An equilibrium and calorimetric study of some transamination reactions

Article abstract of DOI:10.1006/jcht.1998.0342

Apparent equilibrium constants and calorimetric enthalpies of reaction have been measured for the following enzyme-catalysed biochemical reactions at the temperature 298.15 K: L-alanine(aq) + 2-oxoglutarate(aq) = pyruvate(aq) + L-glutamate(aq); L-tyrosine(aq) + 2-oxoglutarate(aq) = 4-hydroxyphenylpyruvate(aq) + L-glutamate(aq); and L-phenylalanine(aq) + 2-oxoglutarate(aq) = phenylpyruvate(aq) + L-glutamate(aq). The results are used to calculate equilibrium constants and standard molar enthalpy, entropy, and Gibbs energy changes for reference reactions involving specific species. Apparent equilibrium constants and standard transformed Gibbs energy changes for these reactions under physiological conditions have also been calculated. The results are discussed in terms of the changes in chemical bonding characteristic of transamination reactions.

Full text of DOI:10.1006/jcht.1998.0342

J. Chem. Thermodynamics 1998, 30 777᎐793
Article No. ct980342
An equilibrium and calorimetric study of
some transamination reactions
Yadu B. Tewari, Nand Kishore,a Robert N. Goldberg
Biotechnology Di¨ision, National Institute of Standards and Technology,
Gaithersburg, MD 20899, U.S.A.
and Tinh N. Luong
Department of Molecular and Cell Biology, Stanley Hall, Uni¨ersity of California,
Berkeley, CA 94720-3206, U.S.A.
Apparent equilibrium constants and calorimetric enthalpies of reaction have been measured
for the following enzyme-catalysed biochemical reactions at the temperature 298.15 K:
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
L-alanine aq q 2-oxoglutarate aq s pyruvate aq q L-glutamate aq ; L-tyrosine aq q 2-
Ž
.
Ž
.
Ž
.
Ž
.
oxoglutarate aq s 4-hydroxyphenylpyruvate aq q L-glutamate aq ; and L-phenylalanine aq
Ž
.
Ž
.
Ž
.
q 2-oxoglutarate aq s phenylpyruvate aq q L-glutamate aq . The results are used to
calculate equilibrium constants and standard molar enthalpy, entropy, and Gibbs energy
changes for reference reactions involving specific species. Apparent equilibrium constants
and standard transformed Gibbs energy changes for these reactions under physiological
conditions have also been calculated. The results are discussed in terms of the changes in
chemical bonding characteristic of transamination reactions.
ᮊ 1998 Academic Press
KEYWORDS: L-alanine; aminotransferase; enthalpy; equilibrium constant; L-glutamate;
4-hydroxyphenylpyruvate; 2-oxoglutarate; L-phenylalanine; phenylpyruvate; pyruvate; L-
tyrosine
1. Introduction
As part of a continuing program of thermodynamic studies on enzyme-catalysed
reactions, we have measured apparent equilibrium constants and calorimetric
enthalpies for the following biochemical transamination reactions:
L-alanine aq q 2-oxoglutarate aq spyruvate aq q L-glutamate aq ,
1
Ž .
Ž
.
Ž
.
Ž
.
Ž
.
L-tyrosine aq q 2-oxoglutarate aq s
Ž
.
Ž
.
4-hydroxyphenylpyruvate aq q L-glutamate aq ,
2
Ž .
Ž
.
Ž
.
L-phenylalanine aq q 2-oxoglutarate aq s
Ž
.
Ž
.
phenylpyruvate aq q L-glutamate aq .
3
Ž .
Ž
.
Ž
.
These three reactions involve the same structural change, namely the transfer of an
aGuest researcher from the Department of Chemistry, Indian Institute of Technology, Bombay, India.
0021᎐9614r98r060777 q 17 $30.00r0
ᮊ 1998 Academic Press

778
Y. B. Tewari et al.
Ž . Ž .
Ž .
FIGURE 1. Structures of the substances in reactions 1 , 2 , and 3 .
Ž
.
amino group from an amino acid to a 2-keto acid see figure 1 . Therefore, to a
first approximation, one would expect that thermodynamic quantities for these
three reactions should be similar. While values of apparent equilibrium constants
Ž1 ᎐ 9.
Ž .
for reaction 1 under a variety of conditions have been reported several times,
Ž .
Ž .
there have been no such results reported for reactions 2 and 3 . Also, a recent
surveyŽ10. of the literature on these reactions shows that there have been no
calorimetric studies for any biochemical transamination reactions. Additionally, the
only available molar enthalpy in the literature on transamination reactions is the
approximate value that can be calculated from the temperature dependency of
the apparent equilibrium constants reported by GuynnŽ11. for the conversion of
Ä
Ž
.
Ž
.
4
Ä
Ž
.
Ž
.4
L-alanine aq q hydroxypyruvate aq to L-serine aq q pyruvate aq .
Interest in transamination reactions stems from their ubiquitous occurrence in

