Reaction Specificity of Native and Nicked
3,4-Dihydroxyphenylalanine Decarboxylase*
Mariarita
Bertoldi,
Paolo
Frigeri,
Maurizio
Paci
, and
Carla Borri
Voltattorni§
From the Dipartimento di Scienze Neurologiche e della Visione,
Sezione di Chimica Biologica, Facoltà di Medicina e
Chirurgia, Università degli Studi di Verona, Strada Le Grazie, 8,
37134 Verona and the
Dipartimento di Scienze e
Tecnologie Chimiche, Università di Roma Tor Vergata, 00173 Roma, Italy
 |
ABSTRACT |
3,4-Dihydroxyphenylalanine (Dopa) decarboxylase
is a stereospecific pyridoxal 5'-phosphate (PLP)-dependent
-decarboxylase that converts L-aromatic amino
acids into their corresponding amines. We now report that reaction of
the enzyme with D-5-hydroxytryptophan or D-Dopa
results in a time-dependent inactivation and conversion of
the PLP coenzyme to pyridoxamine 5'-phosphate and
PLP-D-amino acid Pictet-Spengler adducts, which have been
identified by high performance liquid chromatography. We also show that
the reaction specificity of Dopa decarboxylase toward aromatic amines
depends on the experimental conditions. Whereas oxidative deamination occurs under aerobic conditions (Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol.
Chem. 271, 23954-23959; Bertoldi, M., Dominici, P., Moore,
P. S., Maras, B., and Borri Voltattorni, C. (1998)
Biochemistry 37, 6552-6561), half-transamination and
Pictet-Spengler reactions take place under anaerobic conditions.
Moreover, we examined the reaction specificity of nicked Dopa
decarboxylase, obtained by selective tryptic cleavage of the native
enzyme between Lys334 and His335. Although this
enzymatic species does not exhibit either decarboxylase or oxidative
deamination activities, it retains a large percentage of the native
transaminase activity toward D-aromatic amino acids and
displays a slow transaminase activity toward aromatic amines. These
transamination reactions occur concomitantly with the formation of
cyclic coenzyme-substrate adducts. Together with additional data, we
thus suggest that native Dopa decarboxylase can exist as an equilibrium
among "open," "half-open," and "closed" forms.
 |
INTRODUCTION |
Recombinant pig kidney
Dopa1 decarboxylase (DDC; EC
4.1.1.28) is a homodimer containing two molecules of pyridoxal
5'-phosphate (PLP) per protein dimer (1). Its primary structure has
been determined (2), and according to the classification of Sandmeier et al. (3), DDC is a group II decarboxylase. This allows the identification of some functionally important residues of the enzyme by
multiple sequence alignment with other group II decarboxylases combined
with site-directed mutagenesis (4). The enzyme is a stereospecific
-decarboxylase that catalyzes the conversion of
L-aromatic amino acids into their corresponding amines (5). D-Aromatic amino acids have also been shown to bind to the
active site of the enzyme. Whereas aromatic amino acids with a
catechol-related structure in the D form have been shown to
inhibit enzymatic activity and to form stable intermediate complexes
with the enzyme absorbing at 430 nm, the indole-related D
forms exert a time-dependent inactivation and form
intermediate complexes absorbing at 430 nm which undergo time-dependent modification (5). The behavior of the latter amino acids has been ascribed to a half-transamination reaction, although the stoichiometry of this reaction appeared to be abnormal (5). This is similar to the situation with 5-hydroxytryptophan (5-HTP)
and
-methyl-dopa (6) for which a
decarboxylation-dependent transamination had been proposed
(7, 8). However, it has been demonstrated recently that in the case of
serotonin (5-HT), dopamine, and
-methyldopamine this minor reaction
catalyzed by DDC is actually an oxidative deamination and not a
decarboxylation-dependent transamination. Indeed, ammonia
and the corresponding aldehyde or ketone are produced in equivalent
amounts, while O2 is consumed in a 1:2 molar ratio with
respect to these products (9, 10). In the light of these results, a
reinvestigation of the interaction of DDC with D-aromatic
amino acids appears to be necessary.
Previous studies have provided evidence that partial trypsinolysis of
DDC leads to the exclusive cleavage of the
Lys334-His335 peptide bond (2). The nicked and
native proteins have identical spectroscopic features and coenzyme
content. The partially proteolyzed protein will bind aromatic amino
acids in L- and D forms but lacks decarboxylase
activity (11). Loss of decarboxylase activity would appear to be
incompatible with the apparent preservation of the three-dimensional
structure of the active site of the proteolyzed enzyme. However, it has
been observed recently that the binding of aromatic amino acid methyl
ester analogs to DDC in the native and nicked forms varies depending on
both the analog structure and the enzyme form. This would suggest that
the binding of these analogs to the nicked enzyme does not occur as to
the native enzyme (12). To gain some insight into the nature of the
interaction of substrates and substrate analogs with the nicked enzyme,
spectral and kinetic studies of the interaction of this enzymic species with various ligands have been performed and compared with those of
native enzyme.
