On the L-DOPA and carbidopa reactivity against pyridoxal 5′-phosphate. A kinetic study

Article abstract of DOI:10.1246/bcsj.75.545

The apparent rate constants of the formation (k₁) and hydrolysis (k₂) of Schiff bases formed by pyridoxal 5′-phosphate (PLP) with L-3,4-dihydroxyphenylalanine (L-DOPA) at a variable pH, 25 °C and an ionic strength of 0.1 M was determined. The individual rate constants for the formation and hydrolysis of Schiff bases corresponding to the different chemical species present in the medium as a function of its acidity were also determined, as were the pK_a values for the Schiff bases. The formation and hydrolysis rate constants of the Schiff bases were compared with those of the reaction of PLP with carbidopa (CD), showing that the reactivity of L-DOPA and carbidopa on PLP are the same over the whole pH range studied, and that the hydrolysis rate is somewhat greater for the Schiff bases between PLP and CD than those between PLP and L-DOPA.

Full text of DOI:10.1246/bcsj.75.545

c. Jpn.
© 2002 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 75, 545–549 (2002)
545
e Chemical
ciety of Ja-
n
e Chemical
ciety of Ja-
n
On the L-DOPA and Carbidopa Reactivity against Pyridoxal
5ꢀ-Phosphate. A Kinetic Study
Schiff Bases of PLP with L-Dopa and Carbidopa
G. R. Echevarría Gorostidi
Gerardo R. Echevarría Gorostidi,* José G. Santos,† Julia Figueroa,† and Francisco García Blanco††
02
5
9
Department of Physical Chemistry, University of Alcalá, E-28871 Alcalá de Henares, Spain
†Faculty of Chemistry, Pontificia Universidad Católica de Chile, Casilla 306, Santiago 22, Chile
††Department of Physical Chemistry, Faculty of Pharmacy, Complutensian University, E-28040 Madrid, Spain
SJA8
09-2673
296
(Received August 27, 2001)
01
01
The apparent rate constants of the formation (k1) and hydrolysis (k2) of Schiff bases formed by pyridoxal 5ꢀ-phos-
phate (PLP) with L-3,4-dihydroxyphenylalanine (L-DOPA) at a variable pH, 25 °C and an ionic strength of 0.1 M was de-
termined. The individual rate constants for the formation and hydrolysis of Schiff bases corresponding to the different
chemical species present in the medium as a function of its acidity were also determined, as were the pKa values for the
Schiff bases. The formation and hydrolysis rate constants of the Schiff bases were compared with those of the reaction
of PLP with carbidopa (CD), showing that the reactivity of L-DOPA and carbidopa on PLP are the same over the whole
pH range studied, and that the hydrolysis rate is somewhat greater for the Schiff bases between PLP and CD than those
between PLP and L-DOPA.
.1
4
Pyridoxal 5ꢀ-phosphate (PLP) is one form of vitamin B6 that
plays a central role as a coenzyme in a wide range of reactions
(e.g. transaminations, transiminations, dealdolations, elimina-
tions, and decarboxylations) involved in amino acid metabo-
lism.1–3 Its action relies on the formation of a α-hydroxy
amine intermediate by bonding of its carbonyl group to the ε-
amino group of the L-lysine residue in the peptide chain.1,4
The α-hydroxy amine then releases one molecule of water to
give the Schiff base in an acid-catalyzed process.4,5
In virtually all PLP-dependent enzymes, the first step of the
process is a transimination reaction, viz. conversion of the
PLP-lysine imine into a PLP-substrate imine;6 in other words,
the covalent linkage in the Schiff base must be broken during
the course of the catalytic cycle and a new base be formed be-
tween the coenzyme and the substrate (usually an amino
acid).1
carboxylation yields dopamine. Dopamine generated in the in-
testine can not cross the blood-brain barrier; nevertheless, L-
DOPA can without difficulty get to the brain by means of an
active transport system; consequently, L-DOPA is transported
into the brain where it is decarboxylated by dopa decarboxy-
lase to dopamine.7 A strategy frequently used is to adminis-
trate the patient L-DOPA in combination with carbidopa (3-
(3,4-dihydroxyphenyl)-2-hydrazino-2-methyl propionic acid,
Scheme 1) in order to permit that L-DOPA gets as such to the
brain.7
In the literature there are references to Schiff bases formed
by PLP and compounds bearing amino groups such, as amines,
amino acids, and polypeptides.8–10
In this work, the kinetics of formation and hydrolysis of the
Schiff bases of pyridoxal 5ꢀ-phosphate with L-DOPA (viz. the
PLP–DOPA system) was determined and the results compared
with those previously obtained for the reaction of PLP with
carbidopa (PLP–CD system),10 in order to shed additional light
on the use of carbidopa in the L-DOPA formulations in Parkin-
son’s disease. The results for both systems were examined in
terms of the individual rate constants for those species in-
volved in the process (see Scheme 2).
Parkinson disease is caused by a decrease of 3,4-dihydroxy-
phenethylamine (dopamine) in the central nervous system;7 in
order to increase it, the cerebral level, L-3,4-dihydroxyphen-
ylalanine (L-DOPA, Scheme 1), a dopamine precursor, is used.
L-DOPA reacts with a PLP-dependent enzyme (dopa-decar-
boxylase), forming a corresponding Schiff base which by de-
Scheme 1.