Thermodynamics of transamination reactions
779
nature in metabolic pathways involving amino acids and amines. In microorganisms,
Ž .
Ž .
reactions 2 and 3 are the final steps in the shikimic acid pathway that leads to
the biosynthesis of L-phenylalanine and L-tyrosine.Ž12. This pathway is currently
receiving much attention due to its possible use for the large scale production of
aromatic amino acids.Ž13,14. We also note that transamination reactions lead to
optically pure substances, an important advantage for many applications.
The principal aim of this study is to determine apparent equilibrium constants
Ž . Ž .
Ž .
and calorimetric molar enthalpies for reactions 1 , 2 , and 3 . These results are
then used to calculate equilibrium constants, standard molar enthalpies, and
standard molar entropies for reference reactions that involve specific species.
These thermodynamic quantities can be used to calculate the position of equilibrium
of the corresponding biochemical reactions as a function of temperature, pH, and
ionic strength. Apparent equilibrium constants and standard transformed Gibbs
energy changesŽ15. for biochemical reactions 1 , 2 , and 3 under physiological
conditions are also calculated.
Ž . Ž .
Ž .
2. Experimental
Pertinent information on the substances used in this study is given in table 1. The
purities of the biochemical substances were also examined by using the
chromatographic procedures described below and were found to be consistent with
Ž
the values given in table 1. The mass fractions of water in these substances see
.
column 5 in table 1 were measured with a Metrohm Model 633 automatic Karl
Fischer titrator, which was calibrated with a water-saturated octanol solution.Ž16.
These mass fractions of water were used in all subsequent calculations that
involved the amounts of these substances.
Ž
.
The alanine aminotransferase EC 1.6.1.2 was obtained from Sigma in the form
of a lyophilized powder that had been isolated from porcine heart. The tyrosine
Ž
.
aminotransferase EC 2.6.1.5 was prepared by using the E. coli aminotransferase-
deficient strain MG204Ž17. and was purified according to the method of Herold and
Kirschner Ž18. as modified by Onuffer et al.Ž17. The enzyme was stored at the
y3
.
y4
Ä
Ž
.
q pyridoxal 5-phosphate
temperature 277.15 K in a K2 HPO4 0.010 mol dm
y5
y3
y3
.
.
.
.
Ž
.
Ž
.
q ethylenediamine-
2.0 10 mol dm
q dithiothreitol 5.0 10 mol dm
y3
.
Ž
.4
solution at pH s 7.5. The mass fraction of the
tetraacetic acid 0.001 mol dm
tyrosine aminotransferase in this solution was f0.015.
ŽL-alanine q 2-oxoglutarate q pyruvate q L-glutamate q pyridoxal
The mixture
.
5-phosphate was separated by using a Hewlett-Packard 1100 h.p.l.c equipped with
a u.v. detector set at the wavelength ␭ s 215 nm and a Hewlett-Packard Hypersil
Ž
.
C18 column 4.5 mm i.d., 250 mm long thermostatted at T s 313 K. The mobile
Ž .
I
Ä
Ž
phase consisted of
tetrabutyl ammonium hydroxide concentration c s
q ammonium phosphate c s 0.025 mol dm
y3
y3
.
.
.
Ž
.
adjusted to pH s
0.01 mol dm
4
Ž .
5.3 with H 3PO4 and II acetonitrile. The following gradient of these two mobile
Ž . Ž . Ž .
Ž .
phases was formed: volume fraction ␾ I s 0.85 and ␾ II s 0.15 at t s 0; ␾ I s
Ž .
Ž .
0.80 and ␾ II s 0.20 at t s 10 min; ␾ I s 0.70 and ␾ II s 0.30 at t s 20 min.
The flow rate was 0.0133 cm sy1. Approximate retention times were: L-alanine,
3
.