Here, we present spectral and kinetic evidence for the occurrence of
concomitant half-transamination and Pictet-Spengler reactions during
the interaction of native or nicked DDC with D-aromatic amino acids. We also report data showing that half-transamination and
Pictet-Spengler reactions take place when the native enzyme reacts with
aromatic amines under anaerobic conditions and when the nicked enzyme
reacts with these ligands under aerobic conditions. In Scheme
I the Pictet-Spengler reaction of PLP
with 5-HTP or Dopa is reported. Moreover, protection against limited
tryptic proteolysis of native DDC by aromatic amino acids and amines
has been examined as well as the effect of these ligands on the
spectral properties of bound PLP. Together, these data suggest that DDC forms different intermediate complexes, possibly resulting in the
stabilization of different conformational states. Herein, the possible
relationship of these conformations with the reaction specificity of
DDC is discussed.
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EXPERIMENTAL PROCEDURES |
Materials--
D-5-HTP, D-tryptophan,
D-Dopa, 5-HT, dopamine, PLP, pyridoxamine 5'-phosphate
(PMP), indole-3-pyruvic acid, bovine liver L-glutamic dehydrogenase, and horse liver alcohol dehydrogenase were purchased from Sigma. All other chemicals were of the highest purity available.
Enzyme Purification--
Recombinant DDC was purified to
homogeneity from Escherichia coli expressing pKKDDC
4
3'
as described (1, 9) and was used throughout. The enzyme concentration
was determined using a molar extinction coefficient of 1.30 × 105 M
1 cm
1 (13).
Limited tryptic digestion of DDC was performed as described (11).
Enzyme Assays and Inactivation Assays--
DDC activity was
measured as described by Sherald et al. (14), as modified by
Charteris and John (15). The inactivation incubation mixtures contained
native DDC (10 µM) and freshly diluted inhibitor
(D-5-HTP or D-Dopa) at varying concentrations
(0.05-5 mM) at 25 °C in 100 mM potassium
phosphate, pH 7.5. At various time intervals, aliquots were removed and
assayed for residual decarboxylase activity. Production of ammonia or
5-hydroxyindolacetaldehyde by the reaction of DDC with 5-HT was
determined by spectroscopic assays using the coupled system with
glutamate dehydrogenase or alcohol dehydrogenase, respectively, as
already described (9). Reaction of DDC with L-5-HTP,
L-Dopa, or 5-HT under anaerobic conditions was performed
using 1-ml Reacti-Vials (Aldrich) as already described (10).
SDS-Polyacrylamide Gel Electrophoresis--
SDS electrophoresis
on 12.5% polyacrylamide slab gels was performed according to Laemmli
(16).
Kinetic Measurements--
The rates of the reaction of DDC with
D-Dopa or D-5-HTP in 100 mM
potassium phosphate buffer, pH 7.5, at 25 °C were obtained by
observing the decrease of the 430 nm absorbance band. The kinetic measurements were performed at several D-aromatic amino
acid concentrations using an excess of D-aromatic amino
acid over enzyme concentration (2 µM). For each
D-5-HTP concentration, the time course was recorded. The
apparent pseudo first-order rate constants,
kobs, were calculated by nonlinear least squares
regression fitting of a single exponential function
|
(Eq. 1)
|
where A is the measured absorbance at 430 nm,
kobs is the observed rate constant, t
is time, A
is an offset to represent a
nonzero base line, and A0 is the absorbance
associated with kobs. The dependence of
kobs on D-5-HTP concentrations
exhibits a saturation behavior, and a hyperbolic fit gives the value of apparent dissociation constant, KD, and the maximum
value of rate constant, kmax.
Spectral Measurements--
All of the spectral measurements were
carried out using 100 mM potassium phosphate, pH 7.5, at
25 °C. Absorption spectra were recorded in a Jasco V-550
spectrophotometer. Circular dichroism (CD) measurements were carried
out in a Jasco J-710 spectropolarimeter at a protein concentration of
10 µM. Spectra were recorded at a scan speed of 50 nm/min
with a band width of 2 nm and averaged automatically except where
indicated. NMR spectra were obtained on a Bruker AM-400 machine
operating at 400.131 MHz. A spectral width of about 20 ppm was used,
and a repetition rate of 4.5 s was applied. After a 90° pulse
(6.2 µs) free induction decays were stored on 16,000 bytes of memory
and then, after multiplication for a sine bell shifted of
/3, were
Fourier-transformed. For every experiment a typical average of 32 scans
was obtained. The ppm scale was referred on tetramethylsilane on the
basis of the resonance frequency of water at 300 K. Resonance caused by
the residual water protons present in deuterated water was suppressed by presaturation irradiating for 1.66 s before pulsing. In the time-dependent experiments time was taken as the mean
between the start and the end of averaging using the same number of scans.
Characterization of the Reaction of PLP with D-5-HTP
or D-Dopa and Chemical Synthesis of Pictet-Spengler
Adducts--
The addition of D-5-HTP or D-Dopa
at varying concentrations (1-10 mM) to 20 µM
PLP in 100 mM potassium phosphate buffer, pH 7.5, results
in marked absorption spectral changes that consist of an immediate
shift of the maximum at 388 nm of free PLP to an absorption maximum at
about 410 nm followed by a time-dependent decrease in the
388 nm and 410 nm absorbance bands. The reactions, monitored at 388 nm,
follow pseudo first-order kinetics with second-order rate constants of
47.52 ± 0.003 M
1 min
1 and
of 28.98 ± 0.02 M
1 min
1
for the reaction of PLP with D-5-HTP and
D-Dopa, respectively (data not shown).
