(Received for publication, August 7, 1996, and in revised form, December 7, 1996)
From the Fakultät für Biologie, Universität
Konstanz, D-78434 Konstanz, Germany and Facoltà di
Scienze III, Università di Milano,
via Ravasi 2, 21100 Varese, Italy
The kinetic mechanism of the reaction of
D-amino acid oxidase (EC 1.4.3.3) from Trigonopsis
variabilis with [-1H]- and
[
-2H]phenylglycine has been determined. The pH
dependence of Vmax is compatible with
pKa values of
8.1 and >9.5, the former of which
is attributed to a base which should be deprotonated for efficient
catalysis. The deuterium isotope effect on turnover is
3.9, and the
solvent isotope effect
1.6. The reductive half-reaction is biphasic,
the first, fast phase, k2, corresponding to
substrate dehydrogenation/enzyme flavin reduction and the second to
conversion/release of product. Enzyme flavin reduction consists in an
approach to equilibrium involving a finite rate for
k
2, the reversal of
k2. k2 is 28.8 and 4.6 s
1 for [
-1H]- and
[
-2H]phenylglycine, respectively, yielding a primary
deuterium isotope effect
6. The solvent deuterium isotope effect on
the apparent rate of reduction for [
-1H]- and
[
-2H]phenylglycine is
2.8 and
5. The rates for
k
2 are 4.2 and 0.9 s
1 for
[
-1H]- and [
-2H]phenylglycine,
respectively, and the corresponding isotope effect is
4.7. The
isotope effect on
-H and the solvent one thus behave multiplicatively consistent with a highly concerted process and a
symmetric transition state.
The k2 and k2 values
for phenylglycines carrying the para substituents F, Cl,
Br, CH3, OH, NO2 and OCH3 have been
determined. There is a linear correlation of k2
with the substituent volume VM and
with
+; k
2 correlates best
with
or
+ while steric parameters have little
influence. This is consistent with the transition state being
structurally similar to the product. The Brønsted plot of
G
versus
G0 allows the estimation of the intrinsic
G0
as
58 kJ·M
1. From the linear free energy
correlations, the relation of
G
versus
G0 and according to the theory of Marcus it
is concluded that there is little if any development of charge in the
transition state. This, together with the recently solved
three-dimensional structure of D-amino acid oxidase from
pig kidney (Mattevi, A., Vanoni, M.A., Todone, F., Rizzi, M.,
Teplyakov, A., Coda, A., Bolognesi, M., and Curti, B. (1996)
Proc. Natl. Acad. Sci. U. S. A. 93, 7496-7501), argues
against a carbanion mechanism in its classical formulation. Our
data are compatible with transfer of a hydride from the substrate
C-H to the oxidized flavin N(5) position, although, clearly,
they cannot prove it.
D-Amino acid oxidase (EC 1.4.3.3,
DAAO)1 is the paradigm of flavin enzymes.
It was the second flavoprotein to be uncovered, and probably it is the
most studied member of this superfamily. In addition to the classical
protein from mammalian kidney, recently related DAAOs have been
described from various yeasts (1, 2). A common feature of all these
enzymes is the dehydrogenation of D-amino acids to yield
-imino and, upon subsequent hydrolysis,
-ketoacids. The terminal
redox acceptor is dioxygen. In spite of the innumerable studies, the
molecular mechanism by which this enzyme brings about substrate
dehydrogenation is far from being solved. Mechanistic proposals revolve
around possible modes by which the substrate
C-H bond is being
broken in the step critical for catalysis.
The most prominent proposal is the carbanion mechanism, which is
characterized by initial abstraction of the -H as H+
leading to an intermediate in which the
-carbon carries a negative charge. Evidence in its favor has been discussed in various review articles (3, 4). So called "hydride mechanisms" in which a
H
is expulsed from
C-H also have been discussed at
various occasions but have not been proposed explicitly until most
recently by Mattevi et al. (5). From Miura and Miyake (6)
stems a proposal in which "the lone-pair electrons of the neutral
amino group of the substrate are transferred to the flavin in a
concerted manner with the abstraction of the
-proton." (For
schematic representations of the mechanisms and structures see Denu and
Fitzpatrick (7).)
An approach to investigate the molecular mechanisms of enzymes consists
in the correlation of reactivities (reaction rates) with the properties
of substrate substituents which influence the steric or electronic
properties of the latter. This approach was advocated originally by
Hammett (8) for chemical systems and was extended by Hansch and Leo
(9). Klinman and co-workers (10, 11) have pioneered its use in the
study of enzymatic reactions. Recently Walker and Edmondson (12) have
used it to study monoamine oxidase. As early as in 1966 Neims et
al. (13) have employed substituted phenylglycines for probing the
mechanism of pkDAAO; however, the results were contradictory. In
retrospect the reason for this is clear: these authors did rely on the
correlations of Vmax data hoping to probe the
reductive half-reaction. With pkDAAO the rate-limiting step in turnover
is, in general, product release (14). Later, Porter et al.
(15) using a series of substituted phenylalanines have correlated the
rate of the reversal of the dehydrogenation step of pkDAAO with the
Hammett n coefficient. They interpret their positive
(the numerical coefficient of
) as compatible with a carbanion
mechanism. Effects of the substituents on the rate of enzyme reduction,
on
G0 and, by reflection of the latter on
G
, did not get addressed.