546 Bull. Chem. Soc. Jpn., 75, No. 3 (2002)
Schiff Bases of PLP with L-Dopa and Carbidopa
Scheme 2.
Crison pH-meter equipped with a Metrohm EA120 electrode that
was previously calibrated with aqueous buffers at 25.0 °C.
The overall reaction between an aldehyde and an amine can be
schematized as follows:
Material and Methods
L-DOPA was purchased from Sigma Chemical Co. Pyridoxal
5ꢀ-phosphate and all other chemicals used were of reagent-grade
and purchased from Merck.
k1
Acetate, phosphate, and carbonate buffers were used over ap-
propriate pH ranges. The buffer concentration used was typically
0.02 M and the ionic strength was maintained at 0.1 M by adding
appropriate amounts of KCl to the medium.
PLP solutions were made in appropriate buffers and stored in
the dark. Their exact concentrations were determined by dilution
with 0.1 M HCl,11 and were found to be in the region of 5 × 10−5
M. L-DOPA solutions spanning the concentration range from 5 ×
10−4 to 2 × 10−2 M were also prepared on a daily basis by dilut-
ing appropriate amounts of stock solutions in the corresponding
buffer.
R1 – CHO + NH2 – R2
R1 – CHwN – R2 + H2O,
(1)
k2
where k1 and k2 are the overall rate constants for the formation and
hydrolysis, respectively, of the Schiff base. The procedure used to
calculate these two constants is described in detail elsewhere.12
Nucleophilic rate constants (kN) were obtained from the slopes of
linear plots of kobs vs free amine concentration, or by dividing the
k1 values by the corresponding free amine molar fractions.
Results and Discussion
Kinetic measurements were made at various pH by using a
Hewlett–Packard 8453 diode array spectrophotometer furnished
with thermostated cells of 1-cm light path. In each case, the reac-
tion was started by adding a known volume of PLP buffered solu-
tion to prethermostated L-DOPA solutions at (25 0.1) °C. The
difference between the initial and final pH in the reaction cell nev-
er exceeded 0.03 units. pH measurements were made by using a
The kinetic law obtained in the present reactions is given by
Eq. 2, where kobs is the pseudo-first-order rate coefficient, k1
and k2 are the rate constants for the formation and hydrolysis
of the Schiff base, respectively, and [L-DOPA] represents the
total L-DOPA concentration. The values of k1 and k2 were ob-
tained as the slope and intercept, respectively, of linear plots of

