Thermodynamical characteristics of the reaction of pyridoxal-5'-phosphate with L-amino acids in aqueous buffer solution

Article abstract of DOI:10.1134/S003602441101002X

The reaction of pyridoxal-5'-phosphate with L-isomers of alanine, lysine, arginine, aspartic acid, glutamic acid, and glycine in phosphate buffer solution was studied by absorption spectroscopy and the calorimetry of dissolution at physiological acidity of the medium (pH 7.35). The formation constants of Schiff bases during reactions and changes in Gibbs energy, enthalpy, and entropy were determined. It was shown that the formation constant of the Schiff base and its spectral properties depend on the nature of the bound amino acid. The progress of the reaction with a majority of amino acids is governed by the entropy factor due to the predominant role of the dehydration effect of the reaction center of amino acids during chemical reactions. The intramolecular electrostatic interaction of an ionized phosphate group with the positively charged amino group on the end of the chain of amino acid residue stabilizes the Schiff bases formed by lysine and arginine. The extinction coefficient of the base, equilibrium constant, and the exothermic effect of the reaction then increase. The excess negative charge on the end of the chain of amino acid residues of aspartic and glutamic acids destabilizes the molecule of the Schiff base. In this case, the equilibrium constant decreases and the endothermic effect of the reaction increases. Pleiades Publishing, Ltd., 2011.

Full text of DOI:10.1134/S003602441101002X

ISSN 0036ꢀ0244, Russian Journal of Physical Chemistry A, 2011, Vol. 85, No. 1, pp. 16–20. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © V.P. Barannikov, V.G. Badelin, E.A. Venediktov, I.N. Mezhevoi, S.S. Guseinov, 2011, published in Zhurnal Fizicheskoi Khimii, 2011, Vol. 85, No. 1,
pp. 20–24.
CHEMICAL THERMODYNAMICS
AND THERMOCHEMISTRY
Thermodynamical Characteristics of the Reaction
of Pyridoxalꢀ5'ꢀPhosphate with LꢀAmino Acids
in Aqueous Buffer Solution
V. P. Barannikov, V. G. Badelin, E. A. Venediktov, I. N. Mezhevoi, and S. S. Guseinov
Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russia
eꢀmail: vpb@iscꢀras.ru
Received December 17, 2009
Abstract—The reaction of pyridoxalꢀ5'ꢀphosphate with Lꢀisomers of alanine, lysine, arginine, aspartic acid,
glutamic acid, and glycine in phosphate buffer solution was studied by absorption spectroscopy and the calꢀ
orimetry of dissolution at physiological acidity of the medium (pH 7.35). The formation constants of Schiff
bases during reactions and changes in Gibbs energy, enthalpy, and entropy were determined. It was shown that
the formation constant of the Schiff base and its spectral properties depend on the nature of the bound amino
acid. The progress of the reaction with a majority of amino acids is governed by the entropy factor due to the
predominant role of the dehydration effect of the reaction center of amino acids during chemical reactions.
The intramolecular electrostatic interaction of an ionized phosphate group with the positively charged amino
group on the end of the chain of amino acid residue stabilizes the Schiff bases formed by lysine and arginine.
The extinction coefficient of the base, equilibrium constant, and the exothermic effect of the reaction then
increase. The excess negative charge on the end of the chain of amino acid residues of aspartic and glutamic
acids destabilizes the molecule of the Schiff base. In this case, the equilibrium constant decreases and the
endothermic effect of the reaction increases.
Keywords: solutions, amino acids, UV spectroscopy, calorimetry, Schiff bases.
DOI: 10.1134/S003602441101002X
INTRODUCTION
EXPERIMENTAL
In many processes of the metabolism of amino
Electronic absorption spectra were obtained on a
acids, pyridoxalꢀ5'ꢀphosphate (POP) serves as a coenꢀ Specord Mꢀ40 twoꢀbeam spectrophotometer in the
zyme [1]. It is known that the product of reaction with range of wavelengths from 337 to 600 nm. The studied
solutions were placed in quartz cells
1
×
1 cm in size.
amino acids is a Schiff base [1–3]. The effectiveness of
binding amino acids by pyridoxal phosphate coenꢀ
zyme depends on the structure of the amino acid molꢀ
ecules and their state in solution. The chemical proꢀ
cesses with the participation of amino acids proceeds
under the strong influence of the acid–base equilibꢀ
rium in the system. The purpose of this study was to
obtain the thermodynamical characteristics of the
reaction of pyridoxalꢀ5'ꢀphosphate with various
amino acids in aqueous buffer solution at physiologiꢀ
cal acidity of the medium pH 7.35. Under these conꢀ
ditions, the predominant ionic forms of the existence
of the studied amino acids differ substantially: glycine
and alanine are zwitterions, aspartic and glutamic acid
are anions, and lysine and arginine are cations [4].