It has been pointed out elsewhere (16-18) that the dehydrogenation of
amino acids by DAAOs should be mechanistically related to that of
-OH acids as catalyzed, e.g. by lactate monooxygenase or
flavocytochrome b2, to name only the two most
prominent members of this family. This assumption has been
substantiated nicely by Mattevi et al. (5), who have shown
that the active sites of pkDAAO and flavocytochrome
b2 are mirror images resulting probably from
convergent evolution. If these assumptions are correct, then the
mechanistic arguments from the two subfamilies should be usable reciprocally. A major argument against a simple carbanion mechanism is,
as discussed earlier (16), the finding of incorporation of the
substrate
-H into the 5-deazaflavin position C(5) both in the case
of pkDAAO and of
-OH acid oxidases (16, 19, 20). If a carbanion
mechanism was to be operative, this would require additional steps or
intermediates, since the H+ originating from the
C-H
cannot be transferred to the flavin N(5) or C(5) concomitantly with its
abstraction, unless the flavin position N(5) (or C(5) in 5-deazaflavin)
would be the "abstracting base" itself, a most unlikely
alternative. From this, the importance of the question about the
concertedness of the reaction, as stated and studied e.g. by
Denu and Fitzpatrick (7), becomes apparent.
During our recent studies on the catalytic mechanism of the two yeast
DAAOs from Rhodotorula gracilis and Trigonopsis
variabilis (21) using aliphatic D-amino acids, major
differences compared with pkDAAO have emerged. Importantly, the yeast
enzymes are more tolerant of variations in the chain of the amino acid;
they have, overall, much higher turnover rates and have a different
rate-limiting step, e.g. with alanine as substrate it is the
reductive half-reaction, compared with product release in the case of
pkDAAO (14). In view of this we have attempted to apply the concepts of
linear free energy relationships (LFER) using p-substituted
phenylglycines to probe the mechanistic questions mentioned above. The
rationale behind the choice of phenylglycine is that the electronic and inductive effects of substituents on the aromatic ring should be more
substantial compared with those in substituted phenylalanines as
studied by Porter et al. (15). We have worked out the
detailed kinetic mechanism for phenylglycines since we consider this to be an indispensable basis for linear free energy interpretations. Concomitantly, we have studied the primary deuterium isotope effect (on
C-H) and the solvent deuterium isotope effect on the dehydrogenation step of phenylglycine in order to establish whether, with
TvDAAO, it occurs in a concerted fashion or via
intermediates. The results are interpreted in view of the recently
solved three-dimensional structure of pkDAAO, the coordinates of which
were made available to us prior to publication by Mattevi et
al. (5).
D- and DL-phenylglycine,
p-F-, and p-OH-phenylglycine derivatives were
from Sigma. Phenylglyoxylic acid and its nitro-derivative were from
Lancaster. DL-[-2H]Phenylglycine was
prepared according to Ref. 22, and
p-NO2-phenylglycine according to Refs. 23 and
24. p-Cl-, p-Br, p-CH3-,
and p-CH3O-phenylglycine were synthesized from
the corresponding substituted benzaldehydes according to Ref. 25. The
purity of the synthesized compounds was checked by NMR and mass
spectrometry. All other reagents were of the highest purity
commercially available. DAAO from T. variabilis was provided
by Boehringer Mannheim and was further purified according to Pollegioni
et al. (26). Enzyme concentration was determined using an
455 = 10,800 M
1
cm
1 (27).
Steady state activity measurements were carried out polarographically at 25 °C in 90 mM Tris-HCl buffer, pH 8.3. All assay solutions contained 10 µM FAD and were air-saturated ([O2] = 0.253 mM). Enzyme was used in the 0.02-0.2 µM concentration range. Coenzyme and substrate solutions were freshly prepared daily. Rates were estimated from the initial velocities obtained from the linear portion of the traces using Grafit (Erithacus Software).
Rapid Reaction (Stopped-flow) MeasurementsThe experiments
were performed at 25 °C in 50 mM Tris-HCl buffer, pH
8.3, containing 100 mM KCl. All concentrations mentioned in
the context of the stopped-flow experiments are those of the reagent
after mixing, i.e. 1:1 dilution, and refer to the
D-isomer of the substrate. All the experiments were
performed in a thermostatted stopped-flow spectrophotometer which has a
2-cm path length cell and which is equipped with a diode array detector
(Spectroscopy Instruments, Gilching) interfaced with a MacIntosh IIcx
computer using a POSMA 2.3K data acquisition program (27). The
photometric response of the diode array is 80%, and indicated
absorbance values should be corrected accordingly in order to obtain
the correct values. Rapid reactions were routinely recorded in the 300-650 nm wavelength range using the normal scan mode with a scan
time of 10 ms/spectra and with a resolution of 2 pixels/nm. For fast
reactions (kobs > 20 s
1) a so
called "fast access" routine was used, which has an acquisition time of 1.0 ms/spectrum and a resolution of 5 nm.
For the reoxidation experiments the enzyme was reduced with a 1.5-fold
excess of substrate under anaerobic conditions in the presence of 100 or 400 mM NH4Cl and 50 mM
phenylglyoxylate which can lead to formation of the reduced
enzyme-ligand complex. Different [O2] in the reoxidation
mixture were obtained by equilibration of the buffer solutions,
containing NH4Cl and -ketoacid, with air (21%
O2), and with commercially available
N2/O2 mixtures (90/10, 50/50, v/v) and pure
O2. Anaerobiosis was obtained by repeated cycles of
evacuation and flushing with O2-free argon. Prior to experiments, oxygen was scrubbed from the stopped-flow apparatus using
the following procedure. The thermostatting solution was flushed with
N2 at 25 °C, and the syringes were incubated with a
solution of glucose and glucose oxidase (25 mM and 1 µM, respectively) for 10 h and then rinsed with
deoxygenated buffer.
