Kinetic and thermodynamic study of the reaction of pyridoxal 5′-phosphate with L-tryptophan

Article abstract of DOI:10.1002/poc.893

The apparent rate constants for formation (k₁) and hydrolysis (k₂) of the Schiff bases formed by reaction of pyridoxal 5′-phosphate with L-tryptophan were determined at various pH values, at different temperatures and at constant ionic strength (0.1 M). Also obtained were the elementary rate constants for formation and hydrolysis of the Schiff bases corresponding to the different chemical species present in the media, and the pK values of the Schiff's bases. The activation and thermodynamic parameters for the formation and hydrolysis of the Schiff's bases also were determined. Some of the ΔH⁰ and ΔS⁰ values for the individual processes were found to be positive. In basic media the enthalpic factor is unfavorable but the entropie contribution leads to a negative ΔG⁰. Copyright

Full text of DOI:10.1002/poc.893

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY
J. Phys. Org. Chem. 2005; 18: 546–551
Published online 15 December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/poc.893
Kinetic and thermodynamic study of the reaction
of pyridoxal 50-phosphate with L-tryptophan
1
2
2
3
´
´
Gerardo R. Echevarr´ıa, * Jose G. Santos, Andrea Basagoitia and Francisco Garcıa Blanco
1
´
´
Department of Physical Chemistry, University of Alcala, E-28871 Alcala de Henares, Spain
2
´
Faculty of Chemistry, Pontificia Universidad Catolica de Chile, Casilla 306, Santiago 22, Chile
3Department of Physical Chemistry, Faculty of Pharmacy, Complutensian University, E-28040 Madrid, Spain
Received 1 July 2004; revised 6 October 2004; accepted 27 October 2004
ABSTRACT: The apparent rate constants for formation (k1) and hydrolysis (k2) of the Schiff bases formed by reaction
of pyridoxal 50-phosphate with L-tryptophan were determined at various pH values, at different temperatures and at
constant ionic strength (0.1 M). Also obtained were the elementary rate constants for formation and hydrolysis of the
Schiff bases corresponding to the different chemical species present in the media, and the pK values of the Schiff’s
bases. The activation and thermodynamic parameters for the formation and hydrolysis of the Schiff’s bases also were
determined. Some of the ÁH0 and ÁS0 values for the individual processes were found to be positive. In basic
media the enthalpic factor is unfavorable but the entropic contribution leads to a negative ÁG0. Copyright # 2004
John Wiley & Sons, Ltd.
KEYWORDS: Schiff base; L-tryptophan; PLP
INTRODUCTION
constants for the species involved in the process (see
Scheme 1).
An important number of enzymes related to amino acid
metabolism require pyridoxal 50-phosphate (PLP) as
coenzyme.1–3 The first step in the formation of a Schiff
base from PLP involves an aminomethanol intermediate
by attack of the amino group on the carbon atom of the
carbonyl group of PLP. Dehydration of the aminometha-
nol gives the Schiff base in an acid-catalyzed process.4,5
Although the kinetics and mechanism of formation and
hydrolysis of the Schiff’s bases formed by PLP and various
compounds bearing amino groups, such as primary
amines, hydrazine derivatives, amino acids and polypep-
tides, have been the subject of many reports,6 very few
kinetic studies have been carried out on the reactions of
PLP or its analogues (e.g. 50-deoxypyridoxal) with amines
or amino acids as a function of pH and temperature.7–13
This paper reports the results of a study on the kinetics
of formation and hydrolysis of the Schiff bases formed by
PLP and L-tryptophan (PLP–TRP system) at variable pH
values and temperatures. The activation parameters for
both processes and the thermodynamic parameters for the
reaction are examined in terms of the elementary rate
EXPERIMENTAL
L-Tryptophan was purchased from Sigma Chemical Co.
and pyridoxal 50-phosphate and all other chemicals used
were reagent grade and purchased from Merck.