Therefore, at constant acidity of the medium, the conꢀ
tribution from the electrostatic interactions of a recepꢀ
tor with various amino acid ions should differ substanꢀ reagents were
All measurements were performed at 293 ± ꢀ03 K. The
reaction between amino acids and coenzyme took
place in a buffer solution containing monobasic
sodium phosphate NaH PO4 (0.04165 M) and dibasic
2
sodium phosphate Na HPO4 (0.20492 M), pH 7.35.
2
The use of buffer solution maintained the constant
acidity of the medium and the stability of the ionic
forms of reagents during reaction. The measurements
of dissolution enthalpies of amino acids in buffer soluꢀ
tion and analogous solution with the addition of POP
were performed on a calorimeter with an isothermal
shell at 293 ± ꢀ0ꢀꢀꢁ K. The technical characteristics of
calorimeter and the technique of measurements were
described in [5]. The error in measuring the heat effect
–
2
was
1
×
1ꢀ J.
During spectral studies, the concentrations of
–
ꢁ
4
×
1ꢀ mol/l for POP and up to 108
×
–
3
tially in the order of amino acids, determining the 1ꢀ mol/l for amino acids; during calorimetric invesꢀ
–
3
⎯ 2
hierarchy of the “recognition” of the given substrates tigations, they were
in accordance with their charge distribution. respectively. The mean values of dissolution enthalpy
1
×
1ꢀ and
1
×
1ꢀ mol/kg,
16

THERMODYNAMICAL CHARACTERISTICS
17
Δ
solH from 4–5 measurements were used as the stanꢀ
43ꢀ0ꢁ
Δ
D
dard dissolution enthalpy solН . Lꢀisomers of amino
Δ
°
ꢀ
0ꢀꢁꢀ
0ꢀ2ꢁ
ꢀ
acids (all Sigma–Aldrich, 98–99% purity) and pyriꢀ
doxalꢀ5'ꢀphosphate (Sigma–Aldrich, 98%) were dried
prior to the experiments for 48 h under vacuum and
used without further purification. Concentrations of
substances in the solution were calculated by test porꢀ
tions.
3
2
1
ꢀ
λ
, nm
RESULTS AND DISCUSSION
−
ꢀ0ꢀ2ꢁ
In electronic absorption spectra of POP in aqueous
buffer solution at pH 7.35, the absorption maximum is
observed at 387 nm with an extinction coefficient of
37ꢁ
Fig. 1. Differential electronic absorption spectra of 3099
1ꢀ M aqueous solution of pyridoxalꢀ5'ꢀphosphate in
phosphate buffer at pH 7.35 and 293 K in the presence of
×
–1
–1
–ꢁ
6
250 cm l mol . This band could be related to the
absorption of a bipolar ion or anion of POP [3]. With
an addition of an amino acid, the band widens and
undergoes a bathochromic shift. Changes in the specꢀ
trum indicate the formation of a Schiff base [2]. The
progress of the reaction between the amino acid and
POP was observed by changes in the differential elecꢀ
tronic absorption spectrum of POP upon various addiꢀ
tions of amino acid, recorded relative to the solution of
a single POP with a same concentration. A reduction
in the intensity of absorption at 375 nm and a rise in
the intensity of absorption of the Schiff base at 430–
–
4
various additives of Lꢀarginine: (
1
) 4.0 × 1ꢀ , (2) 10.1 ×
–4
–4
) 18.2 ×
1ꢀ mol l–1.
1
ꢀ
, and (
3
D43ꢀ0ꢁ
0ꢀ8
Δ
ꢀ
5
ꢀ
0ꢀ6
4
3
4
36 nm are observed in the spectrum upon the binding
of the amino acid (Fig. 1). The molar absorption coefꢀ
ficient in the band maximum of the Schiff base was
found from the linear correlation of changes in optical
densities and the molar extinction coefficients for two
bands of the differential spectrum: the absorption of
POP at 375 nm (
Schiff base at 430–436 nm (
ꢀ0ꢀ4
ꢀ0ꢀ2
2
1
3
7ꢁ 37ꢁ
Δ
D
,
ε
) and the absorption of the
ShB ShB
Δ
D
,
ε
).