780
Y. B. Tewari et al.

Thermodynamics of transamination reactions
781
2.4 min; L-glutamate, 3.6 min; pyruvate, 5.1 min; pyridoxal 5-phosphate, 8.6 min;
and 2-oxoglutarate, 9.5 min.
ŽL-tyrosine q 2-oxoglutarate q 4-hydroxyphenylpyruvate q L-
The mixture
.
glutamate was separated by using a Hewlett-Packard 1100 h.p.l.c. equipped with a
u.v. detector set at ␭ s 210 nm and a Hewlett-Packard Hypersil C18 column
Ž .
Ž
thermostatted at T s 313 K. The mobile phase consisted of I KH 2 PO4 c s
y3
.
.
Ž .
at pH s 3.3 and II acetonitrile. The following gradient of these
0.01 mol dm
Ž .
Ž .
two mobile phases was formed: ␾ I s 1.00 at t s 0; ␾ I s 1.00 at t s 7 min;
3
y1
.
Ž .
Ž .
␾ I s 0.50 and ␾ II s 0.50 at t s 20 min. The flow rate was 0.0117 cm s
.
Approximate retention times were: L-glutamate, 3.0 min; 2-oxoglutarate, 3.6 min;
L-tyrosine, 6.7 min; pyridoxal 5-phosphate, 9.3 min; and 4-hydroxyphenylpyruvate,
13.4 min. The same chromatographic procedure was used to separate the mixture
ŽL-glutamate q 2-oxoglutarate q pyridoxal
5-phosphate q L-phenylalanine q
.
phenylpyruvate . The respective retention times for these substances were 3.0 min,
3.6 min, 9.3 min, 13.6 min, and 15.9 min.
The aim in the equilibrium measurements was to measure the molalities of the
substrates in solution with equilibrium being approached from both directions of
Ž .
reaction. Thus, for reaction 1 , the solution used for the forward direction of
reaction contained L-alanine and 2-oxoglutarate in a phosphate buffer containing
pyridoxal 5-phosphate; the solution used for the reverse direction of reaction
Ž
contained pyruvate and L-glutamate in the same phosphate buffer q pyridoxal
.
5-phosphate solution. The enzyme alanine aminotransferase was added to the two
Ž .
Ž .
solutions. For the experiments involving reactions 2 and 3 , L-glutamate was also
added to the solutions used for the measurements from the forward direction of
reaction. The reason for doing this was the fact that L-glutamate has a smaller
molar absorption coefficient than the other substrates. Thus, by increasing the
amount of L-glutamate in these solutions, the fractional uncertainty in its measured
molality is lowered. The solutions used in the equilibrium measurements were
3
Ž
contained in 20 cm Teflon capped glass bottles and gently shaken back and forth
.
motion at f50 r.p.m. in a water-bath thermostatted at T s 298.15 K. Following
equilibration for f24 h, the molalities of the substrates in these solutions were
measured by using the chromatographic procedures described above. Response
factors were determined by using solutions which were prepared so that the
molalities of the substrates in these solutions would be close to the molalities of
the substrates in the actual reaction mixtures.
Three heat-conduction microcalorimeters were used for the enthalpy of reaction
measurements. They were calibrated electrically with a high stability d.c. power
supply, calibrated digital voltmeter, standard resistor, and time-interval counter.
Descriptions of the microcalorimeters and their performance characteristics, the
data acquisition system, and the computer programs used to treat the results are
Ž
.
Ž .
given in references 19 and 20 . The sample vessels, fabricated from high-density
polyethylene, contained two compartments that held f0.55 cm3 and f0.40 cm3 of
3
Ž
.
solution. The substrate solution placed in the 0.55 cm compartment contained
2-oxoglutarate and either L-alanine, or L-tyrosine, or L-phenylalanine dissolved in a
phosphate buffer containing pyridoxal 5-phosphate. The enzyme solution consisted