G. R. Echevarría Gorostidi et al.
Bull. Chem. Soc. Jpn., 75, No. 3 (2002) 547
Fig. 1. Plot of log k1 vs pH for the PLP–DOPA (ꢀ) system.
Curves (—) for PLP–DOPA and (----) for PLP–CD sys-
tems were calculated using Eq. 3 and data from Table 1.
Fig. 3. Plot of log kN vs pH for the PLP–DOPA (ꢀ) system.
Curves (—) for PLP–DOPA and (----) for PLP–CD sys-
tems were calculated using Eq. 5 and data of Table 1.
The apparently increased reactivity of carbidopa with L-
DOPA relative to PLP below pH 9 (see Fig. 1) arises from the
differential values of pKa for the amino group in the two com-
pounds. One way to compare their reactivity is by making the
formation rate constants for the Schiff bases independent of
pKa of the amino group. Figure 3 shows the variation of the
nucleophilic rate constant (kN) with the pH; as can be seen,
both compounds show practically the same reactivity through-
out the pH range studied.
As shown in Scheme 2, the overall rate constants of forma-
tion and hydrolysis of the Schiff bases can be described in
terms of the individual constants for the different chemical
species present in the medium at each pH.
Fig. 2. Plot of log k2 vs pH for the PLP–DOPA (ꢀ) system.
Curves (—) for PLP–DOPA and (----) for PLP–CD sys-
tems were calculated using Eq. 4 and data from Table 2.
i
i
Thus, k1 and k2 (with i = 0, 1, 2, or 3) are the individual
rate constants of formation of the Schiff bases and of their hy-
2
drolysis by H2O, respectively, and kOH is the rate constant of
hydrolysis of species B2 (a Schiff base with a net charge of -2)
by OH− ions. Pi (with i = 0, 1, 2 or 3) denotes the different
chemical species of PLP, and pK3P, pK2P, and pK1P the different
pKa values that relate them. Bi (with i = 0, 1, 2, or 3) are the
different chemical species of the Schiff bases, and pK3B, pK2B,
and pK1B the pKa values that relate them. Finally, KN is the
kobs vs [L-DOPA] at constant pH,
kobs = k1[L-DOPA] + k2.
(2)
Figures 1 and 2 show the experimental results in the form of
the variation of the logarithmic overall rate constants of forma-
tion (k1) and hydrolysis (k2) for the Schiff bases of PLP with L-
DOPA as a function of pH. The figures also include the calcu-
lated curve for the Schiff bases of the PLP–CD system.8,10
Reaction measurements could only be made up to pH 10.5
because more alkaline media resulted in the oxidation of L-
DOPA,13 and thus hindering the reaction and any precluding
examination beyond this pH.
Figure 1 shows that log k1 increases with increasing pH for
the PLP–DOPA system; nevertheless, for the PLP–CD system,
log k1 increases to a maximum at pH = 7 and then decreases
again as the pH increases. Below pH = 9, the k1 values are
greater for the latter system.
+
deprotonation equilibrium rate constant of the NH3 group in
L-DOPA. The experimental k1 and k2 values for the Schiff
bases were fitted to Eqs. 3 and 4, derived from Scheme 2,12
kO2HKW
where a = 10−pH
,
, and KW is the ionic
kOH = k23 +
K3B
product of water.
k12a
K3P
k11a2
k10a3
k13 +
+
+
K3PK2P
K3PK2PK1P
(3)
k1 =
a
a
a2
a3






1 +
1 +
+
+




KN
K3P
K3PK2P
K3PK2PK1P
k22a
k21a2
k20a3
On the other hand, k2 was greater for the PLP–CD system
than the PLP–DOPA system throughout the pH range studied
(Fig. 2). Both systems show a maximum at pH = 7 in the log
k2 vs pH plot.
kOH
+
+
+
+
(4)
K3B
a
K3BK2B
a2
K3BK2BK1B
a3
k2 =
1 +
+
K3B
K3BK2B
K3BK2BK1B