ꢀ0ꢀ1
ꢀ0ꢀ2
ꢀ0ꢀ3
ΔD
3
7ꢁ
ShB
375
ShB
375
ΔD
ΔD = ε
ε
0
(1)
DShB and D37ꢁ at various conꢀ
centrations of amino acid are related by a linear correꢀ
lation, as is shown in Fig. 2 for solutions with the addiꢀ
tion of arginine. For the system under consideration,
Fig. 2. Interdependence of changes in optical density at
wavelengths of 430.5 and 375 nm in the differential specꢀ
trum of pyridoxalꢀ5'ꢀphosphate in phosphate buffer at
pH 7.35 and 293 K in the presence of various additives of
The change in values
Δ
Δ
Lꢀarginine: (
1) 0.0002, (2) 0.0004, (3) 0.001, (4) 0.0014,
–1
43ꢀ0ꢁ
37ꢁ
Δ
and ( ) 0.0018 mol l
5
.
the ratio
Δ
D
/
D
was 2046 ± ꢀ01ꢁ. The extinction
coefficient in the band maximum of the Schiff base
formed by arginine was found using Eq. (1) and
37ꢁ
387
–1
–1 43ꢀ0ꢁ
librium concentrations of Schiff bases found from
spectrophotometric data:
approximation ε ≈ ε = 6250 cm l mol :
ε
=
–1
–1
43ꢀ0ꢁ
ε
1
7500 cm l mol . The resulting
was used in the
spectrophotometric determination of the equilibrium
concentration of the Schiff base [ShB] by the Buger–
Lambert–Beer equation
R'CH=O + NH R''
2
↔
R'CH=NR'' + H O0 (3)
2
The total equilibrium concentrations for all forms and
ionic states of the reagents and products of the reacꢀ
tion were used in the expression for formation conꢀ
stant:
D43ꢀ0ꢁ
43ꢀ0ꢁl[ShB]0
Δ
=
ε
(2)
As follows from the obtained spectrophotometrical
data, the equilibrium concentration of the Schiff base
–
6
⎯
6
–1
increases from ꢀ08
×
1ꢀ to 403
×
1ꢀ mol l when
K = K '
[
H O
]
=
[
ShB] [POP][AA
]
,
(4)
2
the initial concentration of arginine is increased from
–
4
⎯
4
–1
2
0ꢀ
×
1ꢀ to 1802
×
1ꢀ mol l . The method for where [ShB], [POP], and [AA] are the equilibrium
determining the equilibrium concentrations of the concentrations of the Schiff base, pyridoxalꢀ5'ꢀphosꢀ
Schiff bases with other amino acids is analogous to the phate, and amino acid, respectively. Equilibrium conꢀ
procedures described above. The formation constants centrations [ShB] were determined from spectrophoꢀ
of the Schiff bases were calculated on the basis of equiꢀ tometric data by Eqs. (1) and (2). Equilibrium conꢀ
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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18
BARANNIKOV et al.
Table 1. Positions of the absorption maximum, extinction residue leads to a rise in wavelength in the absorption
–1
–1
coefficients ( , cm l mol ), and formation constants of maximum and a reduction in the molar absorption
ε
the Schiff bases (
K
, l/mol) with various amino acids
coefficient of the Schiff base. The binding constant
tends to decrease. Residues of aspartic and glutamic
acids having even longer chains break the conjugation
even more strongly. The binding constants of these
amino acids are considerably lower than those for glyꢀ
cine and alanine. The positive inductive effect of the
additional carboxylic group on the ends of the chains
of amino acid residues manifests itself in an increase in
the extinction coefficient of the Schiff bases with their
participation relative to the base formed by alanine.