Acetate, phosphate and carbonate buffers were used
over appropriate 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.
The PLP solutions were prepared in appropriate
buffers and stored in the dark. Their exact concentra-
tions were determined by dilution in 0.1 M HCl and
subsequent measurement of its absorbance at 295 nm
(" ¼ 6700 l molꢀ1 cmꢀ1 14
)
and were found to be in the
region of 2 ꢁ 10ꢀ5 M. The L-tryptophan solutions in the
concentration range 5 ꢁ 10ꢀ4–2 ꢁ 10ꢀ2 M were also
prepared, on a daily basis, by diluting appropriate
amounts of stock solutions in the corresponding buffer.
Kinetic measurements were carried out at various pH
values by using a Hewlett-Packard 8453 diode array
spectrophotometer and an Uvikon 941-Plus spectrophot-
ometer furnished with thermostated cells of 1 cm light
path. In each case, the reaction was started by adding a
known volume of PLP buffered solution to prethermo-
stated L-tryptophan solutions at the desired temperature
and pH.
´
*Correspondence to: G. R. Echevarrıa, Department of Physical
Chemistry, University of Alcala, E-28871 Alcala de Henares, Spain.
´
´
E-mail: gerardo.echevarria@uah.es
Contract/grant sponsor: DGI; Contract/grant numbers: BQ2003-
07281; BQ2000-0787.
Contract/grant sponsor: FONDECYT; Contract/grant numbers:
1990551; 2990006.
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2005; 18: 546–551

REACTION OF PYRIDOXAL 50-PHOSPHATE WITH L-TRYPTOPHAN
547
Table 1. Kinetic parametersa for the formation and hydrolysis of the Schiff bases formed by reaction of PLP and L-tryptophan
10 ꢂC
20 ꢂC
30 ꢂC
37 ꢂC
pH
k1
k2 ꢁ 102
pH
k1
k2 ꢁ 102
pH
k1
k2 ꢁ 102
pH
k1
k2 ꢁ 102
2.80
3.81
4.56
6.04
6.95
7.64
7.91
9.06
9.48
9.75
10.30
—
—
—
30.0
30.0
18.0
36.0
24.0
12.0
5.40
2.30
2.00
2.20
6.30
—
2.66
3.91
3.91
4.96
5.80
6.76
7.36
7.99
8.76
9.16
9.47
10.17
—
50.4
46.8
47.7
52.5
77.4
55.5
30.5
5.70
2.50
2.80
4.60
34.8
2.88
4.00
4.96
5.76
6.79
7.42
8.06
8.92
9.78
—
—
162
96.7
96.7
138
90.6
42.6
16.8
8.50
28.3
—
3.95
4.89
5.64
6.62
7.18
7.94
8.67
9.34
9.48
9.83
9.89
—
20.0
25.6
54.6
169
190
228
241
218
172
181
193
—
126
138
216
122
72.0
15.6
—
19.2
36.0
66.0
66.0
—
6.06
5.40
11.7
29.8
73.7
83.9
9.72
18.2
43.5
5.27
20.8
35.8
43.3
46.0
71.6
69.2
60.3
48.5
—
118
150
153
165
140
—
108
112
121
110
—
—
—
—
—
—
80.3
a Units: molꢀ1 minꢀ1 for k1 and minꢀ1 for k2. The estimated errors of k1 and k2 are not greater than 5%.
Pseudo first-order kinetic curves were observed (L-
tryptophan concentration was more than 10-fold larger
than PLP) by monitoring the absorbance at 435 nm due to
the Schiff base. The observed rate constants, kobs, were
determined by means of the spectrophotometer kinetic
software for first-order reactions. The difference between
the initial and final pH in the reaction cell never exceeded
0.03 units; pH measurements were made by using a
Crison pH-meter equipped with a Metrohm EA120
electrode that was calibrated previously with aqueous
buffers at 25 ꢂC.
rate constants for formation (k1) and hydrolysis (k2) of
the Schiff bases of PLP with L-tryptophan.
