. Both rapid
kinetic and steady-state results can be readily explained by a proposed
kinetic mechanism involving two alternative catalytic cycles depending
on the relative availability of substrate and electron acceptor. We
conclude that the kinetic behavior of the enzyme can be adequately
described from a consideration of the known properties of the enzyme
without invoking a second substrate-binding site.
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EXPERIMENTAL PROCEDURES |
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Enzyme Purification--
M. methylotrophus
W3A1 was grown on trimethylamine as sole carbon
source and TMADH was purified essentially as described by Steenkamp and
Mallinson (1) with the exception that Sephacryl S-200 rather than
Sephadex G-200 was used for the gel filtration step in the
purification. Enzyme concentrations were determined from the 442 nm
absorbance of oxidized enzyme using an extinction coefficient of 27.3 mM1 cm
1 (6). The enzyme was
found to be stable over the pH range 6-11 used in the present study.
Chemicals-- Trimethylamine hydrochloride was obtained from Sigma. TMAC was from Aldrich and dried prior to use employing an indirectly heated drying tube containing phosphorous pentoxide in a side chamber. Phosphate and pyrophosphate buffers were obtained from Sigma and boric acid from Jenneile Chemical Co. (inorganic buffers are necessary since most organic buffers contain substituted amines, and either inhibit the enzyme or serve as substrates). Sodium dithionite was from Virginia Chemicals. Phenazine ethosulfate and 2,6-dichlorophenolindophenol were from Sigma, and benzyl viologen from Aldrich.
Pre-steady-state Experiments--
Kinetic experiments were
carried out using a Kinetic Instrument Inc. stopped-flow apparatus
equipped with an On-Line Instruments Systems (OLIS) model 3920Z data
collection system. Anaerobic solutions of oxidized TMADH were prepared
in the following way. A concentrated sample of oxidized enzyme was
passed through a Sephadex G-25 column equilibrated with 0.1 M solution of an appropriate buffer adjusted to the desired
pH (phosphate buffer for pH 6.0-7.5, pyrophosphate buffer for pH
8.0-8.5, borate buffer for pH 9.0-11.0). The enzyme solution was then
diluted with buffer to give a final concentration of 10-20
µM, placed into a tonometer equipped with a side arm cuvette, and made anaerobic by repeated evacuation and flushing with
O2-free argon. Trimethylamine hydrochloride solutions
prepared in the same buffer were placed in 20-ml glass syringes and
made anaerobic by bubbling with O2-free argon for at least
15 min. Kinetic transients were monitored as transmittance voltages
collected by a high speed A/D converter and converted to absorbance
changes by OLIS software. All experiments were performed at 10 °C in
order to better observe the first kinetic phase of the reaction, and except where specifically discussed below, three well resolved kinetic
phases were observed. 500-1000 data points were collected for each
kinetic transient and fitted to sums of exponentials using an
interactive nonlinear least squares algorithm based on the expression:
A(t) =
Anexp(-knt) with
An and kn representing the
total absorbance change and observed rate constant, respectively.
Depending on the observation wavelength and substrate concentration,
the kinetic transients were fitted with either a two- or
three-exponential expression, as appropriate, and are designated as
kfast, kint, and
kslow, respectively. At least three independent
measurements were performed for each specific experimental condition
and the mean and standard derivation calculated using Excel 4.0. Rate
constants for each kinetic phase were then plotted versus
substrate concentration. For the fast phase, the hyperbolic substrate
concentration dependence was fitted with Equation 2 to obtain the
corresponding limiting rate constant (klim) and
dissociation constant (Kd) (20).
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(Eq. 2) |
pH profiles for the kinetic parameters klim and klim/Kd were constructed and the data fitted to Equations 3 and 4, respectively, to obtain the relevant pKa values.
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(Eq. 3) |
EH and E being the catalytic activity of the protonated and unprotonated form of the ionization group, respectively.
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(Eq. 4) |
Tmax being the theoretical maximal value of klim/Kd.