Amino acid
λ
, nm
ε
log
К
± ꢀ0ꢀꢁ
Glycine
43ꢀ0ꢁ
4360ꢁ
43ꢀ0ꢀ
43ꢀ0ꢁ
4340ꢀ
4340ꢀ
816ꢀ
343ꢀ
1078
LꢂAlanine
LꢂLysine
LꢂArginine
1074
1083
1092
1061
1048
1786ꢀ
17ꢁꢀꢀ
8ꢁ9ꢀ
LꢂGlutamic acid
LꢂAspartic acid
62ꢁꢀ
Maximal binding with the Schiff base is observed
for arginine and lysine, the predominant form of
which is the cation at pH 7.35. It is evident that the
intramolecular electrostatic attraction between the
positively charged amino group on the end of the
amino acid residue and the negatively charged phosꢀ
phate group stabilizes the molecules of the Schiff base,
as can be seen from Scheme (I) for a Schiff base with
the contribution of lysine. The degree of conjugation
of the aldimine and oxypyridine group increases due to
the reduction in the degree of freedom of the amino
acid residue. The rapid growth of the molar absorption
centrations [POP] and [AA] were calculated using the
expressions
0
[POP] =
c
– [ShB],
POP
0
[AA] =
c
– [ShB],
AA
0
0
AA
where
c
and
c
are the initial molar concentraꢀ
POP
tions of the starting compounds. The calculated forꢀ
mation constants were averaged for the studied conꢀ
–
4
centration range of amino acids from 20ꢀ
×
1ꢀ to
–3
–1
108
×
1ꢀ mol l . The resulting spectral characterisꢀ coefficients of the Schiff bases formed by lysine and
tics and averaged formation constants of the Schiff arginine relative to other bases confirms this. The
bases obtained with various amino acids are given in excess negative charge in the side chain of aspartic and
Table 1.
glutamic acid reduces the yield from the reaction of
The data of Table 1 confirm that the formation the formation of the Schiff base. Electrostatic repulꢀ
constant of a Schiff base and its spectral properties sion between the negatively charged carboxylic and
depend on the nature of the bound amino acid. As folꢀ ionized phosphate group in the molecules of the formꢀ
lows from a comparison of the data on glycine and alaꢀ ing Schiff bases is an additional factor in the breaking
nine, the breaking of the conjugation of the aldimine of the conjugation of the aldimine group with the oxyꢀ
group CH=N with aromatic oxypyridine chroꢀ pyridine ring (Scheme (II) for a Schiff base with the
mophore under the effect of the more branched alanyl participation of glutamic acid).
−
−
COO
COO
O
O
+
H N(CH ) HC
C(CH ) HC
3
2 4
2 2
NH+
CH
−
NH+
CH
−
−
O
−
O
−
POH C
O
POH C
O
2
2
HO
HO
N
CH3
N
CH3
(I)
(II)
It is interesting to note the correlation between the tion enthalpy of a number of compounds (amino acids
logarithm of the formation constant of the Schiff base and amides) with the sum of the bond lengths in the
and the hydration enthalpy of the amino acids (Fig. 3). polyatomic molecule [8].
The hydration enthalpies of the studied amino acids
The dissolution enthalpies of crystalline amino
acids in the buffer solution upon the addition of pyriꢀ
were calculated from the values of the dissolution
enthalpies [6]. The sublimation enthalpies of glycine
and alanine are taken from [7]. The sublimation doxalꢀ5'ꢀphosphate were determined by calorimetry
enthalpies of the Lꢀisomers of lysine, arginine, asparꢀ
( H ). The measured value includes the contribuꢀ
Δ
sol 2
tic, and glutamic acid were estimated on the basis of tion from amino acid dissolution, determined in a
the correlation between the specificꢀvolume sublimaꢀ blank experiment in the absence of coenzyme
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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THERMODYNAMICAL CHARACTERISTICS
19
characteristics of the reaction between the coenzyme
and various amino acids are given at physiological
acidity of the medium (pH 7.35). As follows from
Table 2, the progress of the interaction of the coenꢀ
zyme with the majority of amino acids is governed by
the entropy factor, due to the predominant role of the
dehydration effect of the amino acid’s reaction center
during the chemical reaction. The contribution from
enthalpy in the cases of alanine, glutamic, and aspartic
acid prevents the interaction from progressing. Two
exceptions are lysine and arginine containing posiꢀ
tively charged amino groups in the side chain of the
amino acid. In reactions with their participation, the
exothermic enthalpy effect dominates over the
entropy contribution to the change in Gibbs energy.
log
K
08
06
04
Arg
Lys
Gly
1
Ala
Glu
1
Asp
4ꢀ
1
1
13ꢀ
12ꢀ
11ꢀ 1ꢀꢀ
hydrH, kJ/mol
−
Δ
Fig. 3. Correlation of the formation constants of the Schiff
bases in reaction (3) with the hydration enthalpies of
Lꢀamino acids.