782
Y. B. Tewari et al.
of the appropriate enzyme dissolved in the same buffer containing pyridoxal
5-phosphate that was used for the substrate solution. The enzyme solution was
3
Ž . Ž .
placed in the 0.40 cm compartment. The limiting reagents in reactions 1 , 2 , and
Ž .
3
were, respectively, 2-oxoglutarate, L-tyrosine, and L-phenylalanine. It was
Ž .
Ä
4
lower than the
necessary to keep the molality of the L-tyrosine reaction
2
Ž .
Ž .
molalities of either L-alanine or L-phenylalanine used in reactions 1 and 3 ,
respectively. The reason for this was that use of higher molalities of
2-oxoglutarate led to formation of an unidentified precipitate.
ŽL-tyrosine q
.
The vessels and their contents were allowed to equilibrate in the
microcalorimeters for f60 min before the enzyme and substrate solutions were
mixed. Following reaction in the microcalorimeter for f30 min, the reaction
vessels were removed from the microcalorimeters and their contents were promptly
analysed with the h.p.l.c. In this way the amount of substance that had undergone
reaction was obtained. The ‘‘blank’’ enthalpy changes for the mixing of the
substrate solutions with the buffer ranged from y0.04 mJ to 0.68 mJ; for the mixing
of the enzyme solutions with the buffer these enthalpies ranged from y0.55 mJ to
.
Ž
.
0.60 mJ. The pooled uncertainty 2 s in these ‘‘blank’’ enthalpies was 0.58 mJ.
These ‘‘blank’’ enthalpies of mixing were applied as corrections to the measured
Ž
.
calorimetric enthalpies ⌬r H cal which were f20 mJ, f5 mJ, and f33 mJ for
Ž . Ž .
Ž .
reactions 1 , 2 , and 3 , respectively.
Measurement of pH was done with an Orion Model 811 pH meter and a
Radiometer combination glass microelectrode. The pH meter was calibrated with
Ž
Radiometer standard buffers and with a standard phosphate buffer pH s 7.42 at
.
T s 298.15 K . This latter buffer was prepared from KH 2 PO4 and Na2 HPO4,
standard reference materials 186-Id and 186-IId, respectively, from the National
Institute of Standards and Technology.
3. Results and discussion
The apparent equilibrium constants corresponding to the overall biochemical
Ž . Ž .
Ž .
reactions 1 , 2 , and 3 are, respectively,
.
KЈ s m pyruvate m L-glutamate r
Ž
.
Ž
.
.
Ä
m L-alanine m 2-oxoglutarate
4
,
4
Ž .
Ž
.
Ž
.
.
KЈ s m 4-hydroxyphenylpyruvate m L-glutamate r
Ž
.
Ž
.
.
Ä
m L-tyrosine m 2-oxoglutarate
4
,
5
Ž .
Ž
.
Ž
.
.
KЈ s m phenylpyruvate m L-glutamate r
Ž
.
Ž
.
.
Ä
m L-phenylalanine m 2-oxoglutarate
4
.
6
Ž .
Ž
.
Ž
.
Ž . Ž .
Ž .
The molalities m in equations 4 , 5 , and 6 are the total molalities of the various
charged and uncharged species that are formed from the dissociation of the various
substances in solution. It will be seen later that there is a predominant species
present in each case.