Kinetic difference spectra were obtained at pH 8.0 over the range 300-600 nm using enzyme and substrate concentrations of 18 and 500 µM, respectively (conditions that ensured adequate kinetic resolution of the three kinetic phases). The spectral change associated with the fast phase of the reaction was corrected for the dead-time of the stopped-flow apparatus (approximately 1.0 ms) (18).
Steady-state Experiments--
Steady-state experiments were
performed anaerobically at 10 °C, in 0.1 M pyrophosphate
buffer, pH 8.0, using a protocol based on the dye-linked assay of Colby
and Zatman (21). The buffer solution was placed in the side arm cuvette
first and made anaerobic by flushing with O2-free argon.
Anaerobic phenazine ethosulfate and 2,6-dichlorophenolindophenol stock
solutions were then injected into the side arm cuvette through a rubber
septum with gas tight Hamilton syringes to get final concentrations of
2 mM and 100 µM, respectively. Ten
microliters of enzyme solution was then injected to give a final
concentration of about 40 nM. The reaction was initiated by
injection of a concentrated solution of trimethylamine hydrochloride to
give the desired final concentration, and followed at 600 nm for the
reduction of 2,6-dichlorophenolindophenol ( = 21.5 mM
1 cm
1) (22) using a
Hewlett-Packard 8452A single beam diode array spectrophotometer. The
observed rate constants were corrected for the slow autoreduction of
the dye in the absence of both enzyme and substrate.
Optical/EPR Experiments--
Oxidized TMADH (in 0.1 M pyrophosphate buffer, pH 8.0) was mixed with a
stoichiometric amount of TMAC to give a final concentration of about
500 µM each. Trace amount of benzyl viologen (0.5 µM) was also included in the reaction mixture to avoid
formation of a sulfite adduct during dithionite reduction (15). The
enzyme/TMAC solution was placed in a tonometer equipped with a side arm
cuvette, made anaerobic by repeated evacuation and flushing with
O2-free argon, and then titrated with sodium dithionite to
the level of two-electron reduced enzyme. Enzyme samples were removed
through the rubber septum using a long needle Hamilton syringe and
placed in serum-stoppered quartz EPR tubes which had been previously flushed with O2-free argon. Different amounts of anaerobic
buffer were added to the EPR tubes to obtain different concentrations of enzyme and TMAC, ranging from 488 to 10 µM; the
samples were then frozen by hand in liquid nitrogen. A separate EPR
sample containing 200 µM TMADH and 10 mM
trimethylamine (in which formation of the spin-interacting state was
complete) was prepared as an integration standard. X-band EPR spectra
were recorded using a Brüker ER 300 EPR spectrometer equipped
with a ER035M gaussmeter and a Hewlett-Packard 5352B microwave
frequency counter. EPR parameters were as follows: microwave frequency,
9.45 GHz; microwave power, 1.00 mW; modulation amplitude, 10.084 G;
temperature, 15 K. Ten scans were taken for each sample to facilitate
quantitation of the signals. Relative intensities of EPR signals were
determined by the double integration method using Brüker
Instruments software.
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RESULTS |
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Reductive Half-reaction Kinetics of TMADH with
Trimethylamine--
The reductive half-reaction of TMADH with
trimethylamine has been studied by stopped-flow spectroscopy at
10 °C over the pH range 6.0-11.0. The reaction is found to consist
of three kinetic phases: a fast process followed by two slower ones
(hereafter designated fast, intermediate, and slow, respectively).
Kinetic transients observed at 450 nm consist principally of the fast kinetic phase throughout the pH range examined here, although a small
absorbance change associated with the two slower phases can also be
seen (Fig. 1A). The fast phase
of the reaction at 10 °C is sufficiently slow as to permit accurate
determination of the rate constants for this phase. At 365 nm, the
kinetic transients consist essentially of the two slower phases with
only a very small spectral change associated with the fast phase (Fig.
1B), while kinetic transients obtained at 520 nm contain
substantial contributions from all three kinetic phases (Fig.
1C).