Reaction measurements could be carried out only at the
pH values shown in Table 1 because more alkaline media
resulted in intramolecular cyclization of the secondary
imine by attack of the indole group on the azomethine
carbon, which hindered the reaction and precluded ex-
amination beyond this alkalinity level.6b
As shown in Scheme 1, the overall rate constants for
formation and hydrolysis of the Schiff bases can be des-
cribed in terms of the elementary constants for the
different chemical species present in the medium at
each pH.
The overall reaction between an aldehyde and an
amine can be depicted as follows:
Table 2 gives the elementary rate constants for forma-
k1
i
i
---
tion (k1 ) and hydrolysis (k2 , kOH) and the pKiB obtained
by fitting the experimental k1, k2 and KpH values to Eqns
(2), (3) and (4) derived from Scheme 1:7
R1---CHO þ NH2--- R 2 R ---CH--- N --- R þ H O ð1Þ
Ð
1
2
2
k2
where k1 and k2 are the overall rate constants for forma-
tion and hydrolysis, respectively, of the Schiff base. The
procedure used to calculate these two constants is de-
scribed in detail elsewhere.7 Their ratio coincides with
the equilibrium constant (KpH ¼ k1/k2).
k13 þ k12 ꢃ a=K3P þ k11 ꢃ a2=ðK3P ꢃ K2PÞ þ k10 ꢃ a3=ðK3P ꢃ K2P ꢃ K1P
Þ
k1 ¼
k2 ¼
ð1 þ a=KNÞ½1 þ a=K3P þ a2=ðK3P ꢃ K2PÞ þ a3=ðK3P ꢃ K2P ꢃ K1PÞꢄ
(2)
The pKN values for L-tryptophan at the different
temperatures studied were determined potentiometrically
by titration of a solution of L-tryptophan (0.01 M) and HCl
(0.01 M) with NaOH (0.1 M) using a Radiometer autoti-
trator equipped with a PHM-62 pH-meter, an ABU-11
autoburette, a TTT-60 titrator, an REA-160 recorder, a
TTA-60 thermostatic support, a G-2040 glass electrode
and a K-4040 calomel electrode. The experimental con-
ditions used were the same as those for the kinetic
measurements. The pKN values thus obtained were 9.6,
9.3, 9.0 and 8.8 at 10, 20, 30 and 37 ꢂC, respectively, and
were confirmed by semi-neutralization.
kOH þ k22 ꢃ a=K3B þ k21 ꢃ a2=ðK3B ꢃ K2BÞ þ k20 ꢃ a3=ðK3B ꢃ K2B ꢃ K1B
Þ
1 þ a=K3B þ a2=a2ðK3B ꢃ K2BÞ þ a3=ðK3B ꢃ K2B ꢃ K1B
Þ
(3)
½1 þ a=K3B þ a2=ðK3B ꢃ K2BÞ þ a3=ðK3B ꢃ K2B ꢃ K1BÞꢄKM
KpH
¼
ð1 þ a=KNÞ½1 þ a=K3P þ a2=ðK3P ꢃ K2PÞ þ a3=ðK3P ꢃ K2P ꢃ K1PÞꢄ
(4)
where kOH ¼ k23 þ kO2 HðKW=K3BÞ, a ¼ 10ꢀpH, KW is the
ionic product of water and KM is the equilibrium constant
of formation of the Schiff’s base at very high pH
À
ꢀ Á
¼ k13 k23 .
RESULTS
Note that, owing to intramolecular cyclization of the
Schiff base,6b only few experimental points were deter-
mined in the alkaline pH range; therefore, values of pK3B
and kOH are affected by a significant error.
Table 1 shows the experimental results obtained at
different pH values and temperatures for the overall
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2005; 18: 546–551

´
G. R. ECHEVARRIA ET AL.
548
Scheme 1
DISCUSSION
increases with increasing pH) and of the sequence
k10 > k11 > k12 > k13 (the reactivity decreases with increas-
ing pH). The net balance between these opposing effects
leads to the maxima in the k1 values.