The reaction was also studied with NMR spectroscopy. The assignment of
resonances was straightforward on the basis of the NMR spectra of
isolated compounds. Upon addition of a substoichiometric amount (1:10)
of PLP to D-5-HTP or D-Dopa the NMR spectrum
changes markedly. A very fast decrease up to the complete disappearance of the resonance because of the formyl proton of PLP was observed in
both reactions. Moreover, in the same time course the resonances caused
by the protons of D-5-HTP or D-Dopa decreased,
and new resonances became clearly visible. These resonances display a spectral shape similar to those of D-5-HTP or
D-Dopa, a low intensity similar to that of PLP, and are
shifted slightly with respect to those of the reagents. All of these
signals are consistent with the rapid and nearly complete formation of
a Schiff base between PLP and the
-NH2 group of the
amino acid. Subsequently these resonances decreased, and new ones
arose, indicating that a nucleophilic attack of the C-2 or C-6 of
D-5-HTP or D-Dopa, respectively, to the imine
carbon atom of PLP had occurred (data not shown). Comparison of these
spectral features and of the chemical shift with those for the reaction
of PLP with Dopa and tryptophan in the L form (17, 18)
indicates that the compounds formed are cyclized Schiff bases, the
result of Pictet-Spengler condensation (Scheme I).
These cyclic adducts were prepared by mixing 1 mM PLP with
10 mM D-5-HTP or D-Dopa in 100 mM potassium phosphate buffer, pH 7.5. After a 3-h
incubation at 25 °C, the mixtures were processed by HPLC (see
below). With 5-HT or dopamine, PLP forms cyclized Schiff bases that had
been isolated using the HPLC methods described below and characterized
by means of absorbance spectroscopy.
HPLC Detection of PLP, PMP, Pictet-Spengler Adducts, and
Indole-3-pyruvic Acid--
The Pictet-Spengler adducts, obtained by
synthesis as described above, were isolated from unreacted reagents
using a 5-µm Pinnacle ODS (250 × 4.6 mm) (Restek) column
connected to a Waters 625 LC HPLC control system. The eluent was 50 mM potassium phosphate buffer, pH 2.2, at a flow rate of
0.6 ml/min. A Waters UV detector set at 295 nm was employed. In
addition to peaks corresponding to the retention time of PLP and
D-5-HTP or D-Dopa, the analysis revealed the
appearance of an additional peak. For the reaction of PLP with
D-5-HTP, this peak has a retention time of 44 min and an
absorption spectrum with maxima at 275, 295, and 325 nm. For the
reaction of PLP with D-Dopa, the peak has a retention time
of 10.7 min and an absorption spectrum with maxima at 280 and 325 nm
(data not shown).
Native or nicked DDC (8 µM) was incubated with 5 mM D-5-HTP in 100 mM potassium
phosphate buffer, pH 7.5, at 25 °C. Aliquots were removed at time
intervals, and trichloroacetic acid was added to a final concentration
of 5% (v/v). The quenched solutions were centrifuged to remove
protein, and the supernatants were analyzed using the above
chromatographic system. Peaks, corresponding to PLP, PMP, and
PLP-D-5-HTP adduct, were integrated with a Waters 745B data
module. In the same manner, detection and quantification of these
coenzymatic forms were carried out for reaction of native or nicked DDC
with other ligands (D-Dopa, D-tryptophan,
L-5-HTP, or 5-HT). Standard curves of peak area as a
function of concentration of coenzyme or coenzyme adducts were prepared
using commercially available PLP and PMP or coenzyme adducts obtained
by synthesis.
The separation of indole-3-pyruvic acid formed during the reaction of
DDC with D-tryptophan was done isocratically on the 5-µm
Pinnacle ODS column. The solvent was H2O/methanol/acetic acid (81.5/18/0.5, v/v/v) containing 1 mM octanesulfonic
acid at a flow rate of 0.6 ml/min, and detection was at 295 nm. After incubation of 8 µM native DDC with 3 mM
D-tryptophan for 15 h in 100 mM potassium
phosphate, pH 7.5, at 25 °C, the reaction was stopped by adding
trichloroacetic acid to a final concentration of 5% and then
centrifuged to remove the precipitated protein. The supernatant and
appropriate blanks were run. For quantification of indole-3-pyruvic
acid, the area of the peak was measured and converted to absolute
amounts by using a standard reference curve.
 |
RESULTS |
Reaction of Native DDC with D-Aromatic Amino
Acids--
As already reported for DDC purified from pig kidney (2),
when recombinant DDC (10 µM) is preincubated with
increasing concentrations of D-5-HTP (0.05-5
mM), the decarboxylase activity decreases as a function of
time and D-5-HTP concentration following a pseudo
first-order kinetics. Decarboxylase activity can be restored completely
by the addition of exogenous coenzyme to the reaction mixture (data not shown).