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Under the pseudo-first order conditions used in the present
experiments, the rate constant of the fast phase exhibits a hyperbolic substrate concentration dependence at all pH values examined (data not
shown), consistent with the formation of a Michaelis complex between
enzyme and substrate prior to bleaching of the flavin (20). The data
can be fitted to the hyperbolic equation: kobs = klim [S]/(Kd + [S]) to
obtain kinetic parameters klim and
Kd (as described under "Experimental
Procedures"). The pH dependence of klim and
klim/Kd are shown in Fig. 2, A and B. It is
seen that this phase is quite rapid, with extrapolated klim values greater than 1000 s1
above pH 8.5 even at
10 °C.2 The pH dependence
of klim, reflecting the progress of the
enzyme-substrate complex through the first irreversible step of the
reaction (20), exhibits simple sigmoidal behavior with a single
pKa of 7.1 ± 0.2.3 By contrast, the pH
profile of klim/Kd is
bell-shaped and a fit using the general equation for such a profile
("Experimental Procedures," Equation 4) yields
pKa values of 9.3 ± 0.1 and 10.0 ± 0.1. Since klim/Kd tracks the
reaction of free substrate and free enzyme (20) and the latter
pKa value agrees well with that for free
trimethylamine (9.81; Ref. 23), we thus attribute the latter to the
ionization of free substrate and the former to an ionizable group in
the enzyme active site. The present results are similar to those
obtained previously with diethylmethylamine as substrate, although,
significantly, the pKa associated with free
substrate was not identified in this earlier work as the data were not
extended to sufficiently high pH (18). To confirm the bell-shaped
behavior in the klim/Kd plot,
we have reexamined the reaction of TMADH with diethylmethylamine to
include data for pH 9.5 and 11.0. The results are shown in Fig. 2,
C and D, and the ionization of substrate is
clearly evident in the pH profile for
klim/Kd as the descending
limb of the bell-shaped curve at high pH.
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Several difficulties are associated with analysis of the intermediate
phase of the reaction. Below pH 7.0, the spectral change associated
with this phase is small at most wavelengths, making it difficult to
resolve the two phases (given the large spectral change associated with
the fast phase, there is little compromise in the determination of its
rate constant). Also, above pH 9.5 the rate constants for the
intermediate and slow phases approach each other at high substrate
concentrations and prevent reliable determination of rate constants for
either phase. As a result, only data over the range pH 7.5-9.5 can be
analyzed (the result for pH 8.0 is shown in Fig.
3A). At all pH values
examined, the observed rate constant for intermediate phase is
substrate concentration dependent, but exhibits a pronounced substrate
inhibition pattern.4 The
maximum apparent rate constant is essentially independent of pH (about
20 s1). At a given pH, the rate constant decreases as
substrate concentration increases (except for pH 7.5 where the rate
constant slightly increases as the substrate concentration increases at
[TMA]
500 µM). The degree of apparent substrate
inhibition increases with pH, with the concentration required to give a
50% reduction in rate constant decreasing from ~2 mM at
pH 7.5 to ~0.3 mM at pH 9.5.
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As with the intermediate phase, it is difficult to obtain reliable rate
constants for the slow phase of the reaction. Generally, the magnitude
of the spectral change associated with the slow phase decreases as pH
increases, and as a result there is poor resolution from the
intermediate phase at high pH. Therefore, we were only able to
determine the substrate concentration dependence of the slow phase
between pH 6.0 and 8.0. The results of the experiment at pH 8.0 are
shown in Fig. 3B, where it is evident that the observed rate
constant again exhibits substrate inhibition. At each pH, the rate
constant increases at low substrate concentrations, then decreases at
higher concentrations. The maximum rate constant increases with pH
(from ~0.03 s1 at pH 6.0 to ~2 s
1 at pH
8.0) while the substrate concentration that gives the maximum rate
constant decreases (ranging from ~20 mM at pH 6.0 to ~1
mM at pH 8.0). Inhibition by excess substrate is more
dramatic at higher pH, as seen for the intermediate phase.