Table 1 shows that at each temperature, as the pH
increases, the k1 value increases up to a maximum and
then decreases. The maxima in the k1 values are the result
of the pKN for the amino group (the fraction of free amine
The rate-determining step of the formation of a Schiff
base is known to be the dehydration of an aminomethanol
Table 2. Best kinetic constants and pK values (Scheme 1) obtained by fitting of experimental values of k1, k2 and KpH to
Eqns (2), (3) and (4)
10 ꢂC
20 ꢂC
30 ꢂC
37 ꢂC
log k10
log k11
log k12
log k13
6.73 ꢅ 0.03
4.91 ꢅ 0.02
2.91 ꢅ 0.03
1.60 ꢅ 0.05
6.49 ꢅ 0.03
5.08 ꢅ 0.02
3.16 ꢅ 0.03
1.85 ꢅ 0.05
6.57 ꢅ 0.05
5.06 ꢅ 0.03
3.22 ꢅ 0.06
2.07 ꢅ 0.07
6.73 ꢅ 0.04
4.96 ꢅ 0.03
3.24 ꢅ 0.04
2.22 ꢅ 0.04
a
pK1P
pK2P
pK3P
3.61
6.32
3.77
6.12
3.46
6.02
3.39
6.05
a
a
8.68
8.37
8.16
8.13
0
log k21
log k22
log k2
log kOH
pK1B
pK2B
pK3B
log kM
pKN
ꢀ0.74 ꢅ 0.08
ꢀ0.23 ꢅ 0.04
ꢀ1.85 ꢅ 0.08
0.041 ꢅ 0.10
5.85 ꢅ 0.18
6.88 ꢅ 0.17
11.7 ꢅ 2.5
1.68 ꢅ 0.03
9.60
ꢀ0.23 ꢅ 0.1
0.13 ꢅ 0.1
ꢀ1.37 ꢅ 0.13
0.46 ꢅ 0.17
5.85 ꢅ 1.1
6.55 ꢅ 1.1
11.3 ꢅ 4.2
1.72 ꢅ 0.08
9.30
0.02 ꢅ 0.1
0.54 ꢅ 0.13
ꢀ1.05 ꢅ 0.2
0.21 ꢅ 0.28
5.73 ꢅ 0.28
6.23 ꢅ 0.28
10.7 ꢅ 1.0
1.76 ꢅ 0.03
9.00
0.03 ꢅ 0.09
0.80 ꢅ 0.07
ꢀ0.94 ꢅ 0.2
0.44 ꢅ 0.07
5.89 ꢅ 0.32
6.05 ꢅ 0.30
10.46 ꢅ 0.42
1.86 ꢅ 0.04
8.80
a From refs 7 and 15.
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2005; 18: 546–551

REACTION OF PYRIDOXAL 50-PHOSPHATE WITH L-TRYPTOPHAN
549
Figure 1. Brønsted plots for the PLP–TRP system at different temperatures
intermediate formed by attack of the amine on the
carbonyl group.4,5 The increase in k1i with decreasing
pH observed at all the temperatures used (Table 2) is due
to the involvement of all the protonable groups in PLP in
intramolecular acid catalysis on the formation of the
Schiff base. The linear Brønsted plot (Fig. 1) confirms
the intramolecular acid catalysis; the values of ꢀ ¼ 0.71–
0.75 are similar to those reported for the formation of
Schiff bases of PLP with amines and amino acids.6,7,13,16
On the other hand, experiments carried out by Llor
et al.17,18 exclude the possible influence of general
acid catalysis on this reaction by the buffer systems. A
similar observation was made by Auld and Bruice19,20 in
the Schiff base formation from 3-hydroxypyridine-4-
carbaldehyde.