Upon addition of D-5-HTP to DDC, an increased absorption
centered at 430 nm appears immediately, and both the 420- and 335 nm
dichroic bands decrease. These spectral changes can be attributed to
the formation of the external aldimine. The 430 nm absorption and the
420 nm and 335 nm dichroic bands decrease with time, concomitant with
the increase of the absorption in the 330 nm region (Fig. 1, A and B). During
the reaction the absorption change at 430 nm follows a pseudo
first-order behavior, and the apparent rate constant
(kobs) shows dependence on the
D-5-HTP concentration in a hyperbolic manner (Fig.
1A, inset). The value of the apparent dissociation constant and the maximum value of the rate constant are
1.07 ± 0.16 mM and 0.086 ± 0.003 min
1, respectively. To determine if this reaction
converted PLP into PMP or other coenzyme forms, at various times
aliquots were withdrawn from a reaction mixture containing 8 µM DDC and 5 mM D-5-HTP in 100 mM potassium phosphate buffer, pH 7.5, and subjected to
analysis by HPLC after total denaturation. These analyses confirmed
that freshly purified native enzyme had all of its coenzyme in the PLP
form. Moreover, time-dependent inactivation of DDC by
D-5-HTP is accompanied by a decrease in PLP and a
concomitant increase in PMP and PLP-D-5-HTP Pictet-Spengler
adduct (Fig. 2A). This latter
adduct has been identified as Pictet-Spengler type in that it has a
spectrum (maxima at 325, 295, and 275 nm) and a retention time
identical to those obtained with an authentic sample of
PLP-D-5-HTP adduct formed by Pictet-Spengler reaction, as
described above (data not shown).

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Fig. 1.
Time-dependent spectral changes
occurring upon addition of D-5-HTP to DDC. The enzyme
(10 µM) in 100 mM potassium phosphate buffer,
pH 7.5, was incubated with 5 mM D-5-HTP at
25 °C. Absorbance (panel A) and CD (panel B)
spectra were recorded at the indicated times. The absorbance and CD
spectra of 10 µM DDC are included (broken
line) for reference. Inset, plot of
kobs as a function of the D-5-HTP
concentration.
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Fig. 2.
Coenzyme content and activity during the
reaction of DDC with D-5-HTP. Native (panel
A) or nicked (panel B) DDC (8 µM) was
incubated with 5 mM D-5-HTP in 100 mM potassium phosphate buffer, pH 7.5, at 25 °C. At the
indicated times aliquots were removed and denatured with
trichloroacetic acid. After removal of the precipitated protein by
centrifugation, the supernatants were subjected to HPLC analysis as
described under "Experimental Procedures." , PLP; , PMP; ,
PLP-D-5-HTP adduct. represents the residual
decarboxylase activity measured at the indicated times as described
under "Experimental Procedures" on aliquots of reaction mixture
containing native DDC.
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The addition of D-Dopa to DDC causes the immediate increase
of the absorbance at 430 nm with a concomitant decrease at 335 nm.
However, it has been noted that in contrast to a previous report (5),
these absorbing species are not stable intermediate complexes but
undergo very slow time-dependent changes consisting of a
decrease of the absorption at 430 nm and an increase of the 335 nm
absorbance band. The apparent rate constant, measured following the 430 nm absorbance change at 5 mM D-Dopa, is
0.019 ± 0.01 min
1. When DDC was preincubated with 5 mM D-Dopa in 100 mM potassium phosphate buffer, pH 7.5, at 25 °C, a slow loss of decarboxylase activity as a function of time was observed with a rate constant of
inactivation of 0.019 ± 0.013 min
1. After a 15-h
reaction of DDC with D-Dopa, the solution was denatured, and fractionation of the supernatant was then performed by HPLC under
the same experimental conditions described above. In addition to
D-Dopa, peaks were found corresponding to 32% PLP, 16%
PMP, and 52% PLP-D-Dopa adduct, with respect to the
original coenzyme content of the enzyme (Table
I). The latter peak has been identified as a Pictet-Spengler adduct, PLP-D-Dopa, in that its
retention time and its absorbance spectrum are identical to those of
the authentic compound.
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Table I
PLP, PMP, and Pictet-Spengler adduct content during the reaction of
native and nicked DDC with various substrates
Native or nicked DDC (8 µM) was incubated with several
substrates in 100 mM potassium phosphate buffer, pH 7.5, at
25 °C. At the indicated times, aliquots were removed and denatured
with trichloroacetic acid. After removal of the precipitated protein by
centrifugation, the supernatants were subjected to HPLC analysis as
described under "Experimental Procedures." Numbers represent
percentage of the original PLP content of the enzyme. ND, not
determined.
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In previous studies it was observed that inactivation of DDC by
D-5-HTP does not parallel the conversion of PLP into PMP, and an appreciable fraction of the coenzyme remained in an unidentified form. This led to the suggestion that the inactivation could be explained, at least in part, on the basis of transamination (5). The
finding that in addition to PMP, a Pictet-Spengler adduct is formed
during the reaction of DDC with D-aromatic amino acids has
allowed for a more complete understanding of this reaction.