To assess the effect of pH on the activity of TvDAAO
different buffer solutions, all containing 100 mM KCl, were
used as follows: 50 mM potassium phosphate below pH 7.7, 50 mM Tris-HCl around pH 8.3, and 50 mM sodium
carbonate at pH >8.9. The pH value was measured in the waste solution
after the shot. Enzyme-monitored turnover data were analyzed according
to the method of Gibson et al. (28). Program A (Dr. D. P. Ballou, University of Michigan) and KaleidaGraph (Synergy Software)
were used for fittings of the kinetic traces and for data analysis and
Specfit (Spectrum Software Association, Chapel Hill, NC) for
deconvolution analysis. Substituent parameters (,
,
+,
, and Es) were from Ref. 9 and
VM from Ref. 29. Fitting of
correlations of rates with substituent parameters (Equation 4, see
below) were done using Origin (Microsoft) and Statview (Abacus) and
using a maximum of two variable proportionality factors.
For the study of solvent isotope effects on the reaction with D-phenylglycine, solutions were prepared by dissolving the dry substrate and buffer chemicals directly in 2H2O and by diluting a concentrated enzyme solution 16-fold in the deuterated buffer. The pD value was taken as the reading of the pH electrode plus 0.4 (30), and the pD of the solution was adjusted with DCl to the desired value.
Catalytic Mechanism of Yeast DAAO with D-Phenylglycine
Polarographic MeasurementsThe kinetic parameters
Vmax and Km (AA)
for D-phenylglycine as substrate were estimated with the
polarographic assay at air saturation only ([O2] = 0.253 mM). The presence of the substrate L-isomer at
various concentrations was found to have no effect on the measured
rates, allowing the use of either the D-form or of the
racemic DL-mixture. The apparent steady state parameters
obtained for [-1H]- and
[
-2H]phenylglycine are reported in Table
I.
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The enzyme-monitored turnover method (28) was used at
different concentrations of both [-1H]- and
[
-2H]phenylglycine, and at an initial
[O2] = 0.253 mM in the range pH 5.5-9.15 by
recording continuously the absorbance spectra of TvDAAO in
the 350-650 nm wavelength range. The time course of the 454 nm
absorbance shows a first rapid decrease corresponding to 10-25% of
the total change, which is followed by a steady state and subsequently
by a final large decrease to yield the fully reduced enzyme. This
behavior indicates that the overall process of (re)oxidation of the
reduced DAAO forms with O2 is faster than that involving
the reductive half-reaction; it was observed at all pH values studied.
At pH 8.3 and with [
-1H]phenylglycine the enzyme is
present at 78% and with [
-2H]phenylglycine at
85%
in the oxidized form indicating a corresponding ratio of
1:4 and
1:6 for the reductive and oxidative half-reactions. Significant
amounts of the red anionic radical are formed, especially at low pH,
when the reaction time is >100 s, resulting in an alteration of the
absorption trace. In such a case the analysis was done at multiple
wavelengths to minimize the spectral contribution of the radical. In
spite of this, an overall uncertainty up to 15% has to be taken into
account for single parameters (Table II). The
Lineweaver-Burk plots of such traces show a set of converging lines at
all pH values; this is in contrast to the parallel line patterns
observed using D-alanine and D-valine as
substrate (21). These lines intersect in the lower left quadrant,
similarly to the situation reported for pkDAAO with
D-phenylalanine (15). These data were analyzed assuming an
ordered BiBi mechanism; plotting the slope and the intercept values of
the regression lines as function of the D-phenylglycine
concentration allows the estimation of
Km (AA),
Km (O2)), and
Ks (AA). The corresponding steady state
coefficients for [
-1H]- and
[
-2H]phenylglycine, respectively, and at pH 8.3, are
reported in Table II. The estimated
Km (O2) is very
low and differs from the values previously determined with
D-alanine and D-valine as substrate (0.2-0.8
mM) (21). The corresponding Dalziel coefficient for oxygen,
1/
O2, is not in agreement with the measured rate of the
O2 reaction with the reduced enzyme, for which a
second-order rate constant of 1.4 × 10
4 M
1 s
1 was
estimated (see below). As an example, at 2.5 mM
[substrate] and from the
AA and
O2
coefficients reported in Table II, corresponding rates of 16 and 102 s
1 for [
-1H]phenylglycine and of 6 and
38 s
1 for [
-2H]phenylglycine can be
estimated, in good agreement with the extent of initial flavin
reduction to approach the steady state phase mentioned above.
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The only other substrate investigated using the enzyme-monitored turnover method at pH 8.3 was p-F-phenylglycine. With this substrate a set of converging lines in the Lineweaver-Burk plot is again observed, and the corresponding steady-state coefficients are reported in Table II. The values determined with this substrate are in reasonable agreement with the polarographic ones, although the measurements were performed at different oxygen and enzyme concentrations (compare Tables I and II).
The effect of pH on the
kcat/Km (AA)
ratio with D-phenylglycine is depicted in Fig.
1. This dependence, using the
-1H-substrate, is consistent with the presence of two
functions with apparent pKa values of 8.1 ± 0.08 and >9.5 (the second one cannot be estimated accurately based on
the present measurements), where the first must be unprotonated and the
second protonated for activity. With
[
-2H]phenylglycine, where, due to the low activity,
only a limited number of measurements could be done at pH <7.5, only
one pKa 7.9 ± 0.1 can be estimated. The
DV/Km (AA)
value, the ratio of
Vmax/Km (AA) for
[
-1H]- and [
-2H]phenylglycine, is
6.1 ± 1.2 in the pH range 7-9.15 but does not show a definite
trend.
Polarographic Measurements with para-Substituted Phenylglycines
The data listed in Table I show the influence of the para substituent on Vmax and Km (AA). We have attempted to use CF3-phenylglycine as substrate; however, because of its extremely low solubility, no reliable data were obtained.