consistent with expectation because the pK values for
phosphoric acid are known to be independent of tem-
perature.23 However, pK2B decreases with an increase in
temperature. This behavior corresponds to the deprotona-
tion of pyridinic nitrogen, as reported for the Schiff’s
bases of PLP with n-hexylamine,7 PLP with GABA13 and
50-deoxypyridoxal with n-hexylamine.8
The Arrhenius and Eyring plots of the rate constants k1i
and k1i obtained (Fig. 3) allow calculation of the energy of
activation (Ea) and activation parameters (ÁHꢅ and
ÁSꢅ ) of the elementary processes of formation and
The rate constant k22 is the smallest among the hydro-
lysis constants obtained at each temperature (Table 2).
Accordingly, species B2 is the most stable against hydro-
lysis by water: because its imine nitrogen is protonated, it
forms a hydrogen bond with the 3-phenoxy group on the
pyridine ring.21 In other words, the incorporation of a
proton in B3 stabilizes the Schiff base; on the other hand,
the incorporation of the second proton to form B2
facilitates its hydrolysis by water. Figure 2 shows the
calculated curves of log k2 vs. pH at three different
temperatures. It can be observed that the minimum shifts
from pH 9.2 at 10 ꢂC to pH 8.5 at 37 ꢂC; this shift can be
due to a greater shift for pK3B than for pK2B with
temperature (see Table 2), in the same way as the shift
described by solvent polarity.22
Figure 2. Plot of log k2 vs. pH for the Schiff bases of
pyridoxal 50-phosphate and L-tryptophan at different tem-
peratures. Points calculated using Eqn (3) and data from
Table 2
Table 2 also shows the pK values for the various forms
of the Schiff’s bases. As can be seen, pK1B is very similar
throughout the temperature range studied. This is quite
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2005; 18: 546–551

´
G. R. ECHEVARRIA ET AL.
550
Figure 3. Arrhenius and Eyring plots for the formation (k1i ) and hydrolysis (k2i ) of the Schiff bases of pyridoxal 50-phosphate and
L-tryptophan: (&) i ¼ 0; (*) i ¼ 1; (~) i ¼ 2; (!) i ¼ 3
hydrolysis of the Schiff bases (see Table 3). The k23 values
were obtained from the relation KM ¼ k13=k23 (see Scheme
although a catalytic effect due to the carboxylic group has
been disregarded.6b Nevertheless, it should be noted that,
although dehydration of the aminomethanol intermediate
is the rate-determining step, there are several previous
equilibrium steps that can be affected by the carboxylate
group. Our values are also lower than those reported by
Wiesinger and Hinz for the reactions of PLP with "-
aminocaproic acid and L-serine.9
0
1). As can be observed in Table 2, the k1 values are
nearly constant in temperature, suggesting very low
Ea(k10) and ÁHꢅ (k10) values in acid media.
As can be seen, for the formation process, Ea(k1i ) and
ÁHꢅ (k1i ) increase with the increasing number of nega-
tive charges in the PLP molecule. In neutral media the
values are lower than those reported for the PLP–n-
hexylamine system7 and PLP–GABA system,13 but in
basic media they are greater. This behavior can be due to
the presence of the carboxylate anion in the ꢀ-position,
With regard to hydrolysis of the Schiff base, the Ea(k2i )
and ÁHꢅ (k2i ) values for species B0 in the PLP–TRP
system are smaller than those for the Schiff base of n-
hexylamine7 or GABA.13 On the other hand, these para-
meters for species B1 and B2 are very similar in all cases;
for B3 in hydrolysis of the Schiff base of L-tryptophan the
values are larger than in the PLP–GABA system.13
Table 3 also shows that, as in the reactions of PLP with
Table 3. Energy of activation and activation parametersa for
the elementary processes (i ¼ 0–3)
¼
n-hexylamine7 and GABA,13 the ÁS values are nega-
0
1
2
3
tive in all cases, in accordance with the decreased number
of degrees of freedom in the transition state: the removal
of a water molecule from the aminomethanol intermedi-
ate requires the presence of a catalyst, and hydrolysis of
the Schiff base is a bimolecular process.