5-Hydroxyindolepyruvic acid or 3,4-dihydroxyphenylpyruvic acid produced
during the reaction of DDC with D-5-HTP or
D-Dopa, respectively, could not be identified because
standard samples are not available. However, indole-3-pyruvic acid
produced after 15 h of reaction of DDC with
D-tryptophan has been identified by comparing its retention
time on HPLC and its absorption spectrum with those of standard sample
and quantitatively determined. 0.5 nmol of ketoacid is produced, a
quantity nearly equimolar with the PMP (0.4 nmol) formed under the same
experimental conditions (data not shown).
Protection against Limited Proteolysis of Native DDC by Aromatic
Amino Acids and by Aromatic Amines--
Limited trypsin digestion of
DDC yields two fragments of 38 and 14 kDa produced by cleavage of the
Lys 334-His335 peptide bond (11). When DDC was
preincubated with D-5-HTP or D-Dopa, the
tryptic digestion proceeded at a rate similar to that of the free
enzyme. In fact, for unliganded DDC and the D-5-HTP·DDC or D-Dopa·DDC complexes, the gradual disappearance of the
52-kDa band (corresponding to the undigested enzyme) and the
concomitant appearance of the 38- and 14-kDa bands (corresponding to
the proteolytic fragments) have similar time courses that lead to the
near complete disappearance of the 52-kDa band within 10 min. On the
other hand, the cleavage was remarkably retarded by
L-5-HTP, dopamine, or 5-HT. In the presence of saturating
concentrations of L-5-HTP, dopamine, or 5-HT, the band
corresponding to the intact enzyme persists up to 30 min (data not
shown). These ligands were used at concentrations at least 10-fold
higher than their KD values. These results indicate
a difference in accessibility of the enzyme and of the enzyme-ligand
complexes to trypsin.
Functional Properties of Trypsin-treated DDC and Comparison with
Those of the Native Enzyme--
Absorption and CD (Fig.
3) spectra of nicked inactivated DDC
(<1% of residual decarboxylase activity) in the UV-vis region are
essentially identical to those of the native enzyme. CD spectra taken
in the far UV region were superimposable for the native and nicked DDC
(data not shown). These data indicate that no alterations in the
tertiary or secondary structures are produced by limited tryptic
digestion. As for native DDC, the addition of L-5-HTP to
nicked enzyme causes an increase of the absorbance at 430 nm with a
concomitant decrease at 335 nm. However, although binding of
L-5-HTP to native DDC results in the inversion of the 420 nm CD signal, i.e. disappearance of the original positive CD
and its replacement by a negative CD shifted to 440 nm and in the increase of the 335 nm dichroic band, binding of L-5-HTP to
nicked DDC results in a marked decrease of both the positive 420- and 335 nm dichroic bands (Fig. 3). Moreover, after a 1-h reaction, 55% of
the original PLP content of the nicked enzyme was converted into
PLP-L-5-HTP Pictet-Spengler adduct. Upon addition of
D-5-HTP to the nicked enzyme, absorption and CD spectral
changes are nearly identical to those observed with the native enzyme
described above. The change in the 430 nm absorbance with time follows
a pseudo first-order behavior at each fixed concentration of
D-5-HTP. A plot of the apparent rate constant
(kobs) against the concentration of
D-5-HTP fits a hyperbolic curve. The KD
determined for D-5-HTP was 0.64 mM ± 0.21 mM, with a kmax of 0.039 ± 0.003 min
1 (data not shown). This latter value is 2-fold
lower than that determined for native enzyme. By HPLC we observed that
during the reaction of the nicked enzyme with D-5-HTP, the
decrease of the original PLP content of the enzyme corresponds to its
conversion into PMP and PLP-D-5-HTP Pictet-Spengler adduct
(Fig. 2B).

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Fig. 3.
CD spectral changes of native (solid
line) and nicked (broken line) DDC upon
reaction with L-5-HTP. Curve 1 represents
native DDC (10 µM) in 100 mM potassium
phosphate buffer, pH 7.5. To this mixture, 5 mM
L-5-HTP was added and the CD spectrum immediately recordred
(curve 2). Similarly, curve 1' represents nicked
DDC (10 µM) in the same buffer as above, and curve
2' is the CD spectrum after the addition of 5 mM
L-5-HTP.
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Reaction of the nicked enzyme with D-Dopa is also
characterized by absorbance and CD spectral changes similar to those
observed for the native enzyme. After a 15-h reaction in 100 mM potassium phosphate buffer, pH 7.5, at 25 °C, the PLP
content of the enzyme was 47% of the original amount. Of the remaining
53%, 10% was found as PMP and 43% as PLP-D-Dopa adduct
(Table I). Thus, the nicked enzyme retains a large percentage of the
native transamination activity. Moreover, the Pictet-Spengler reaction
occurs to nearly the same extent in both the native and nicked species.
After the addition of 5-HT or dopamine, the CD spectrum of the native
enzyme displays a positive band corresponding to the 400 nm absorbance
region. Both absorbance and dichroic bands decrease gradually with time
(Fig. 4, A and B).