Steady-State Kinetics of TMADH with Trimethylamine-- To correlate with the rapid kinetic work, the steady-state kinetic behavior of TMADH with trimethylamine has been studied at pH 8.0, 10 °C. The substrate concentration dependence of initial velocity over a TMA concentration range of 5 µM to 5 mM is shown in Fig. 3C. As seen previously (14), excess substrate inhibition is observed with the initial velocity increasing at low substrate concentration (up to approximately 50 µM) and then decreasing as substrate concentration further increases. The concentration at which substrate inhibition is observed, however, is much lower than is the case for the reductive half-reaction experiments described above.
Spectral Changes Associated with the Reaction of TMADH with
Trimethylamine--
The spectral changes associated with each kinetic
phase seen in the reductive half-reaction have been determined by
stopped-flow spectroscopy with the results shown in Fig.
4. Under the present experimental
conditions ([TMADH] = 18 µM, [TMA] = 500 µM, 0.1 M pyrophosphate buffer, pH 8.0, 10 °C), the three kinetic phases are well resolved
(kfast = 500 s1;
kint = 17 s
1;
kslow = 1.6 s
1) and the spectral
change associated with each phase readily
determined.5 That for the
fast phase agrees well with that reported previously by Beinert and
co-workers obtained at pH 7.7 (10) and is consistent with either true
reduction of the flavin cofactor or formation of a substrate-flavin
covalent intermediate, as proposed previously (18). As seen previously
with diethylmethylamine (18), the spectral changes associated with the
intermediate and slow phases are essentially the same, only the
relative amplitudes of the two phases change as a function of pH (18).
The total absorbance change associated with the two phases seen here is
also comparable to that reported by Beinert and co-workers (Ref. 10;
working at pH 7.7).
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EPR Studies--
Formation of the spin-interacting state in TMADH
is associated primarily with the slow phase of the reductive
half-reaction (13-15), and has previously been interpreted as
involving a redistribution of reducing equivalents within two-electron
reduced enzyme as product dissociates and a second equivalent of
substrate binds (18). More recently, however, it has been ascribed to
the binding of a second equivalent of substrate to a discrete
inhibitory site on the enzyme (19). In order to investigate whether a
second substrate-binding site exists on TMADH whose occupancy is
required for formation of the spin-interacting state, the following
experiment has been performed, taking advantage of the well known
observation that binding of the substrate analog TMAC to partially
reduced TMADH elicits the spin-interacting state (24). Several
different 1:1 mixtures of two-electron reduced TMADH and TMAC ranging
in concentrations from 10 µM to about 500 µM were prepared and the half-field EPR spectra of each
sample recorded (Fig. 5). The EPR signals
were quantitated by double integration and compared with that of a
control sample (see "Experimental Procedures"). The extent of
triplet state formation were estimated to be 71, 69, and 64% for 488, 300, and 200 µM 1:1 mixtures, respectively. The data are
well fit assuming only a single binding site for TMAC, with an
associated Kd of 40 ± 5 µM. We
note that, were 2 equivalents of substrate required for formation of
the triplet state, only 30 to 40% of the enzyme would have been
converted to the triplet state under the present conditions.
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DISCUSSION |
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We have examined the reductive half-reaction of TMADH with
trimethylamine as a function of pH. The pH profile for
klim/Kd from the fast phase
of the reaction is bell-shaped and fits to the data yield
pKa values of 9.3 ± 0.1 and 10.0 ± 0.1, which we attribute to a basic residue on the free enzyme and the ionization of the free substrate, respectively. The implication is that
substrate must be protonated to bind to the protein, an observation
consistent with the proposal that TMADH binds its substrate through
cation- bonding interactions (25). Examination of the x-ray crystal
structure of TMADH indicates the possible candidates for the basic
residue are Tyr-169, Tyr-60, and His-172 (24). Tyr-169 may be excluded
as a Y169F mutant of TMADH still exhibits the observed ionization (see
the Ref. 33), as does an H172Q mutant (although the limiting rate
constant at high pH is <10% that seen with wild-type
enzyme).6 This leaves Tyr-60,
which with Trp-355 and Trp-264 constitutes the substrate-binding site
for the enzyme (25). Given the only modest pH dependence to
klim (see below), with the principal basis for
the pH dependence of klim/Kd
arising from the pH dependence of Kd itself. This
being the case, ionization of a group in the substrate-binding site
such as Tyr-60 is reasonable to account for the effects observed here.