Table 4 shows the thermodynamic parameters for the
elementary processes as obtained from the activation
parameters corresponding to each elementary process.
Ea(k1i )
—
—
3 ꢅ 8
1 ꢅ 1
20 ꢅ 6
18 ꢅ 6
40 ꢅ 1
38 ꢅ 1
¼
ÁH (k1i )
¼
ÁS (k1i ) ꢀ140 ꢅ 40 ꢀ160 ꢅ 30 ꢀ140 ꢅ 20 ꢀ100 ꢅ 4
Ea(k2i )
49 ꢅ 11
46 ꢅ 10
65 ꢅ 2
62 ꢅ 2
57 ꢅ 7
54 ꢅ 8
19 ꢅ 3
16 ꢅ 3
¼
ÁH (k2i )
¼
ÁS (k2i ) ꢀ110 ꢅ 40
ꢀ46 ꢅ 7 ꢀ100 ꢅ 30 ꢀ200 ꢅ 10
a Units: Ea and ÁH in kJ molꢀ1; ÁS in J molꢀ1 Kꢀ1
.
¼
¼
Table 4. Thermodynamic parameters for the elementary processes (i ¼ 0–3) and equilibrium constants obtained from kinetic
(Table 2) and thermodynamic parameters (Table 3)a
ꢁ
ꢂ
Process i
ÁH0
ÁS0
ÁG0
logKeq,298 K log Kpi H ¼ k1i =k2i
298 K
0
1
2
3
ꢀ46 ꢅ 10b
ꢀ61 ꢅ 10
ꢀ42 ꢅ 14
22 ꢅ 46
ꢀ30 ꢅ 80
ꢀ110 ꢅ 40
ꢀ40 ꢅ 50
100 ꢅ 15
ꢀ37
6.5
4.7
5.2
1.5
7.1
4.8
4.5
1.7
ꢀ27
ꢀ30
ꢀ8.6
a Units: ÁH0 and ÁG0 in kJ molꢀ1; ÁS0 in J molꢀ1 Kꢀ1
.
b Assuming ÁH (k10) ¼ 0 (see text).
¼
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2005; 18: 546–551

REACTION OF PYRIDOXAL 50-PHOSPHATE WITH L-TRYPTOPHAN
551
It also shows the corresponding ÁG0 values calculated
for the reaction at 25 ꢂC, and the log K values obtained
from the ÁG0 and k1i and k2i values.6b As can be seen, the
reaction is exothermic in acid and neutral media but
endothermic in basic media, similar to the reaction of
PLP with n-hexylamine7 and also in accordance with
ÁH0 < 0 measured calorimetrically for the reaction of
PLP with n-hexylamine and poly-L-lysine24 at pH 7.0.
The ÁS0 value is negative in neutral and acid media but
positive in alkaline media. The significance of the en-
tropic factor in alkaline media is noteworthy because it
offsets the unfavorable enthalpic factor in ÁG0, which is
thus rendered negative. Because of the entropic contribu-
tion to the reaction, the formation of the Schiff base is
more favorable in an acid than in a neutral or alkaline
medium. No significant differences are observed between
the equilibrium constant obtained from thermodynamic
parameters and that derived from k1i =k2i .
4. Jencks WP. In Catalysis in Chemistry, and Enzymology. McGraw-
Hill: New York, 1969; 490–496.
5. Rosenberg S, Silver SM, Sayer JM, Jencks WP. J. Am. Chem. Soc.
1974; 96: 7986–7998.
´
6. (a) Echevarrıa Gorostidi GR, Basagoitia A, Pizarro E, Goldsmitd
R, Santos JG, Garcıa Blanco F. Helv. Chim. Acta 1998; 81:
´
´
837–844; (b) Echevarrıa Gorostidi GR, Santos JG, Basagoitia
A, Garcıa Blanco F. Bull. Chem. Soc. Jpn. 2002; 75: 2471–2476;
´
´
(c) Echevarrıa GR, Martın Perez MP, Santos JG, Garcıa Blanco F.