It has been shown that these spectral modifications are related to a
reaction producing 5-hydroxyindolacetaldehyde or
3,4-dihydroxyphenylacetaldehyde and ammonia in equimolar amounts, which
consumes O2 in a 1:2 molar ratio with respect to the
products. Concurrent with this reaction, 5-HT or dopamine inactivates
DDC with a kinact of 0.023 min
1
and 0.0059 min
1, respectively. Under anaerobic
conditions, these products are formed in amounts less than 5% with
respect to those found under aerobic conditions (9, 10). We have now
obtained the following results. 1) The reaction of the native enzyme
with 4 mM 5-HT under anaerobic conditions is characterized
by a decrease of bound PLP and a concomitant increase of PMP and
PLP-5-HT adduct with time (Table I). 2) The reaction of DDC with
L-5-HTP under aerobic conditions gives a plot of 5-HT
versus time nearly linear for at least 25 min. However,
under anaerobic conditions the decarboxylation rate dropped to 40% of
its initial value within 25 min. The addition of free PLP at that time
restored the rate to its initial value. Likewise, the same behavior was
observed with L-Dopa (data not shown). Taken together,
these results indicate that under anaerobic conditions DDC undergoes
half-transamination of aromatic amines accompanied by a Pictet-Spengler
reaction and provide evidence that the observed loss of decarboxylase
activity during the time course under anaerobic conditions is caused by
the conversion of PLP into nonfunctional coenzyme forms (PMP and
Pictet-Spengler adduct). The spectral changes observed upon addition of
5-HT or dopamine to nicked DDC consist of a modest absorption increase at 430 nm and no relevant change of the 430 nm dichroic peak. A very
slow 420 nm absorbance decrease with time following a pseudo first-order behavior was observed for both of the aromatic amines (data
not shown). At 2 mM 5-HT or dopamine the
kobs was 0.009 ± 0.004 min
1.
After a 15-h reaction of nicked DDC with 5-HT or dopamine, the original
PLP was converted into PMP and PLP-5-HT or PLP-dopamine adducts,
respectively (Table I). Neither ammonia nor aldehyde formation could be
observed when the reaction of the nicked enzyme with 5-HT or dopamine
was coupled with glutamate dehydrogenase or with alcohol dehydrogenase,
respectively, in the presence of NADH. Moreover, no O2
consumption could be detected. These data suggest that the truncated
enzyme species is unable to catalyze oxidative deamination of aromatic
amines, even at O2 concentrations higher than atmospheric
(data not shown). Taken together, these results indicate that, unlike
the native enzyme, the nicked enzyme undergoes a slow transaminase
reaction of aromatic amines accompanied by a Pictet-Spengler reaction
occurring between the ligand and the PLP-bound enzyme.

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Fig. 4.
Spectral changes occurring upon addition of
5-HT to the native enzyme. The native enzyme (10 µM)
in 100 mM potassium phosphate buffer, pH 7.5, was incubated
with 2 mM 5-HT at 25 °C. Absorbance (panel A)
and CD (panel B) spectra were recorded at the indicate
times. The broken line represents the absorbance
(panel A) and CD (panel B) spectra of untreated
10 µM native DDC.
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 |
DISCUSSION |
Along with the main reaction, almost all PLP-dependent
enzymes catalyze many side reactions. In this regard, DDC is not an exception in that in addition to catalyzing decarboxylation of L-aromatic amino acids, it has been demonstrated recently
that the enzyme catalyzes oxidative deamination of aromatic amines (9,
10). Furthermore, the results reported here clearly indicate that DDC
is also able to catalyze half-transamination of D-aromatic amino acids, either with an indole-related (e.g.
D-5-HTP) or with a catechol-related (e.g.
D-Dopa) structure. In addition, it was found that binding
of both D-5-HTP and D-Dopa to the active site of DDC is characterized not only by transamination, but also by a
Pictet-Spengler reaction occurring between the D-aromatic
amino acid and bound PLP.
These findings allow construction of a functional model of the active
site of DDC, according to Dunathan's proposition (19). Assuming that
the side chain R of a D-aromatic amino acid is oriented in
the same relative position as the corresponding L form, it can be argued that the subsite A (see
Scheme II) is the one that maintains the
scissile bond perpendicular to the plane of the pyridine ring and
therefore is the site at which all bond making or breaking events
occurring at C
will take place. The B and C subsites accept groups
linked to C
which do not directly participate in the chemical events
(i.e. side chain and
-hydrogen in L-aromatic amino acid decarboxylation (conformation I, Scheme II) and side chain and the
-carboxylate group in D-aromatic
amino acids transamination (conformation II, Scheme II).
This active site model of native DDC implies a control of reaction
specificity by a proper location in the external aldimines of yet
unidentified residues which could regulate the orientation of
substrates and/or that of the pyridine ring of the coenzyme.
It is of interest to point out that tryptic cleavage at
Lys334 produces an enzymatic species that retains
transaminase activity but is devoid of decarboxylase and oxidative
deamination activity. A similar, altered reaction specificity has also
been observed in truncated mitochondrial aspartate aminotransferase,
obtained by limited tryptic proteolysis (20).