Efforts are presently under way to prepare a Y60F TMADH mutant of TMADH
so that this can be tested experimentally.
The pH dependence of klim for the fast phase exhibits a pKa of 7.1 ± 0.2 which may be due to the same enzyme residue as seen in the klim/Kd profile (possibly Tyr-60) with a pKa shift upon substrate binding. This decrease in pKa upon binding substrate would tend to deprotonate the active site base, thereby putting this group in the proper ionization state to facilitate the reaction, but we cannot at present exclude the possibility that this ionization arises from another active site residue in the enzyme-substrate complex. We emphasize that the low pH asymptote to the klim profile is non-zero for both trimethylamine and diethylmethylamine (present study and Ref. 18), indicating that the basic residue observed in the klim plot, although important, is not strictly required for catalysis; indeed, it accelerates catalysis only about 6-fold. Even were the protonation step to be fully reversible and this factor of six to underestimate the intrinsic effect (due to a significant equilibrium effect), it is nevertheless evident from the present work that protonation of the responsible group does not seriously compromise breakdown of the E·S complex.
The mechanism of amine oxidation as catalyzed by flavoproteins is an
issue of considerable controversy at present, particularly as regards
the mechanism of action of monoamine oxidase. On the basis of the above
discussion, a mechanism based on proton abstraction (Scheme
1a) seems unlikely in the case
of TMADH, as protonation of the active site base has only a relative
small effect on the observed rate of decay of the enzyme-substrate
complex. Based on the behavior of a variety of mechanism-based
inhibitors and with the chemical precedent of nonenzymatic mechanisms
of amine oxidation, Silverman (26) has advocated a mechanism for
monoamine oxidase in which substrate is initially oxidized by single
electron transfer to the enzyme flavin to give an amminium cation
radical (and anionic flavin semiquinone). The preponderance of the
evidence is considered to support a mechanism in which this radical
pair first recombines to form a covalent adduct which then decays by -elimination, as indicated in Scheme 1b. Alternatively,
Edmondson (27) has considered a hydrogen atom abstraction mechanism to be preferable for monoamine oxidase (Scheme 1c), based
principally on the absence of a significant electronic influence on
reaction rate in a homologous series of benzylamine derivative (in
conjunction with kinetic isotope work suggesting that the transition
state is late rather than early in the course of the reaction). We note here that all proposed mechanisms for monoamine oxidase begin with
neutral substrate rather than the protonated form as is shown to be the
case here with TMADH. On first principles, we consider it unlikely that
the reaction is initiated by a single electron transfer from substrate
to the enzyme flavin when substrate is already positively charged, even
if the large uphill driving force for the reaction (corresponding to a
difference in reduction potentials for donor and acceptor on the order
of +1 V) were somehow accounted for. Similarly, a mechanism involving
direct nucleophilic attack of the nitrogen lone-pair of substrate on
the flavin 4a-carbon (Scheme 1d, also considered in the case
of monoamine oxidase; Ref. 28) can be eliminated as a possibility, at
least for TMADH, again on the basis of our observation that the
reactive form of substrate is protonated. Thus, although the present
data do not directly address the mechanism by which C-H bond cleavage
occurs, the fact that substrate must be protonated essentially rules
out two of the mechanisms shown in Scheme 1, at least in the case of
TMADH. Only a mechanism initiated by hydrogen atom abstraction appears
to remain fully consistent with the present results. Here, however, the
problem is that (as in the case of monoamine oxidase) there is no
obvious candidate for the hydrogen atom acceptor. Tyr-60 is part of the
substrate-binding site and well situated to act in this capacity, but
no tyrosyl (or tryptophanyl) radical has ever been observed with the
enzyme, even when treated with strong oxidants such as ferricenium ion.
Clearly, much additional work is required to address this issue, and to
this end mechanism-based inhibitor studies along the lines of those
done previously for monoamine oxidase are presently being pursued with
TMADH.