Helv. Chim. Acta 1999; 82: 769–778.
´
´
´
´
´
´
7. Garcıa del Vado MA, Donoso J, Mun˜oz F, Echevarrıa GR, Garcıa
Blanco F. J. Chem. Soc. Perkin Trans 2 1987: 445–448.
´
8. Vazquez MA, Donoso J, Mun˜oz F, Garcıa Blanco F, Garcıa del
Vado MA, Echevarrıa GR. Helv. Chim. Acta. 1990; 73: 1991–
´
´
´
1998.
9. Wiesinger H, Hinz HJ. Arch. Biochem. Biophys. 1984; 235:
34–40.
10. Wiesinger H, Hinz HJ. Biochemistry 1984; 23: 4921–4928.
11. Wiesinger H, Hinz HJ. Biochemistry 1984; 23: 4928–4934.
12. Giartosio A, Salerno C, Franchetta F, Turano C. J. Biol. Chem.
1982; 257: 8163–8170.
´
13. Echevarrıa Gorostidi GR, Castellanos MG, Martın Perez MP,
Santos JG, Garcıa Blanco F. Bull. Chem. Soc. Jpn. 2003; 76: 523–
´
´
´
528.
14. Peterson EA, Sober HA. J. Am. Chem. Soc. 1954; 76: 169–175.
´
´
´
´
15. Echevarrıa GR, Garcıa del Vado MA, Garcıa Blanco F, Menendez
M, Laynez J. J. Solution Chem. 1986; 15: 151–156.
Acknowledgments
´
´
16. Vazquez MA, Mun˜oz F, Donoso J, Garcıa Blanco F. Int. J. Chem.
Kinet. 1990; 22: 905–914.
´
´
Financially supported by DGI (Spain: Projects BQ2003-
07281 and BQ2000-0787) and FONDECYT (Chile:
Projects 1990551 and 2990006).
17. Sanchez Ruiz JM, Rodrıguez Pulido JM, Llor J, Cortijo M.
J. Chem. Soc. Perkin Trans. 2. 1982: 1425–1428.
´
´
18. Llor J, Sanchez Ruiz JM, Rodrıguez Pulido JM, Cortijo M.
An. Quim. 1984; 80: 27–32.
19. French TC, Auld DS, Bruice TC. Biochemistry 1965; 4: 77–84.
20. Auld DS, Bruice TC. J. Am. Chem. Soc. 1967; 89: 2083–2089.
21. Szpoganicz B, Martell AE. J. Am. Chem. Soc. 1984; 106: 5513–
5521.
REFERENCES
´
22. Echevarrıa Gorostidi GR, Santos JG, Basagoitia A, Castillo M,
´
23. Albert A, Serjeant EP. In The Determination of Ionization
Constants: A Laboratory Manual. Chapman and Hall: London,
1971; 7–8.
Garcıa Blanco F. Bull. Chem. Soc. Jpn. 2003; 76: 335–440.
1. Snell EE. In Vitamin B-6 Pyridoxal Phosphate. Chemical,
Biochemical and Medical Aspects. Part A, Dolphin D, Poulson
R, Avramovic O (eds). John Wiley: New York, 1986; 1–12.
2. Emery VC, Akhtar M. In Enzyme Mechanisms, Page MI, Williams
A (eds). Royal Society of Chemistry: London, 1989; 345–389.
3. Braunstein E. In Transaminases, Christen P, Metzler DE (eds).
John Wiley: New York, 1985; 2–19.
´
24. Garcıa del Vado MA, Echevarrıa GR, Garcıa Blanco F, Santos JG,
Laynez Vallejo J, Garcıa de Paz JL. Helv. Chim. Acta 1991; 74:
´
´
´
1749–1756.
Copyright # 2004 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2005; 18: 546–551