Solvent Isotope Effect on VmaxVmax values were determined with the enzyme-monitored turnover method in solvent of increasing content of 2H2O at pH 8.3. Their dependence from the 2H2O fraction follows a dome-shaped curve (Fig. 2). The experimental data points coincide reasonably with the theoretical curve derived from Equation 1, in which, for a good simulation, a deuterium isotope effect of 3.1 is required solely on the flavin reduction step k2 and none on the rate of product release k6 (see Scheme 1).
![]() |
(Eq. 1) |
The Reductive Half-reaction with D-Phenylglycine
The course of the reductive half-reaction is typically biphasic as shown in Fig. 3 and similar to that found in other cases with DAAO (15, 21, 32). This is interpreted as reflecting the sequence of steps represented by Equation 2.
![]() |
(Eq. 2) |
Starting with oxidized enzyme the traces at 454 nm, which reflect
flavin reduction, are biphasic and were best fitted by two sequential
exponentials, similar to those reported in a preceding paper for the
reaction with alanine and valine (21). The observed rates for the first
phase of reduction, kobs 1, exhibit saturation
with increasing [phenylglycine] and a finite intercept on the
ordinate as shown in Fig. 4. This is a typical case of a
two-step process involving formation of an enzyme-substrate complex
(steps k1, k1, Equation 2) followed by reversible reduction (k2,
k
2), where the y axis intercept
reflects k
2 (35, 36). The value of
k
2 was subtracted from
kobs 1 to estimate k2
and the apparent Kd (AA) using the
usual double-reciprocal representation (Fig. 4, inset). The
estimated kinetic parameters including the deuterium isotope effects
are reported in Table III. The amplitude of the first, fast phase of enzyme reduction, kobs 1,
contributes to
60% of the total absorbance changes and, for
[
-1H]phenylglycine, the separation between
kobs 1 and the ensuing phase is not very large.
As a consequence the estimation of velocities as first-order rates is
somewhat imprecise and the error depends on the substrate
concentration. It should be noted that the apparent values of
Kd (AA) as reflected, e.g. by the abscissa intercept in double-reciprocal plots of
kobs 1 (corrected for
k
2) versus the concentrations of
[
-1H]- and [
-2H]phenylglycine, yield
straight lines extrapolating to a similar but not to the same value
(Fig. 4, inset). This indicates a situation where true
pre-equilibrium conditions (k
1
k2) are not fully satisfied as was the case also
for R. gracilis DAAO and D-alanine (21). In
order to verify this case we have carried out a simulation of the
reactions with [
-1H]- and
[
-2H]Dphenylglycine at all concentrations
used and at selected concentrations of the other substrates. For
obtaining a reasonable duplication of the experimental traces
(cf. Fig. 3), the minimal values required for
k1 and k
1 were 6 × 104 M
1 s
1 and 40 s
1 for all substrates. This indicates that for
[
-1H]phenylglycine k
1
k2 while for the
-2H-analogue the
smaller value of k2 resulting from the isotope effect leads to a true pre-equilibrium condition where
k
1
k2 (35, 36).
The values of the rates required for the simulation are consistent with
k
2 being of the same order as
k2 (Table III). A particular situation concerns
[
-2H]D-phenylglycine where the rates of
k2, k
2, and
k3 are of the same magnitude. For this case the
best simulations were obtained using values of
k2 = 4.9, k
2 = 1.8, and
k3 = 2.4 s
1, which are in good
agreement with the experimentally determined values listed in Table
III. A further point is the absolute rate for k3
using [
-1H]- and
[
-2H]D-phenylglycine, which shows an
apparent isotope effect
2.2. Extensive simulations suggest that this
effect is real; although its molecular origin is unclear at present, it
might be speculated that it results either from coincidence of kinetic
rates or is related to steps subsequent to reduction and involving
product conversion to the ketoacid and/or dissociation.
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The spectral deconvolution of the primary data for the reductive
half-reaction with various substrates and obtained using the
stopped-flow spectrophotometer equipped with a diode array detector was
done with the Specfit program. In all cases three different spectral
forms corresponding to the oxidized, the fully reduced, and to one
intermediate, the Ered~IA complex were found. These spectral species have the same extinction coefficients as those
derived from single wavelength analysis of the experimental data and
which were used for the simulations (see legend of Fig. 3). The value
of k2 is higher than the 1/0 term
determined from steady state conditions. This signifies that the
oxidative half-reaction is partially rate-limiting in the reaction of
TvDAAO with phenylglycine, whereas with
D-alanine and D-valine the reductive
half-reaction is substantially smaller than the oxidative one and thus
0 is <1/k2 (21). With pkDAAO and
D-phenylalanine the bimolecular coefficient is significant
because of the relatively large value of the reverse of the reductive
half-reaction "krev" (15). The isotope
effect of
6 found on k2 is high and similar
to those reported for D-alanine and D-valine
(21). In the case of some substrates such as [
-2H]-
and p-CH3-phenylglycine, the rate of the second
phase of enzyme reduction kobs 2 exhibits some
dependence on the substrate concentration; this is consistent with
k
2 being an important term in the reductive
half-reaction and with k
2 being in the same
order of magnitude as k3 (see Table III). This
leads to a difficult estimation of k
2 for
these substrates. In analogy with our previous study, we assume that
kobs 2 reflects product release as such or in
combination with conversion of IA to the ketoacid (21). The value of
kobs 2 (
6 s
1, Table III) is
too small to be important in turnover of the enzyme (14.0 s
1, Table II); therefore, reoxidation of the reduced
enzyme must result largely from reaction of O2 with the
Ered~IA (or
Ered~ketoacid) complex. The Arrhenius plot of
the reductive half-reaction (k2) of
TvDAAO with phenylglycine as substrate was linear in the
15-35 °C range, allowing to estimate an activation energy of 56 kJ/mol (data not shown).