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Regardless of the precise mechanism for the first step of the reaction
of TMADH with trimethylamine, the present study demonstrates it is
indeed very rapid, with extrapolated klim values
greater than 1000 s1 at pH above 8.5, 10 °C. The
magnitude of these rate constants are generally consistent with the
half-life for the fast phase at pH 7.7 of 1.5-2 ms reported by Beinert
and co-workers (10) when the higher temperature utilized in this
earlier study is taken into account. In a more recent study, however,
the kinetic properties for the reductive half-reaction of TMADH have
been reported that differ significantly from those reported here (19), with limiting rate constants for the fast phase of 230 s
1
at pH 7.5, 30 °C (significantly smaller than those reported here). In addition, the present results indicate that the slower phases of the
reaction exhibit excess substrate inhibition, while this more recent
work reported hyperbolic substrate concentration dependence for both
the intermediate and slow phases. The basis for these discrepancies in
kinetic behavior appear to have to do with the fact that different
substrates are used as carbon source for the cultures from which
protein was purified; trimethylamine was used in the present case while
dimethylamine was used in the other study. It is known that TMADH
isolated from cells grown on dimethylamine exhibits lower specific
activity and also has altered spectral properties (29), which have been
attributed to chemical modification of the enzyme by impurities in
commercially available dimethylamine, and/or incomplete flavinylation
of the enzyme.7 The present
work underscores the importance of working with enzyme isolated from
cells grown on the native substrate for the enzyme. In addition, we
have recently found that ethylene glycol is a pronounced inhibitor of
TMADH,6 and this may also have contributed to the
differences in kinetic behavior exhibited by the enzyme in these two
studies (ethylene glycol was not used in storage of protein used in the
present work).
For the intermediate and slow phases of the reductive half-reaction, we are able to obtain reliable rate constants over only a limited pH range. At pH 7.5, our results are again generally consistent with the previous work of Steenkamp and Beinert (14) done at pH 7.7. A comparison of the rate constants obtained in pre-steady-state studies with kcat from the steady-state analysis indicates that the slow phase is principally rate-limiting in catalysis (although the intermediate phase may also be responsible for the overall catalytic resistance at high substrate concentrations). Excess substrate inhibition is observed in the rapid reaction kinetics for the two slower phases as well as in the steady-state kinetics, but steady-state inhibition is observed at much lower substrate concentrations than either of the two slower phases of the reductive half-reaction (Fig. 3), indicating that other kinetic effects must account for the phenomenon.
By way of understanding the steady-state inhibition of TMADH at high
concentrations of substrate, we have noted that TMADH can utilize two
alternate catalytic cycles (17): oxidized and two-electron reduced
enzyme (an 0/2 cycle) or one- and three-electron reduced enzyme (a 1/3
cycle). This arises from the circumstance that substrate donates two
electrons, while ETF takes up only one electron and the enzyme itself
can take as many as three electrons. Which cycle predominates in the
steady-state depends primarily on the relative concentrations of
reducing substrate and electron acceptor. At low TMA and/or high ETF
concentrations, the 0/2 cycle is expected to predominate, and
conversely at high substrate and/or low ETF concentrations, the 1/3
cycle should be more important. Enzyme turnover in the 1/3 cycle is
expected to be slower than that in the 0/2 cycle since substrate
binding is known to stabilize the semiquinone form of the flavin in
one-electron reduced enzyme (30). Thus, binding of substrate to the
partially reduced enzyme forms that accumulate in the steady-state
under conditions of high substrate concentrations must result in a
redistribution of reducing equivalents in TMADH1e such
that the flavin center becomes reduced. To the extent that this takes
place, oxidation of the bound substrate cannot occur as the flavin is
not able to accept a pair of reducing equivalents from substrate. The
kinetic effect is equivalent to excess substrate inhibition but,
significantly, does not involve a second inhibitory substrate-binding
site. An analogous mechanism has also been shown to account for the
excess substrate inhibition observed with xanthine oxidase (31). We emphasize that this mechanism for excess substrate inhibition in the
steady-state is a necessary consequence of the known properties of
TMADH, and is distinct from a model in which a second, inhibitory, substrate-binding site is present in the enzyme, as has been suggested (19). We note that the x-ray crystal structure of TMADH in complex with
the substrate analog TMAC gives no indication of the presence of a
second substrate-binding site (24, 32). In addition, it has been shown
in gel filtration experiments with [14C]trimethylamine
that no more than 1 equivalent of substrate is bound to the reduced
enzyme (13). The existence of only a single substrate-binding site is
further supported by the present EPR studies demonstrating that 1 equivalent of the substrate analog TMAC is sufficient to generate the
spin-interacting state in two-electron reduced TMADH. Our results are
thus entirely consistent with the x-ray crystallographic data and the
earlier gel filtration experiment indicating the presence of a single
substrate-binding site on the enzyme.