Thermodynamics of transamination reactions
783
␾
␾
␾
␾
␾
␾

784
Y. B. Tewari et al.

Thermodynamics of transamination reactions
785
␾
␾

786
Y. B. Tewari et al.
The results of the equilibrium and calorimetric measurements are given in tables
2 and 3, respectively. In these tables, the uncertainties in the values of the apparent
equilibrium constants KЈ and calorimetrically determined molar enthalpies of
Ž
.
reaction ⌬r Hm cal indicate only the random errors inherent in the measurements
and do not reflect the possible systematic errors which we now consider. We judge
that a reasonable estimate of error due to impurities in the biochemicals used in
.
.
Ž .
Ž .
Ž .
this study is "0.02 KЈ for reactions 1 and 2 and "0.03 KЈ for reaction 3 . We
.
.
.
Ž .
also allow for errors of "0.003 KЈ, "0.01 KЈ, and "0.006 KЈ for reactions 1 ,
Ž .
Ž .
2 , and 3 , respectively, due to uncertainties in the values of the mass fraction
moisture contents of the reactants. These estimates of possible systematic error are
combined in quadrature together with the statistical uncertainties in the measured
values of KЈ, expressed as one estimated standard deviation of the mean s, to
obtain combined standard uncertainties.Ž21. These combined standard uncertainties
are then multiplied by two to arrive at the final set of results for the apparent
Ž
.
Ž
.
equilibrium constants: KЈ s 0.72 " 0.07 and 0.78 " 0.06 , respectively, for the
Ž .
Ž
.
Ž
.
forward and reverse directions of reaction 1 ; KЈ s 0.88 " 0.06 and 0.88 " 0.05 ,
Ž .
respectively, for the forward and reverse directions of reaction 2 ; and KЈ s
Ž
.
Ž .
Ž
.
1.02 " 0.06 and 1.07 " 0.06 , respectively, for the forward and reverse directions
of reaction 3 .
Similarly, we allow for the following possible systematic errors in the values of
.
Ž
.
Ž
.
⌬r Hm cal : "0.01 ⌬r Hm cal due to impurities in the biochemicals;
.
Ž
.
Ž .
"0.007 ⌬r Hm cal for reaction 2 due to the uncertainty in the value of the mass
fraction moisture content of the L-tyrosine; and "0.015 ⌬r Hm cal ,
"0.058 ⌬r Hm cal , and "0.009 ⌬r Hm cal for reactions 1 , 2 , and 3 ,
respectively, due to uncertainties in the ‘‘blank’’ enthalpies of mixing. These
estimates of possible systematic error are combined with the statistical uncertainties
.
Ž
.
Ž .
.
.
Ž
.
Ž
.
Ž . Ž .
Ž
.
in the measured values of ⌬r Hm cal in the same way as was done for the values of
Ž
.
the apparent equilibrium constants. Thus, the final results for ⌬r Hm cal are:
⌬r Hm cal s 5.2 " 0.4 kJ mol
1.4 kJ mol for reaction 2 ; and ⌬r Hm cal s 8.3 " 0.4 kJ mol for reaction
3 . It is seen that the uncertainty in the value of ⌬r Hm cal for reaction 2 has
increased substantially due to the uncertainty in the ‘‘blank’’ enthalpy of mixing
and the fact that the measured enthalpy of this reaction was only a few mJ.
In the discussion of the thermodynamics of these reactions, it is useful to
introduce the following reference reactions that correspond to the above overall
biochemical reactions:
y1
.
Ž
.
.
Ž
.
Ž .
Ž
.
Ž
for reaction 1 ; ⌬r Hm cal s 8.4 "
y1
y1
.
.
Ž .
Ž
.
Ž
.
Ž .
Ž
.
Ž .
L-alanine aq q 2-oxoglutarate2y aq s
Ž
.
Ž
.
pyruvatey aq q L-glutamatey aq ,
7
Ž
.
Ž
.
Ž .
L-tyrosine aq q 2-oxoglutarate2y aq s
Ž
.
Ž
.
4-hydroxyphenylpyruvatey aq q L-glutamatey aq ,
8
Ž .
Ž
.
Ž
.
L-phenylalanine aq q 2-oxoglutarate2y aq s
Ž
.
Ž
.
phenylpyruvatey aq q L-glutamatey aq .
9
Ž .
Ž
.
Ž
.

Thermodynamics of transamination reactions
787
The corresponding equilibrium constants are
y
K s m pyruvatey m L-glutamate
r
.
Ž
.
Ž
.
2y
.
m L-alanine m 2-oxoglutarate
,
10
Ä
Ž
.
4
.
Ž
Ž
Ž
.
Ž
K s m 4-hydroxyphenylpyruvatey m L-glutamate
r
y
.
Ž
.
Ž
.
2y
.
m L-tyrosine m 2-oxoglutarate
,
11
Ä
Ž
.
4
.
.
Ž
K s m phenylpyruvatey m L-glutamate
r
2y
y
.
Ž
.
Ž
.
.
m L-phenylalanine m 2-oxoglutarate
.
12
Ä
Ž
.
4
.
.
Ž
The standard state used in this study is the hypothetical ideal solution of unit
y1
.
Ž
.
molality mЊ s 1 mol kg . Since all of the reactions considered in this study are
symmetric, the values of the equilibrium constants would be the same if either a
concentration or mole fraction standard state had been used.
The p K values and standard molar enthalpies for the proton dissociation
reactions of the reactants and of the other solutes in solution are needed to relate
Ž . Ž .
Ž .
the experimental results for reactions 1 , 2 , and 3 to thermodynamic quantities
Ž . Ž .
Ž .
for the respective reference reactions 7 , 8 , and 9 . These p K values and
standard molar enthalpies are summarized in table 4. In the absence of values from
Ž
.
the literature, we estimated the p K of 4-hydroxyphenylpyruvic acid aq from the
Ž
.
Ž
.
known p K of phenylpyruvic acid aq and L-tyrosine aq . Based upon these p K
values and the structural information given in reference 22 , the predominant
species mole fraction x )0.96 at the pH used in this study are those shown in the
reference reactions 7 , 8 , and 9 . Thus, on the basis of stoichiometry, the
Ž
.
Ž
.
Ž . Ž .
Ž .
q
Ž
.
Ž . Ž .
Ž .
changes in binding of the hydrogen ion ⌬r N H for reactions 1 , 2 , and 3 are
predicted to be close to zero at the pHs used in this study. Thermodynamic
quantities for the dissociation of the phosphate buffer are also needed for the
y8
.
equilibrium calculations to follow. We used Km s 6.23 10
and ⌬r HTm s
yŽ
y1
.
.
4.2 kJ mol at T s 298.15 K and Im s 0 for the dissociation of H 2 PO4 aq :
H 2 POy4 aq s HPO 2y aq q Hq aq .
13
Ž
.
Ž
.
Ž
.
Ž
.
4
These values were calculated from the standard formation properties given in
Wagman et al.Ž23.
We used a previously describedŽ24. equilibrium model to calculate the values of
the ionic strength Im and equilibrium constants K given, respectively, in columns
10 and 12 in table 2. The uncertainties in the calculated equilibrium constants have
two components: errors in the measured values of the apparent equilibrium
constants and possible errors in the quantities used in the equilibrium model. Since
<
<
the difference p K y pH was G1.45 for all reactions studied herein, perturbation
.
Ž
.
of the p K values had only a small effect -"0.011 K on the calculated values of
the equilibrium constants. However, it was found that the uncertainty in the
activity coefficient was the major factor in the component of uncertainty attributable
Ž . Ž .
Ž .
to the equilibrium model. Since reactions 7 , 8 , and 9 are charge asymmetric,