The
time-dependent reduction of oxidized TvDAAO is
biphasic with all substrates used, and the 454 nm traces are best
fitted by two sequential exponential to yield values for
kobs 1 and kobs 2,
respectively. With p-CF3-phenylglycine no useful data could be obtained due to the low solubility of the compound. As
with unsubstituted phenylglycine, with all p-substituted
analogues no appreciable charge-transfer absorption was observed. In
all cases the direct plot of kobs 1
versus [p-X-phenylglycine] resulted in
traces with a definite ordinate intercept similar to that observed with
phenylglycine (Fig. 4), allowing the estimation of
k2, k2, and
Kd (AA) (cf. Equation 2, data in Table III). In all cases, the value of
k2 is higher than 1/
0 determined
under steady state conditions, confirming that the oxidative
half-reaction in turnover is partially rate-limiting. With the
exception of p-NO2-phenylglycine, the
Kd values for all derivatives are within a narrow
range around 0.7 mM (Table III). In contrast, the rates of
enzyme reduction vary
30-fold and those of
k
2
7-fold. Only with
p-OH-phenylglycine k
2 is small
compared with k2, whereas with
p-Cl-phenylglycine k
2 and
k2 have similar absolute values. Because of the
similar magnitude of k3 and
k
2 for many substrates, the values of
k3 reported in Table III can be considered only
an estimation of this rate constant, in agreement with the values used
in simulation of the experimental traces.
The solvent deuterium isotope effect on the
rate of enzyme reduction was estimated by varying the
1H2O/2H2O fraction
between 0 and 93% as shown in Fig. 2B. The experiments were
carried out at a constant and fixed [D-phenylglycine] = 2 mM, which is close to the limit of solubility and
Km. The experiments were carried out within 120 min in order to minimize the time of incubation of TvDAAO in
deuterated solvent; indeed, the variation of the rate of reduction with
the time of incubation was negligible during the 10-15 min required
for the measurements. This is somewhat surprising in that there is no
apparent incorporation of deuterium in the protein in the mentioned
time frame, which would affect the reduction rate. The dependence of
kobs 1 from the
1H2O/2H2O fraction
depicted in Fig. 2B is linear, compatible with the fission
of a single exchangeable H bond during flavin reduction, and
extrapolates to a solvent isotope effect of
2.7 (ratio of left- and
right-hand side ordinate intercepts for
[
-1H]phenylglycine). This is in good agreement with
the value Dk2
3.1 estimated from
turnover experiments (Fig. 2A, cf. above). The
same type of experiment using [
-2H]phenylglycine
yields a solvent isotope effect
4.5 (Fig. 2B). The
deuterium isotope effect for the rupture of the
C-H bond in
H2O is
5 (Fig. 2B, ratio of
left-hand ordinate intercepts). It corresponds to the
6
on kobs 1 (Table III) estimated from the data
of Fig. 4 at saturation. The isotope effect on
kobs 1 for the
C-H bond in
2H2O is
8.5 (Fig. 2B, ratio of
right-hand ordinate intercepts). Finally, the "double isotope
effect" (rates of [
-1H]phenylglycine in
H2O and [
-2H]phenylglycine in
2H2O) is
24. The effect of the pH on the
solvent isotope effect cannot be addressed in the present work; it is
the subject of an ongoing study. The pH could have some effect on the
magnitude of the isotope effects, but we consider it unlikely that it
will modify them substantially and thus affect the overall conclusions. The isotope effects in question are derived from
kobs 1 which is the sum of
k2 and k
2. Thus and
since Dk2 > Dk
2, the intrinsic isotope effects
on k2 are likely to be somewhat higher, as can
also be deduced from comparison of the corresponding values for
kobs, k2, and
k
2 obtained from [substrate] dependencies
and listed in Table III.
The (re)oxidation of reduced enzyme forms with oxygen was investigated using reduced TvDAAO in the presence of NH4+ and phenylglyoxylate. It could be not studied with free Ered, due to its rapid conversion to the radical anion in the presence of light (21). The course of the reaction is clearly biphasic in the presence of 50 mM phenylglyoxylate and of 100 or 400 mM NH4Cl. The experimental traces are best fitted by two exponentials and assuming parallel reactions, with the reoxidation originating from the free Ered, and ligand (L) complexed enzyme according to Equations 3a and 3b.
![]() |
(Eq. 3a) |
![]() |
(Eq. 3b) |
Linear Free Energy Relationships of Yeast D-Amino Acid Oxidase
Structure-Activity Relationships of the Apparent Vmax Using para-Substituted PhenylglycinesCorrelations of Vmax values with
Hammett parameters have been attempted by Hellerman's group (13).
Their correlations were strongly biphasic, comprising branches with
positive and negative slopes, and could not be interpreted
mechanistically. With the substrates listed in Table I correlations
were attempted first with the following substituent parameters (9):
electronic (,
+,
), hydrophobic
(
), and steric parameters (VM = van der Waals molar substituent volume, Es = Taft steric parameter). A general trend is already apparent from
correlations of Vmax with single parameters in
that there is a marked dependence from
VM and a dependence from
with a
having a negative slope (not shown). Much better
correlations were obtained using two-parameter fits and the modified
Hammett equation (Equation 4) as also used by others (11, 12),
![]() |
(Eq. 4) |
The results listed in Table IV indicate that
VM has a much larger effect on
Vmax compared with parameters. (Note that
VM parameters have larger
values compared with
ones.) The magnitude of the obtained
values is small, and their signs as well as that of x for
VM correlation are
negative.
|
The rate constants
(k2) determined for the reductive half-reaction
with various para-substituted phenylglycine analogues (Table III) were correlated with electronic (), hydrophobic (
), and steric (Es and VM) parameters
using Equation 4. Fits were initially attempted with single parameter
correlations, but for reasonable results two parameter fits were
necessary (Table V). In all cases,
p-NO2-phenylglycine shows an anomalous behavior and was not included in further analysis. The plots of the
two-parameter (
+ + VM) correlations of
k2 are shown in Fig. 5,
A and B. The best correlations
were obtained with
+ as compared with
or
, whereby, as in the case of the correlations of
Vmax, the volume term appears to be the most
important factor. In all cases the slope
is negative. For
k
2 remarkably good fits are obtained already
with single parameters and in particular with
+ (not
shown, Table V). The two parameter correlations of
k
2 with either
+ or
together with VM are of similar
quality (Table V) and that with
+ is shown in Fig.
