The Reaction of Trimethylamine Dehydrogenase with Trimethylamine*

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 (k lim) greater than 1000 s−1 at pH above 8.5, 10 °C. The kinetic parameterk lim/K d for the fast phase exhibits a bell-shaped pH dependence, with two pK a 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 fork lim gives a single pK a 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.

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 ϳ2 and an unusually intense half-field g ϳ4 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 Յ 2 ms at 500 M 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 s Ϫ1 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.

EXPERIMENTAL PROCEDURES
Enzyme Purification-M. methylotrophus W 3 A 1 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 mM Ϫ1 cm Ϫ1 (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 M, placed into a tonometer equipped with a side arm cuvette, and made anaerobic by repeated evacuation and flushing with O 2 -free argon. Trimethylamine hydrochloride solutions prepared in the same buffer were placed in 20-ml glass syringes and made anaerobic by bubbling with O 2 -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) ϭ ⌺⌬A n exp(-k n t) with ⌬A n and k n 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 k fast , k int , and k slow , 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 (k lim ) and dissociation constant (K d ) (20).
pH profiles for the kinetic parameters k lim and k lim /K d were constructed and the data fitted to Equations 3 and 4, respectively, to obtain the relevant pK a values.
EH and E being the catalytic activity of the protonated and unprotonated form of the ionization group, respectively.
T max being the theoretical maximal value of k lim /K d . Kinetic difference spectra were obtained at pH 8.0 over the range 300 -600 nm using enzyme and substrate concentrations of 18 and 500 M, 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 stoppedflow 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 O 2 -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 M, 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 (⌬⑀ ϭ 21.5 mM Ϫ1 cm Ϫ1 ) (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 M each. Trace amount of benzyl viologen (0.5 M) 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 O 2 -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 O 2 -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 M; the samples were then frozen by hand in liquid nitrogen. A separate EPR sample containing 200 M 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 Brü ker 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 Brü ker Instruments software.

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).
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 ) to obtain kinetic parameters k lim and K d (as described under "Experimental Procedures"). The pH dependence of k lim and k lim /K d are shown in Fig. 2, A and B. It is seen that this phase is quite rapid, with extrapolated k lim values greater than 1000 s Ϫ1 above pH 8.5 even at 10°C. 2 The pH dependence of k lim , reflecting the progress of the enzyme-substrate complex through the first irre- 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 (⌬⑀ ϭ 12 mM Ϫ1 cm Ϫ1 ), the high enzyme concentrations used in the present study (10 -20 M), and the 2-cm light path of the mixing cell, the observed absorbance change from which k obs 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.  (20), exhibits simple sigmoidal behavior with a single pK a of 7.1 Ϯ 0.2. 3 By contrast, the pH profile of k lim /K d is bell-shaped and a fit using the general equation for such a profile ("Experimental Procedures," Equation 4) yields pK a values of 9.3 Ϯ 0.1 and 10.0 Ϯ 0.1. Since k lim /K d tracks the reaction of free substrate and free enzyme (20) and the latter pK a 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 pK a 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 k lim /K d 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 k lim /K d as the descending limb of the bell-shaped curve at high pH.
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 s Ϫ1 ). 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] Յ 500 M). The degree of apparent substrate inhibition increases with pH, with the concentration required to give a 50% reduction in rate constant decreasing from ϳ2 mM at pH 7.5 to ϳ0.3 mM at pH 9.5.
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 ϳ0.03 s Ϫ1 at pH 6.0 to ϳ2 s Ϫ1 at pH 8.0) while the substrate concentration that gives the maximum rate constant decreases 3 In the accompanying study (33) of the Y169F mutant of TMADH, the k lim pH profile was fitted to two pK a values rather than a single pK a , as here. Given the magnitude of the standard error in the present data, we are not able to unambiguously ascertain whether a second pK a exists for this plot. Also, we do not eliminate the possibility here that the pK a , evident in the pH profile for k lim 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.  Table I) except for the values at pH 9.5 and 11.0, which are obtained in the present study with diethylmethylamine as substrate under comparable experimental conditions (in 0.1 M borate buffer, 25°C). Note that the experiments with trimethylamine were performed at 10°C.
(ranging from ϳ20 mM at pH 6.0 to ϳ1 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 M 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 M) 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 halfreaction 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] ϭ 18 M, [TMA] ϭ 500 M, 0.1 M pyrophosphate buffer, pH 8.0, 10°C), the three kinetic phases are well resolved (k fast ϭ 500 s Ϫ1 ; k int ϭ 17 s Ϫ1 ; k slow ϭ 1.6 s Ϫ1 ) 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). EPR Studies-Formation of the spin-interacting state in TMADH is associated primarily with the slow phase of the reductive half-reaction (13)(14)(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 M to about 500 M 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 M 1:1 mixtures, respectively. The data are well fit assuming only a single binding site for TMAC, with an associated K d of 40 Ϯ 5 M. 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. DISCUSSION We have examined the reductive half-reaction of TMADH with trimethylamine as a function of pH. The pH profile for k lim /K d from the fast phase of the reaction is bell-shaped and fits to the data yield pK a values of 9.3 Ϯ 0.1 and 10.0 Ϯ 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 Ͻ10% 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 k lim (see below), with the principal basis for the pH dependence of k lim /K d arising from the pH dependence of K d 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 k lim for the fast phase exhibits a pK a of 7.1 Ϯ 0.2 which may be due to the same enzyme residue as seen in the k lim /K d profile (possibly Tyr-60) with a pK a shift upon substrate binding. This decrease in pK a 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 k lim profile is non-zero for both trimethylamine and diethylmethylamine (present study and Ref. 18), indicating that the basic residue observed in the k lim 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 E⅐S 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 6  potentials for donor and acceptor on the order of ϩ1 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.
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 k lim values greater than 1000 s Ϫ1 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 Ϫ1 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 commer-SCHEME 1. Proposed reaction mechanisms for flavin-catalyzed oxidation of amines. a, the proton abstraction mechanism initially proposed for TMADH (18); b, the amminium cation radical mechanism for monoamine oxidase proposed by Silverman (26), in this mechanism, several alternate paths for breakdown of the initial flavin semiquinone/amminium radical intermediate have been considered, only the pathway passing through a transient covalent intermediate is shown here; c, the hydrogen atom abstraction mechanism for monoamine oxidase, proposed by Edmondson (27); and d, the nucleophilic attack mechanism for monoamine oxidase, proposed by Kim et al. (28). cially 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 halfreaction, 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 k cat from the steadystate analysis indicates that the slow phase is principally ratelimiting 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 oneand 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 TMADH 1eϪ 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 [ 14 C]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. 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 k lim value for the fast phase is greater than 600 s Ϫ1 at pH 9.0). The fact that recombinant enzyme from an E. coli expression system and native enzyme obtained from M. methylotrophus W 3 A 1 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.