To gain some insight into the structural origin of the reaction
specificity of native and nicked DDC, we have looked at the properties
of the intermediate complexes that these enzymatic species form with
various substrates. The first important observation derives from
protection studies against limited trypsin proteolysis of DDC by these
ligands. In fact, although accessibility of trypsin to the cleavage
site Lys334-His335 decreases significantly upon
binding of L-aromatic amino acids or of aromatic amines, it
does not change appreciably upon binding of D-aromatic
amino acids. These data indicate, even if they do not prove, that
conformational changes occur near residues at the tryptic cleavage
region upon binding of L-aromatic amino acids and their
decarboxylation products. Similar conformational changes do not seem to
take place upon binding of D-aromatic amino acids, although
it cannot be ruled out that some changes may occur elsewhere in the
molecule. Inhibition studies by coenzyme-5-HTP adducts upon
recombination of pig kidney apo-DDC with PLP suggested a different
positioning of L-5-HTP or 5-HT with respect to that of
D-5-HTP at the active site of the enzyme (21). Furthermore, it has been recently proposed by Ishii et al. (22) that in
rat liver DDC the region around Arg334, homologous to the
region around Lys334 in pig kidney DDC, is exposed and
flexible in the unliganded state. During the formation of the Michaelis
complex, the region undergoes a subtle conformational change followed
by a marked conformational change during the transaldimination process.
The finding of lack of protection of DDC by D-aromatic
amino acids against trypsin modification argues against this proposal.
Our results suggest that a substantial conformational change of this region occurs only during a transaldimination that leads to an external
aldimine competent for decarboxylation or for oxidative deamination,
but not for transamination. This view is reinforced by the following
points. 1) Although binding of aromatic amines to the active center of
truncated DDC does occur, as shown by changes in the absorbance and CD
bands at 430 nm, this enzymatic species fails to form an absorbance and
a positive dichroic band around 400 nm. This observed red shift could
indicate a change in the environment which does not necessarily imply a
global change throughout the entire active site region. 2) Although the
addition of saturating concentrations of L-5-HTP to the
native or the nicked forms of the enzyme causes an identical increase
of the absorbance at 430 nm for both enzymic species, it results in
distinctly different changes of their coenzyme dichroic bands (Fig. 3).
This suggests that binding of L-5-HTP to the native enzyme
causes changes in the orientation of the coenzyme, with respect to the
neighboring residues, different from those caused by the binding of the
same ligand to the nicked enzyme. 3) Absorbance and CD spectral
features of the intermediate D-5-HTP-enzyme complexes are
identical for either native or nicked DDC, thus suggesting that binding
of aromatic amino acids in the D form causes similar
changes in the microenvironment of the coenzyme for both of the
enzymatic species, different from that generated upon binding of the
corresponding L-aromatic amino acids to the native enzyme.
On the basis of these results, the occurrence of at least three and two
different conformational states of the native and the nicked DDC
enzymes, respectively, can be predicted (Scheme III). Both of the unliganded enzymatic
species adopt the "open" conformation. Although binding of ligands
leading to decarboxylation or oxidative deamination shifts the
conformational equilibrium to the "closed" form, binding of ligands
leading to half-transamination and Pictet-Spengler reactions shifts the
equilibrium to the "half-open" form. We propose two models of
transition between these conformational states (Scheme III). In the
first model (A), the shift open
closed or open
half-open might take place in one step; in the second (B)
two steps may be required for the transition open
closed. The
closed form may be a compact structure that may be considered a
critical one for catalysis (decarboxylation and oxidative deamination, but not transamination). In this conformational form, the
Pictet-Spengler reaction does not occur. The closing of the active-site
cleft would correspond to a ligand-induced conformational change that cannot occur in the nicked enzyme. Ligand-induced conformational changes closing the active-site pocket have been demonstrated (23-26)
or proposed (27-30) for many PLP-dependent enzymes. Thus in the structural transition from the open to the closed form, a
crucial role appears to be played not only by the region around the
tryptic cleavage site, but also by the chemical nature of the ligand.