6.
|
As pointed out in the Introduction we have worked out the kinetic mechanism with phenylglycines as a basis for the interpretation of LFERs. The general catalytic behavior is similar to that reported previously for D-alanine and D-valine (21). There is, however, a major difference, reflected by the convergent pattern of double-reciprocal plots of steady state analysis, which is indicative of a sequential, ordered BiBi mechanism. According to Dalziel (37) the steady state of this mechanism is described by Equations 5a and 5b which base on the sequences of Scheme 1,
![]() |
(Eq. 5a) |
![]() |
(Eq. 5b) |
This is different from what we reported for D-alanine and
D-valine (21) because the fourth term of Equation 5a is not
0 and 1/0 is < k2,
compatible with k6 being of the same order as
k2 (Scheme 1, see also Tables II and III). Using
the values of k2 and Vmax
for [
-1H]- and [
-2H]phenylglycine
listed in these tables, a k6 value of 20-30
s
1 can be estimated, which is close to that of
k2. A very important difference is the
reversibility of the reductive half-reaction with values of
k
2 being dependent on the nature of the para-substituent and in the range 1.6 to 75% that of
k2 (see Table III).
A further difference between phenylglycine and alanine or valine as
substrates concerns the ratios of the steps k1,
k1, and k2,
i.e. the question about the existence of pre-equilibrium conditions during the reductive half-reaction. The regression curves
shown in Fig. 4 for [
-1H]- and
[
-2H]phenylglycine do not extrapolate to the same
abscissa intercepts (see Table III for
Ks (AA) values). This is interpreted as
reflecting a situation where, with
-1H,
k
1 is not
k2,
whereas this is the case with [
-2H]phenylglycine due
to the deuterium isotope effect on k2.
Consequently the values of Kd (=
k
1/k1) for
[
-2H]phenylglycine can be assumed to be valid also for
the
-1H form, and k2 can be
estimated as described by Porter et al. (15). Note that the
conspicuous isotope effect on k2 (
6) is largely conserved (i.e.
3.9) on turnover in agreement
with the estimation of k6 discussed above.
The rates of reoxidation estimated with the stopped-flow method are
clearly not consistent with the steady state parameters 02 and
AAO2 (see Equations 5a and 5b).
These probably reflect reoxidation of EFlred via
k4 and of EFlred~IA (or
EFlred~ketoacid) via k5
(Equations 3a and 3b) occurring in parallel. Indeed the simulated course of et/v (Fig. 3) obtained using
the rate constants from rapid reaction studies (Table III) and a
k5 = 3 × 105
M
1 s
1 satisfactorily matches
the experimental traces, indicating that this value of
k5 is close to the intrinsic one.
There are thus substantial differences between TvDAAO and
pkDAAO in details of their catalytic mechanisms. These are due, however, to differences in the absolute values of single steps, the
general picture remaining the same. On the other hand important similarities exist such as the coincidence of pK values of
catalytic groups (8.1 and >9.5, Fig. 1) for TvDAAO and
phenylglycine compared with 8.7 and 10.7 or 8.1 and 11.5 for pkDAAO and
serine or alanine, respectively (31), as expressed by the pH
dependences of turnover. Also similar is the requirement of a
deprotonated group with pK 8 is for catalysis. This
coincidence supports the assumption that the yeast and pig kidney
enzymes operate by the same basic catalytic mechanism.
A
cardinal point for the discussion of the mechanism of DAAO is whether
the reductive half-reaction, which involves cleavage of the substrate
C-H and of the
N-H bonds as well as transfer of reducing
equivalents to the (oxidized) flavin cofactor, proceeds in a
synchronous/concerted fashion or whether distinct intermediates occur.
The carbanion mechanism, for instance, is difficult to be formulated
without the occurrence of intermediates. Criteria, based on which a
differentiation should be possible, are e.g. the deuterium
isotope effects observed on breaking either one of the two or both
bonds involved (7, 37-39). In the case of a concerted reaction (single
transition state) the increase in activation energy is due to both
deuterium substitutions and will, at first approximation, be additive
and thus the effect on kobs 1 will be
multiplicative. This is similar to the case of dopamine
-monooxygenase reported by Miller and Klinman (11). In contrast, in
the case of the occurrence of an intermediate, the two isotope effects
should behave additively or only a single one will be expressed. For
TvDAAO the experimentally observed isotope effect on the
fission of the
C-H bond (Fig. 2B) and the solvent ones compute as 2.7·8.5 = 23 and 5·4.5 = 22.5, and this
compares to the experimental "double" isotope effect
24
(
C-1H/2H and
1H2O/2H2O, Fig.
2B). In the case of an occurrence of intermediates a value
of maximally 10-12 might be expected. The reductive half-reaction is
thus most likely concerted/synchronous. The linearity of the dependence
of the rate of kobs 1 from the
2H2O fraction (Fig. 2B) is
compatible with fission of one exchangeable 2H bond during
dehydrogenation; a likely candidate is that of
C-N-2H2/
C-N-2H3+.