CONCLUSIONS
Thermodynamical analysis thus shows that the disꢀ
crimination of amino acids of a given series in a reacꢀ
( solH1), and the effect of the reaction of coenzyme tion with coenzyme pyridoxalꢀ5'ꢀphosphate is deterꢀ
Δ
with amino acid,
mined by the principles of dehydration of their reacꢀ
tion groups. This conclusion is supported by the
correlation between the formation constants of the
Δ
solH2
=
Δ
solH1
+
α ΔrH
r
,
(ꢁ)
where
α
is the degree of conversion of the amino acid, Schiff bases and the hydration enthalpies of the amino
r
and
rH is the change in enthalpy during the reaction, acids, and the domination of the positive contribution
Δ
as calculated for the mole of the product, the Schiff from entropy to the change in Gibbs energy over the
base. The degrees of conversion of the amino acids enthalpy of the reaction with the participation of most
under the given conditions were calculated from the amino acids.
formation constant
Combining the values
to calculate the change in enthalpy for the reaction of
the formation of the Schiff base ( rH) by Eq. (5).
K
of the Schiff bases (Table 1).
Intramolecular electrostatic interaction between
ionized groups in the molecules of the Schiff base is an
additional factor determining the stability of the prodꢀ
ucts of the reaction between the coenzyme and amino
Δ
solH2 solH1, and allows us
,
Δ
α
r
Δ
The calculations for the change in Gibbs energy acid. The reaction between an ionized phosphate
and entropy (in molar scale) during a reaction are perꢀ group and the positively charged amino group on the
formed according to the known correlations from the end of a chain of amino acid residue stabilizes the
formation constants of the Schiff bases and the combiꢀ Schiff bases formed by lysine and arginine. The
nation of the Gibbs energy and enthalpy of the reacꢀ increase in the extinction coefficient, equilibrium
tion:
constant, and the exothermic effect of reaction conꢀ
firms this. The reaction of the ionized phosphate
group with the negatively charged carboxyl group on
the end of a chain of amino acid residue reduces the
stability of the Schiff bases formed by aspartic acid and
Δ
rG
=
–2.303RTlogK,
rH rS
(6)
(7)
Δ
rG
=
Δ
–
T
Δ
0
All of the determined thermodynamic values are glutamic acid. The equilibrium constant declines and
given in Table 2; i.e., the complete thermodynamic the endothermic effect of the reaction increases.
Table 2. Thermodynamic characteristics of the reactions (kJ/mol) of pyridoxalꢂꢁ'ꢂphosphate with various Lꢂamino acids
in phosphate buffer at pH 703ꢁ and 29301ꢁ K
Amino acid
Glycine
Δ
solH1
Δ
solH2
α
–
Δ
rG
Δ
rH
T
Δ
rS
709ꢁ
2ꢁ0ꢁ
r
13089 ± ꢀ0ꢀ2
7023 ± ꢀ0ꢀ2
13081 ± ꢀ0ꢀ2
7077 ± ꢀ0ꢀ2
ꢀ0ꢀ366
ꢀ0ꢀ346
ꢀ0ꢀ424
ꢀ0ꢀ442
ꢀ0ꢀ28ꢁ
ꢀ0ꢀ227
1ꢀ01ꢁ ± ꢀ03
909 ± ꢀ03
1ꢀ08 ± ꢀ03
110ꢀ ± ꢀ03
902 ± ꢀ03
804 ± ꢀ03
–202 ± ꢀ08
1ꢁ06 ± ꢀ09
–902 ± ꢀ07
–707 ± ꢀ07
1203 ± 10ꢀ
1ꢀ06 ± 103
LꢂAlanine
LꢂLysine
–2809ꢀ ± ꢀ0ꢀ2 –29029 ± ꢀ0ꢀ2
–330ꢀ4 ± ꢀ0ꢀ2 –33038 ± ꢀ0ꢀ2
106
303
LꢂArginine
LꢂGlutamic acid
LꢂAspartic acid
2408ꢀ ± ꢀ0ꢀ2
2407ꢀ ± ꢀ0ꢀ2
2ꢁ01ꢁ ± ꢀ0ꢀ2
2409ꢀ ± ꢀ0ꢀ2
210ꢁ
190ꢀ
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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0
BARANNIKOV et al.
2. M. Angees Garcia del Vado et al., J. Mol. Catal. A:
ACKNOWLEDGMENTS
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3
tion for Basic Research, project no. 09ꢀ03ꢀ97510ꢀ
r_tsentr_a.
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. V. P. Barannikov, V. G. Badelin, and M. B. Berezin,
Biofizika 54, 206 (2009) [Biophysics 54, 139 (2009)].
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. V. P. Barannikov, S. S. Guseynov, and A. I. Vyugin,
J. Chem. Thermodyn. 36, 277 (2004).
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