788
Y. B. Tewari et al.
TABLE 4. Thermodynamic quantities for the aqueous proton dissociation reactions of substances
pertinent to this study at T s 298.15 K and Im s 0. These quantities were, with the exception of the
Ž
.
estimated p K values for 4-hydroxyphenylpyruvic acid, taken from reference 22 . When necessary, the
y1r2
p K values were adjusted to Im s 0 by using an ‘‘ion-size’’ parameter of 1.6 kg1r2 mol
in the
.
extended Debye-Huckel equation to estimate the activity coefficients of the aqueous species in solution
¨
⌬f HTm
Reaction
p K
2.34
y1
.
kJ mol
L-alanineqs L-alanine q Hq
L-alanine s L-alanineyq Hq
2.9
10.08
45.2
L-glutamic acidqs L-glutamic acid q Hq
L-glutamic acid s L-glutamateyq Hq
L-glutamateys L-glutamate2yq H q
2.16
4.30
9.96
3.0
1.4
39.8
4-hydroxyphenylpyruvic acid s 4-hydroxyphenylpyruvateyq Hq
f2.5a
4-hydroxyphenylpyruvateys 4-hydroxyphenylpyruvate2yq Hq
f10.5a
2-oxoglutaric acid s 2-oxoglutarateyq Hq
2-oxoglutarateys 2-oxoglutarate2yq Hq
2.24
5.12
L-phenylalanineqs L-phenylalanine q Hq
2.20
9.31
2.0
44.1
L-phenylalanine s L-phenylalanineyq Hq
phenylpyruvic acid s phenylpyruvateyq Hq
2.54
pyridoxal 5-phosphate s pyridoxal 5-phosphateyq Hq
pyridoxal 5-phosphateys pyridoxal 5-phosphate2yq Hq
pyridoxal 5-phosphate2ys pyridoxal 5-phosphate3yq Hq
pyruvic acid s pyruvateyq Hq
4.05
6.46
8.89
16.4
y5.8
23.1
2.49
12.1
L-tyrosineqs L-tyrosine q Hq
L-tyrosine s L-tyrosineyq Hq
L-tyrosine-s L-tyrosine2yq Hq
2.81
9.19
10.47
1.0
39.4
24.8
a Estimated from the p K values of phenylpyruvic acid and L-tyrosine.
this is not unexpected. The effects of these possible errors on the calculated
thermodynamic quantities were determined by perturbing first the measured
Ž
apparent equilibrium constants by the final experimental uncertainties see above
.
discussion and then the ‘‘ion-size’’ parameter used in the activity coefficient model
by " 0.3 kg1r2 moly1r2. The effects due to these perturbations were then combined
.
in quadrature to obtain the final estimated uncertainties assigned to the equilibrium
Ž . Ž .
Ž .
constants for the reference reactions 7 , 8 , and 9 . It is seen that the results
obtained from the forward and reverse directions of reaction are in agreement for
all three reactions. This is consistent with the attainment of equilibrium in the
experiments. We adopt the following weighted averages for the equilibrium
Ž
.
Ž .
Ž
.
Ž .
constants: K s 1.36 " 0.09 for reaction 7 ; K s 1.82 " 0.12 for reaction 8 ;
Ž
.
Ž .
and K s 2.14 " 0.13 for reaction 9 .
The following values of ⌬r HTm were also calculated with the equilibrium model:Ž24.
T
T
y1
y1
.
T
.
Ž
Ž .
.
Ž .
Ž
.
⌬r Hm s 5.9 " 0.3 kJ mol
for reaction 7 ; ⌬r Hm s 10.1 " 1.1 kJ mol
for
y1
.
Ž
.
Ž .
reaction 8 ; and ⌬r Hm s 9.5 " 0.3 kJ mol for reaction 9 . Calculated values
q
Ž
.
of the changes in binding of the hydrogen ion ⌬r N H were y0.0046, 0.0250, and
Ž . Ž .
Ž .
0.0074 for the biochemical reactions 1 , 2 , and 3 , respectively. Thus, the buffer