There is a discrepancy between our results and what was reported by
Denu and Fitzpatrick (7) for pkDAAO and D-serine, who found no solvent isotope effect in studies of
V/Kser versus pH and at pH
>9. In our case a solvent isotope effect was found both in turnover
(Fig. 2A) and, most importantly, on the reductive
half-reaction itself (step kobs 1, Fig.
2B). Differences between pkDAAO and TvDAAO, in
detail, e.g. regarding the concertedness of the reaction,
are conceivable. Thus, the same authors using pkDAAO and
D-alanine as substrate (40) have reported a deuterium
solvent isotope effect
2.1 and
2.6 on Vmax
at pH 6.0 and 10.0 corresponding to 1 and at least 2 proton
inventories, respectively. They attribute these effects to fission of
an X-1H/2H bond during product
release, the rate-limiting step with pkDAAO, and to an equilibrium
between two enzyme species one of these being predominant at the
different pH values.
The single parameter correlation of
Vmax, k2, or
k2 values measured with the
para-substituted phenylglycines resulted always in rather
poor fits, the best statistical correlation being that of
k
2 with the Hammett
+ parameter
(Table V). This is in line with the results of Porter et al.
(15), who found a good correlation of the rate of the reversal of
dehydrogenation of substituted phenylalanines by pkDAAO with
n. Two parameter fits, according to Equation 4, provide much
better statistical correlations when the electronic parameters (
,
+,
) are used in conjunction with the
van der Waals volume VM (Tables
IV and V). The contribution of the steric factor is comparatively large
(VM range is 4-17) compared
with the electronic ones (range
1/+0.2). The reasons for the
requirement of large steric parameters becomes evident upon inspection
of the three-dimensional structure of the active site of pkDAAO
complexed with benzoate (5). The aromatic ring of the latter is
sandwiched between the flavin and Tyr-224, its para-position
is close to the side chains of Leu-51 and Gln-53, and its sides are in
contact with the side chains of Ile-215 and Ile-230. As can be deduced
from a sequence alignment, in TvDAAO groups of comparable
steric requirements are in corresponding positions, namely Tyr-224 > Asp-239, Gln-53 > Leu-52, Leu-51 > Asn-50. There is no
apparent open access to the exterior solvent from the substrate binding
site in the pkDAAO complex with benzoate. We have modeled phenylglycine
at the active center of pkDAAO replacing benzoate (5), the position of
both -COO
being constant. This shows that the aromatic ring
of phenylglycine points toward the solvent and is close to it. The
active site of TvDAAO must be more flexible or open to
solvent since it is able to accommodate a large volume variation at the
p-position of phenylglycine. This is also in agreement with
the finding of little change in the dissociation constants
Kd
(=k
1/k1) of
unsubstituted phenylglycine and of all p-substituted
analogues, with the exception of p-NO2 (Table
III). In view of the startling similarities of the active centers of
pkDAAO and
-OH-acid oxidases uncovered by Mattevi et al.
(5), it is tempting to assume that the active center of
TvDAAO is more similar to that of the latter, where the
substrate chain extends toward and is in contact with the solvent. In
fact, using lactate oxidase or lactate monooxygenase and a series of
mandelates carrying the same substituents as the phenylglycines used in
this work, a similar independence of Kd from the
substituent was observed.2
During dehydrogenation, and irrespective of the involved mechanism, a
conversion of the substrate pyramidal sp3
-carbon center into a planar sp2 product
occurs, and this requires substantial movement of the three
C
substituents. Importantly, in the intermediate transition state a
product-like configuration with sp2 character
will be encountered. Based on this one would predict large steric
effects on k2 and much less pronounced ones, if
any at all, for the "back reaction" k
2.
The experimental findings correspond fully to this expectation (Tables
IV and V, Figs. 5 and 6). In other words, "steric work" (movement
of the substrate substituents and/or active side residues) is necessary
for the interconversion of substrate and transition state, not (or
little) for that of the transition state into product. The
three-dimensional features of DAAO also provide a rationale for the
deviant behavior of p-NO2-phenylglycine. The
-NO2 group is the largest in the series, it is highly
polar, and specific interactions can be expected to occur,
e.g. with Asp-239 or Asn-50. It is worth noting that also in
the case of monoamine oxidase B data obtained with
NO2-substituted substrates do not fit in LFER correlations
(12).
While the validity of Hammett type correlations for the understanding of organic reaction mechanisms is undisputed (41), their interpretations in the case of enzymatic mechanisms is much more difficult due to the uncertainties arising from interactions with the protein. Positive precedents for the use of the LFER approach with flavin enzymes are bacterial luciferase (42) and monoamine oxidase by Walker and Edmondson (12).
We subdivide our analysis of the correlations with electronic
parameters into that of k2, that of
k2 (yielding the
G
, the activation energy for the
interconversion of the substrate and imino acid via the transition
state), and that of the ratio of
k2/k
2 (yielding
G0 the apparent free energy of the reaction,
excluding binding steps).
G
for
k2 can be affected either by substituent induced
changes of the energies of the substrates ground state and/or by
changes of transition state energies (cf. Fig. 8). The
variation of Kd values for formation of the
Michaelis complex within the series of substrates used is from 18.0 to
20.9 kJ·M
1 and thus negligibly small,
i.e.
G0
constant (
19
kJ·M
1). In the case of
p-X-phenylglycines/p-X-phenyliminoglyoxylates, substituent induced differences in ground state free energies are
likely to be much larger on the side of products due to the through
conjugation. For this reason we have normalized the ground state free
energy levels of bound substrates as shown on the left-hand side of
Fig. 8. From the plots of Fig. 5, it is evident that for the
+ correlation of k2
is
negative and has a small value of
0.733. Taken at face value, this
would suggest that p-substituents exert little electronic
effects, and, if at all, a partial positive charge develops in the
transition state.