548 Bull. Chem. Soc. Jpn., 75, No. 3 (2002)
The nucleophilic rate constant kN is
Schiff Bases of PLP with L-Dopa and Carbidopa
Table 1. Best Kinetic Constant Values Obtained in the Fit-
ting of Experimental k1 Values to Scheme 2, and Those
Corresponding to the PLP–GLY,9 PLP–LEU,9 PLP–ILE,9
PLP–CD,10 PLP–PHE,15 and PLP–ALA15 Systems
a

kN = k 1 +
1


KN
3
2
1
0
Log k1
Log k1
Log k1
Log k1
k12a
k11a2
k10a3
k13 +
=
+
+
+
PLP–DOPA
PLP–CD
2.27
1.90
2.50
2.25
2.34
2.23
2.39
3.16
3.32
3.79
3.36
3.54
2.96
3.23
4.90
4.89
5.52
5.05
5.20
4.75
5.01
6.32
6.51
7.30
7.16
7.31
—
K3P
a
K3PK2P
a2
K3PK2PK1P
a3
(5)
PLP–GLY
PLP–LEU
PLP–ILE
PLP–PHE
PLP–ALA
1 +
+
K3P
K3PK2P
K3PK2PK1P
The protonation constants for PLP14 are pK1P = 3.46, pK2P
= 6.02, and pK3P = 8.22, and that for the Schiff bases required
in the initial fitting were estimated from reported data for relat-
ed systems.9,10
—
(k1 in L•mol−1•min−1). PHE and ALA denote L-phenylala-
nine and L-alanine, respectively.
Tables 1 and 2 give individual rate constants of formation
i
i
(k1 ), the individual rate constants of hydrolysis (k2 ) and the pK
values obtained in the fitting. For a comparison, the values for
the Schiff bases of PLP with other amino acids9,15 and of PLP–
CD system10 are also given. The pKN value for L-DOPA ob-
tained in the fitting is 9.33, which agrees with the pK value de-
termined spectrophotometrically at λ = 245 nm, and the same
experimental condition of pK = 9.35
nent role in acid media.16 Each k1 value in the PLP–DOPA
system is very similar to the corresponding one in the PLP–CD
system, resulting in Brönsted plots (log k1(i−1) vs. pKiP) for the
PLP–DOPA and PLP–CD systems being very similar; because
both are linear with α = 0.66, in both reactions the dehydra-
tion step is rate-determining.
i
At this point it is interesting to notice that the reaction
mechanism is through an α-hydroxy amine intermediate, as in
Eq. 6 (R1–CHO is Pi (with i = 0, 1, 2 or 3)) and R1–CHwN–R2
Due to the pH range studied and the expected value for pK3B
2
(about 11), there is no precision in fitting the k2 and kOH rate
constants nor the pK3B values.
i
i
is Bi (with i = 0, 1, 2, or 3); therefore the k1 rate constants in
As can be seen from Table 1, the k1 values for the reactions
Scheme 2 involve the formation of α-hydroxy amine rate con-
stants (kci) and those from the intermediate α-hydroxy amine
of the PLP–DOPA system and those for the reaction of PLP
with different amino acids are quite similar; consequently, the
side chains in the amino acids play no significant role in the
formation of Schiff bases with PLP, as has been described.9 A
to form the Schiff base by water elimination (kdi) and to return
i
to reactants by amine elimination (k−ci), besides k2 = k−di
.
i
comparison of the k1 values obtained for the PLP–CD and
kci
PLP–DOPA systems (Table 1) reveals the absence of signifi-
cant differences in the corresponding k1 . Accordingly, the
(
)
R1 – CHO
NH2 – R2
R1 – CH OH – NH – R2
+
k−ci
kdi
i
greater reactivity expected in L-DOPA than CD, due to the
presence of a hydrazine group in the latter,8,10 is compensated
for the presence of a methyl group in the CD. This effect is
also apparent from a comparison of the kN curves of Fig. 3,
which do not exhibit any appreciable differences.
The rate-determining step for the formation of a Schiff base
is known to be the dehydration of an intermediate α-hydroxy
amine formed by an attack of the amine on the carbonyl
group.4,5 It is also known that, with PLP, the dehydration is
subject to intramolecular acid catalysis, and that the phenol
group at C-3 on the pyridine ring plays an especially promi-
(6)
R1 – CHwN – R2 + H2O
k−di
If Kci = kci/k−ci is defined as the equilibrium formation con-
stant for α-hydroxy amine, provided this is formed and split
fairly rapidly, and this is completely shifted to the reactants,
then k1 = Kcikdi and k2 = k−di. This is in accord with linear
plots of kobs vs [L-DOPA] obtained at different pH values.
The maximum of the k1 curve for the PLP–CD system (Fig.
1) is a result of the pKa of the amino group of carbidopa (pKa
i
i
0
1
2
= 7.2) and of the sequence k1 > k1 > k1 (the reactivity de-
Table 2. Best Kinetic Constant Values Obtained in the Fitting of Experimental k2 Values to
Scheme 2, and pK Values for the Schiff Bases, and Those Corresponding to the PLP–GLY,9
PLP–LEU,9 PLP–ILE,9 and PLP–CD10 Systems
PLP–DOPA
PLP–CD
PLP–GLY
PLP–LEU
PLP–ILE
0
1
2
Log k2
Log k2
Log k2
−1.75
−0.596
—
−4.63
−0.04
−1.32
−3.85
6.97
0.584
0.676
−0.550
1.160
5.46
0.170
0.080
−0.900
1.080
5.68
−0.010
−0.170
−1.000
1.020
5.67
Log kOH
pK1B
—
5.85
6.37
—
pK2B
pK3B
7.15
11.35
6.36
11.35
6.65
11.61
6.66
11.57
(k2 in min−1).