The results presented here provide several important insights into the
mechanism of TMADH and also clarify some discrepancies considering the
kinetic behavior of this enzyme in the literature. Studies of related
TMADH mutants are presently under way to further elucidate the reaction
mechanism of the enzyme and to identify amino acid residues involved in
substrate binding and catalysis.
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ACKNOWLEDGEMENTS |
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We thank Craig Hemann for valuable technical assistance on the EPR studies and Alexander Lazarev for help with the protein purification and pre-steady-state kinetic 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 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 and reprint requests should be addressed. Tel.: 614-292-4964; Fax: 614-292-4118; E-mail: hille.1{at}osu.edu.
2
The dead-time of the stopped-flow apparatus used
in the present study is approximately 1.0 ms and it is estimated that
about 40-60% of the spectral change associated with the fast kinetic phase of the reaction occurs in the dead-time of the rapid mixing apparatus (depending on the pH and/or substrate concentrations used).
Fortunately, with the large spectral changes associated with the fast
phase ( = 12 mM
1 cm
1),
the high enzyme concentrations used in the present study (10-20 µM), and the 2-cm light path of the mixing cell, the
observed absorbance change from which kobs has
been obtained is in the range 0.06 to 0.3 absorbance units. The
reliability of the obtained rate constants are further improved by
performing more than three independent measurements under each
experimental condition and averaging the results.
3 In the accompanying study (33) of the Y169F mutant of TMADH, the klim pH profile was fitted to two pKa values rather than a single pKa, as here. Given the magnitude of the standard error in the present data, we are not able to unambiguously ascertain whether a second pKa exists for this plot. Also, we do not eliminate the possibility here that the pKa, evident in the pH profile for klim may arise from enzyme-bound substrate. This would require that enzyme bind the protonated form of substrate more tightly than the neutral form, but that the Michaelis constant for the latter break down more rapidly.
4 There is a potential concern that a portion of the observed kinetic effect might be due to ionic strength effects on the reaction at high substrate concentration. The reductive half-reaction of TMADH with trimethylamine has also been studied at pH 7.5, 10 °C in the presence of 0.2 N potassium chloride and same kinetic behavior is observed as reported here.
5 The spectral changes associated with each kinetic phases have also been independently determined by kinetic scan using an Applied Photophysics SX.17MV stopped-flow spectrophotometer and the results are essentially identical to those presented here.
6 J. Basran and N. S. Scrutton, unpublished data.
7
Kinetic studies performed on recombinant TMADH
have shown that the obtained rate constants for the fast phase in the
reductive half-reaction of TMADH with trimethylamine are comparable to
those reported here (the experiments on recombinant protein were
performed at 5 °C over a pH range from 6.0 to 9.0; the extrapolated
klim value for the fast phase is greater than
600 s1 at pH 9.0). The fact that recombinant enzyme from
an E. coli expression system and native enzyme obtained from
M. methylotrophus W3A1 grown on
trimethylamine as carbon source exhibit comparable kinetic behavior
suggests that the latter protein has not been modified, as the former
almost certainly has not been.
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ABBREVIATIONS |
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The abbreviations used are: TMADH, trimethylamine dehydrogenase; FMN, flavin mononucleotide; 4Fe/4S, four iron-four sulfur center; ETF, electron transferring flavoprotein; TMAC, tetramethylammonium chloride.
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REFERENCES |
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