For the + (or
) correlation of
k
2, on the other hand,
is positive (0.398 and 0.702, respectively), i.e. opposite as compared with the
same correlation with k2. The similarity of the
absolute values of
for the
+ correlations of
k2 and k
2 satisfies the
principle of microreversibility. For completing the picture,
differences in ground state energies of products (complexed to the
enzyme) have to be considered. Redox potentials are not available for
the series of p-substituted phenylglycines used in this
work. But simple chemical considerations suggest that, qualitatively,
the p-X substituent will exert a strong(er) effect on the ground state free energy of products. Electron
withdrawing substituents should increase it, and donating ones have the
opposite effect (41). In the representation of Fig. 8 dissociation of products from Ered has not been taken into
account. On one hand it was not addressed experimentally; on the other
hand only steps up to Ered~IA (Equation 2) are
of relevance for the intended correlations. The ground state energy
levels of bound products (
G0) have thus been
estimated from the ratio
k2/k
2
Ke, the internal redox equilibrium of the
(de)hydrogenation reaction.
The assessment of the effect of ground state energy levels on
G
is of importance for the discussion of
substituent effects as has been discussed also by Miller and Klinman
(11). In this context, Marcus (43) has derived a relation (Equation 6)
which describes the effect of
G0 on
G
for reactions involving the transfer of
H+,
![]() |
(Eq. 6) |
In our case, and in view of these considerations, we think it is safe
to conclude that in the dehydrogenation of phenylglycines catalyzed by
TvDAAO and as described by Equation 2, there is probably little if any development of (positive) charge in the transition state.
This minimal interpretation is also in agreement with the finding of
the best correlation of k2 and
k2 with
+, the Hammett
parameter for positively charged transient species. The validity of our
reasoning is supported by the analysis of the correlations with
VM (i.e. upon
correction for the electronic effects, Figs. 5B and
6B). In this case a Brønsted correlation with
1.2 is
obtained (not shown), suggesting that the steric requirements of the
substituents have an important effect on the energy level of the
transition state. From the Brønsted plot of Fig. 7 the intrinsic
(i.e. substituent independent or that determined only by
electronic effects) reaction barrier of the dehydrogenation reaction
G0
(intrinsic) can be estimated
as
58 kJ·M
1.
Mattevi et al. (5) have
proposed convincingly that the active site of flavocytochrome
b2 (and thus of the -OH-acid hydroxylase family) can be described as the mirror image of that of DAAO and that
the localization of benzoate in DAAO coincides with that of pyruvate in
flavocytochrome b2. We have done an analysis
similar to the present one using L-lactate oxidase and
L-lactate monooxygenase and a series of
p-substituted mandelic acids.2 In these cases
the rate of flavin reduction correlates with the substrate substituent
Hammett
value, and there is no necessity for correction for steric
parameters. Therefore the assumption mentioned in the introduction that
the two enzyme classes operate by a similar mechanism is reasonable.
Our data are compatible with any mechanism in which little or no charge
is developing in the transition state. The energetic profile of the
reaction should be symmetrical (Fig. 8), the fission of
involved bonds occurring synchronously. This does not exclude
"carbanion mechanisms," as long as intermediates do not carry
substantial negative charge at the substrate
C. This is the case in
the formulation by Miura and Miyake (6) but not in that of Lederer (18)
for flavocytochrome b2 or that for lactate
monooxygenase (44). Clearly, the interpretation of the present data in
terms of a hydride transfer mechanism, as done independently by Mattevi
et al. (5), is much more convincing. The base at the active
center of pkDAAO which might function in H+ abstraction is
Tyr-224, possibly coupled to a H2O molecule. Removal of the
Tyr-OH group by mutagenesis (Tyr-224-Phe) leads to a protein with
substantial activity (45). The same holds for the other tyrosine
residue at the active center (Tyr-228-Phe, (45)). The cases of these
two mutants would lead to the seemingly paradox situation in which just
no functional group capable of acid/base catalysis is left at the
active center of active DAAO, while the enzyme is still active! In a
hydride transfer mechanism and for the Tyr-224-Phe mutant, during the
conversion
NH3+
=NH2+ + H+ (or
NH2
=NH + H+), H+ just
would dissociate into solvent. With normal DAAO removal of the same
H+ would be promoted by H2O linked to Tyr-224
and as shown in Scheme 2. This role would be exerted by
a histidine within the family of
-OH-acid dehydrogenases
(e.g. His-373 in cytochrome b2 (18)). The
difference in functional groups (Tyr versus His) between the two types of "dehydrogenases" would reflect an evolutive adaptation to the different pKa values of the substrate
-XH group (X = O or NH).
We are aware that the conclusions arising from the present work are apparently difficult to reconcile with the carbanion or with other mechanisms proposed previously by others and also by ourselves. An option worth reconsidering might be the occurrence of different mechanisms depending on the type of substrate (16). Finally it is impossible to discuss in the present context all the arguments concerning these mechanisms which were put forward over the years in several dozens of papers; this will have to be done in propri loci.
Dedicated to Prof. Dr. V. Massey, who has been one of the pioneers in the study of flavoproteins, in honor of his 70th birthday.
We thank Drs. Mirella Pilone, Bruce Palfey, D. Edmondson, P. Fitzpatrick, and V. Massey for many suggestions and critical comments and K. Janko for the synthesis of several of the phenylglycines. We are indebted with Dr. W. Tischer, Boehringer Mannheim, for a generous gift of TvDAAO. A special thanks is due to Dr. A. Mattevi and collaborators for providing the three-dimension coordinates of pkDAAO prior to publication and for many fruitful discussions.