G. R. Echevarría Gorostidi et al.
Bull. Chem. Soc. Jpn., 75, No. 3 (2002) 549
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two opposing effects (the free amine increases while the reac-
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1
+
2
V. C. Emery and M. Akhtar, Pyridoxal Phosphate Depen-
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L-DOPA relative to that of carbidopa, consistent with the val-
ues of the individual k2 constants given in Table 2. The pres-
i
3
A. E. Braunstein, Transamination and Transaminases. In
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strate with glycine (GLY) and LEU (Table 2). This effect has
also been observed in the Schiff bases of PLP with poly L-
lysine,17 where the polypeptide chain provides a less-polar en-
vironment, and leads to bases of increased stability. Accord-
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studied here leads to the opposite conclusion, probably due to
other differences in the structure of the corresponding Schiff
base (viz. the amine or hydrazine group).
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4
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6
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In Scheme 2 the hydrolysis reactions of the forms Bi (i = 0,
1) for OH− has been omitted because of the very small concen-
tration of OH− at the pH at which the concentration of Bi (i =
0, 1) is important, as can be obtained from the pK values for
the various forms of the Schiff bases derived from both sys-
tems (Table 2).
8
G. R. Echevarría-Gorostidi, A. Basagoitia, E. Pizarro, R.
Goldsmitd, J. G. Santos, and F. García Blanco, Helv. Chim. Acta,
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9
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i
The conclusions of this study are: i) The k1 and kN values
for PLP–DOPA and PLP–CD systems show the same reactivi-
ty to PLP by both amine group bearer in the whole of the pH
range studied. ii) The greater values for the overall rate con-
stant of formation k1 for the reaction of PLP with carbidopa
than L-DOPA are a consequence of the different pKa of both
amine groups. iii) At physiological pH, the k1 value for the
PLP–CD system is about 100-times greater than that for the
PLP–DOPA system. iv) Probably the same effect can occur at
the enzymatic level (i.e. transimination reaction), thus inhibit-
ing dopa-decarboxylase and, consequently, the carbidopa pres-
ence permits that a greater L-DOPA quantity can get to the
brain.
16 J. M. Sánchez-Ruiz, J. M. Rodríguez Pulido, J. Llor, and
M. Cortijo, J. Chem. Soc., Perkin Trans. 2, 1982, 1425.
17 M. A. García del Vado, G. R. Echevarría, F. García Blanco,
J. G. Santos, M. Blázquez, J. M. Sevilla, and M. Domínguez, J.
Mol. Catal., 68, 379 (1991).
This work was funded by FONDECYT (Chile, Project
1990551), Programa para la Cooperación con Iberoamérica
and DGI (BQU2000-0646 and BQU2000-0787).