Thermodynamics of transamination reactions
789
protonation corrections used to obtain values of ⌬r HTm from the measured values
y1
.
Ž
.
Ž
.
of ⌬r Hm cal were relatively small F0.11 kJ mol . The uncertainties in these
calculated enthalpies were obtained in the same way as was done for the equilibrium
constants. Thus, they reflect the uncertainties both in the experimentally
Ž
.
determined values of ⌬r Hm cal and in the equilibrium model. In this case, it was
y1
.
found that perturbation of the value of B by "0.3 kg mol resulted in changes in
the calculated values of ⌬r HTm of only f"0.07 kJ moly1. The most sensitive
.
Ž
.
quantity was the p K of L-tyrosine aq . In this case, a change of "0.1 in the value
of this p K resulted in a change of "0.33 kJ mol in the calculated value of ⌬r HTm
y1
.
T
Ž .
for reaction 8 . Nevertheless, it is seen that the uncertainties in the ⌬r Hm values
Ž . Ž .
for the reference reactions 7 , 8 , and
uncertainties in the values of ⌬r Hm cal . The results obtained for the reference
reactions are summarized in table 5 where values of the standard molar Gibbs
energy ⌬r GTm and the standard molar entropy ⌬r STm are also given.
Ž .
9 are essentially the same as the
Ž
.
Ž .
Results from the literature for the biochemical reaction 1 are summarized in
table 6. In each case, we used the information given by the researchers on the
compositions of the solutions used in their study to calculate the ionic strengths
given in table 6. A comparison of these results, which were obtained under a
variety of conditions, requires that they be adjusted to a common basis. Therefore,
for each study, we calculated values of the equilibrium constant for the reference
Ž .
reaction 7 at T s 298.15 K and I s 0 from the reported apparent equilibrium
constants with the same equilibrium model that was used in the treatment of our
T
y1
.
own results. The value of ⌬r Hm s 5.9 kJ mol that was obtained in this study for
Ž .
7 was used in the equilibrium model for the treatment of results
reaction
obtained at temperatures other than T s 298.15 K. In these calculations,
Ž
.
thermodynamic quantities p Ks and standard molar enthalpies of ionization for
qŽ 0Ž
.
the ionizations of tris hydroxymethyl aminomethane aq and glycylglycine aq
Ž
.
.
Ž
.
were taken from reference 22 . The results obtained from the early studies of
Cohen,Ž1. Lenard and Straub,Ž2. Darling,Ž3. and Green et al.Ž4. are considered to be
´ ´
approximate. The result of Bulos and HandlerŽ7. was obtained from a kinetic study
and is far outside the range of all the other reported values.
.
Ž
.
The most precise 2s s 0.024 KЈ of the studies from the literature is that of
KrebsŽ5. who used manometry to measure the concentrations of glutamate aq and
Ž
.
Ž
.
2-oxoglutarate aq . However, there is an inconsistency between the concentrations
of the reactants given in Krebs’ tables 2 and 3 and the information given in the text
Ž
of his paper. If we assume that the information given in the text of his paper see
.
.
section on ‘‘Reaction mixture and sampling’’ is correct, we calculate values of the
concentration ionic strength Ic of 0.90 mol kg and 0.95 mol kgy1. However, if
y1
.
Ž
.
Ž
.
we assume that the concentrations in his tables 2 and 3 have units of ‘‘␮M not ␮l
3
y3
.
.
.
Ž
.
substraterml. solution’’ i.e. 1 10 mol dm
we arrive at much higher values of
y3
.
Ic , namely 1.67 mol dm
to 1.82 mol dmy3. In any case, Krebs’Ž5. results were
obtained at much higher ionic strengths than the other results and, because of this,
the adjustment of these results to I s 0 is more uncertain than in the other
Ž
.
studies. Nevertheless, the result obtained in this study, K s 1.36 " 0.09 for the
Ž .
reference reaction 7 at T s 298.15 K and I s 0, is very close to the value of 1.31