Structural elements in the ligands that are required for this
conformational shift can be suggested. The first may involve the
catecholic or the 5-hydroxyindolic ring of L-aromatic amino
acids or aromatic amines, whose specific interactions with the protein
could impose important constraints in the alignment of catalytic groups
at the active site. The fact that L-tyrosine, L-phenylalanine, and L-tryptophan are very poor
substrates for DDC with respect to L-Dopa (31) and the
recent finding that unlike that observed with L-Dopa and
L-tyrosine methyl esters, the binding of
L-phenylalanine methyl ester to the nicked enzyme is
unaltered with respect to the native enzyme (12) are both consistent
with the above view. A second requirement could be the chemical nature
of the group that occupies subsite A (conformation I, Scheme
II), which must be a carboxyl group for decarboxylation to occur. With
regard to oxidative deamination, it is not easy to envisage what
conformation of aromatic amines in the active site of DDC would be
productive for oxidative deamination, in that this latter reaction does
not appear to proceed by any of the known pathways for the principal
types of PLP-catalyzed reactions. Nonetheless, a tentative working
model can be advanced, taking into account that 1) the nicked enzyme
does not catalyze this reaction but does catalyze half-transamination
and Pictet-Spengler reactions of aromatic amines, and that 2) although
native DDC catalyzes oxidative deamination of aromatic amines under
aerobic conditions, it catalyzes half-transamination of these amines
accompanied by a reaction leading to the formation of Pictet-Spengler
adducts under anaerobic conditions. Thus, the proposal that oxidative deamination occurs in the closed state lies on these assumptions, that
the shift of the native enzyme to this form is induced by binding of
both aromatic amines and O2 and that this shift cannot be
achieved without the integrity of the region around the specific tryptic cleavage of DDC. Therefore, according to this view, the active
site of native DDC would be able to accommodate either CO2
(conformation I, Scheme II) or dioxygen molecules at subsite
A. A conformation (III) (Scheme II) orienting the C
-H
bond perpendicular to the plane of the cofactor system is productive
for the transamination half-reaction of aromatic amines. Considering
that the proposed mechanism for the reaction of DDC with 5-HT under
aerobic conditions involves a conformation of the external aldimine
undergoing deprotonation at C
(9), it can be suggested that the
conformation III (Scheme II) could also be productive for
oxidative deamination, provided that in this conformation subsite A is
capable of productively binding molecular oxygen. This hypothesis is
reminiscent of the mutual competition between CO2 and
O2 to the enediol(ate) intermediate formed from
D-ribulose 1,5-bisphosphate at the active site of ribulose-1,5-bisphosphate-carboxylase/oxygenase (32). It is clear that
this view is merely suggestive, and the poorly understood kinetic
mechanism of this reaction makes any attempt to correct interpretation
difficult. However, whatever the mechanism of oxidative deamination may
be, our data indicate a different fate of an L-aromatic amino acid subjected to the action of DDC under aerobic or anaerobic conditions. In fact, in the presence of O2 DDC catalyzes
the conversion of the L-aromatic amino acid into the
corresponding aromatic amine that is cleaved to form aldehyde or ketone
and ammonia. This latter reaction is accompanied by an irreversible
inactivation of the enzyme (9, 10). In contrast, in the absence of
O2 DDC catalyzes decarboxylation of L-aromatic
amino acids followed by half-transamination and Pictet-Spengler
reactions: the final products of these reactions are aldehyde or ketone
and PMP, in equimolar amounts, and a PLP-aromatic amine adduct. These
latter catalytic events cause inactivation of DDC which is readily
reversible provided that free PLP is present. Hence, molecular oxygen
appears to control, although indirectly, the rate of decarboxylation of
L-aromatic amino acids by DDC.
The half-open form of DDC has been attributed to a state of the enzyme
in the native or nicked form, in which in addition to a
half-transamination, a Pictet-Spengler reaction between the ligand and
bound PLP takes place. In the absence of a crystal structure of DDC,
the half-open form of this enzyme can only be presumed to be a looser
structure with respect to the closed one. As for the occurrence of the
Pictet-Spengler reaction, at present it is not possible to distinguish
if the cyclized Schiff base is generated at the active site of the
enzyme or if the external aldimine undergoes subsequent cyclization
after release from the enzyme. Again, it is not easy to envisage why
events leading to the Pictet-Spengler adduct are more frequent than
transamination events with D-Dopa with respect to
D-5-HTP and with dopamine with respect to 5-HT. We can
speculate that different substrates stabilize slightly different
conformers of the enzyme which differ in their preference for the
pathway leading to transamination versus the pathway leading
to cyclization. Nevertheless, the finding that D-5-HTP
forms an external aldimine with either native or nicked enzyme in a
conformation leading to transamination and Pictet-Spengler reactions
indicates that a structural change involving the region around
Lys334 is not required for transition between
conformationally open and half-open states. The behavior resulting from
the binding of aromatic amines to the nicked enzyme is in accord with
this interpretation. Furthermore, the finding that the external
aldimine of L-5-HTP with the nicked enzyme undergoes only a
Pictet-Spengler reaction and not transamination indicates the
nonproductive binding mode for L-5-HTP transamination, as
suggested by the proposed active site model (Scheme II).
In conclusion, spectral and kinetic studies of the reactions of the
native and nicked DDC enzymes with various ligands have allowed the
elucidation of some structural elements of the protein and ligand which
appear to be relevant to the reaction specificity of this enzyme.
 |
FOOTNOTES |
*
This work was supported by funding from the Italian
Ministero dell'Università e Ricerca Scientifica e Tecnologica
(PRIN "Strutture, Meccanismi e Ingegneria Genetica" and "Biologia
Strutturale"), Consiglio Nazionale delle Ricerche, Centro
Interuniversitario per lo Studio delle macromolecole Informazionali
(Milan, Pavia, Verona, Italy).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 39-45-8098-175;
Fax: 39-45-8098-170; E-mail: cborri{at}borgoroma.univr.it.
 |
ABBREVIATIONS |
The abbreviations used are:
Dopa, 3,4-dihydroxyphenylalanine;
DDC, Dopa decarboxylase;
PLP, pyridoxal
5'-phosphate;
5-HTP, 5-hydroxytryptophan;
5-HT, 5-hydroxytryptamine;
PMP, pyridoxamine 5'-phosphate;
HPLC, high performance liquid
chromatography.
 |
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