790
Y. B. Tewari et al.

Thermodynamics of transamination reactions
791
obtained as the average of the earlier results sans that of Bulos and Handler.Ž7.
Ž . Ž .
The apparent equilibrium constants reported herein for reactions
2 and 3
appear to be the first such results reported for these reactions. As mentioned
above, there have been no previous reports of standard molar enthalpies for any of
these reactions.
Ž .
The discussion of how our results for reaction 7 fit into thermochemical cycles
is done in reference to Miller and Smith-Magowan’sŽ25. extensive thermochemical
network analysis of the reactions in the Krebs cycle. In this discussion all values of
⌬r GTm pertain to the standard state used by Miller and Smith-Magowan:Ž25. T s
T
298.15 K and Im s 0.1 mol kgy1. TheyŽ25. used a value of ⌬r Gm s 1.04 "
.
Ž
T
y1
y1
.
.
.
Ž .
Ž
.
0.13 kJ mol for reaction 7 and values of ⌬r Gm s 75.15 " 0.80 kJ mol and
74.80 " 0.80 kJ moly1, respectively, for the following two reactions:
.
Ž
.
L-alanine aq q NADy aq q H O l s
Ž
.
Ž
.
Ž .
2
pyruvatey aq q NHq aq q NADH 2y aq q Hq aq , 14
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
.
4
L-glutamatey aq q NADy aq q H O l s
Ž
.
Ž
.
Ž .
2
2-oxoglutarate2y aq q NHq aq q NADH 2y aq q Hq aq . 15
Ž
.
Ž
.
Ž
.
Ž
.
Ž
4
These values were calculated from the results reported by Krebs,Ž5. Engel and
Ž .
Dalziel,Ž26. and Grimshaw and ClelandŽ27. for reactions 7 , 14 , and 15 ,
Ž
.
Ž .
Ž
.
Ž .
respectively. In reactions 14 and 15 , we used the actual charge numbers for the
oxidized and reduced forms of ␤-nicotinamide-adenine dinucleotide rather than
the standard biochemical abbreviations NADq and NADH, respectively.
T
Combination of the ⌬r GTm values for reactions 14 and 15 leads to ⌬r Gm s
Ž
.
Ž .
T
y1
y1
.
.
Ž
.
Ž .
Ž
.
0.35 " 1.1 kJ mol
Ž .
for reaction 7 . A value of ⌬r Gm s 1.04 " 1.2 kJ mol
Ž25.
for reaction 7 is obtained from Miller and Smith-Magowan’s
solution of the
Ž
thermochemical network see the ‘‘succinate q acetate’’ network solution in
T
their Ž25. tables 9 and 10 , a result that is very close to the value ⌬r Gm s 1.05 "
.
Ž
0.13 kJ mol theyŽ25. obtained from Krebs’Ž5. reported value of KЈ for reaction
y1
.
.
Ž .
1 , and which they used as input data to their network solution. We calculate
T
y1
y1
.
.
Ž
.
⌬r Gm s 0.46 " 0.17 kJ mol at T s 298.15 K and Im s 0.1 mol kg from our
own result for ⌬r GTm for reaction 7 . Thus, it is seen that the result of our study
Ž .
falls within the rather broad uncertainty interval obtained from the thermochemical
y1
network solution of Miller and Smith-Magowan;Ž25. it differs by only 0.09 kJ mol
.
from the value obtained with Miller and Smith-Magowan’s selected values for
Ž
.
Ž .
reactions 14 and 15 . Interestingly, while the thermodynamic pathways involving
Ž .
reaction 7 are relatively extensive, there do not appear to be any thermodynamic
pathways that lead to values of K for reactions 8 and 9 , or values of ⌬r Hm for
Ž . Ž .
reactions 7 , 8 , and 9 .
T
Ž .
Ž .
Ž .
It is also desirable to have values of apparent equilibrium constants KЈ for these
reactions under approximately physiological conditions. Here, physiological
conditions are taken to be:Ž28. T s 311.15 K, pH s 7.0, pMg s 3.0, and Im s
0.25 mol kgy1. With the thermodynamic quantities obtained in this study for the
.

792
Y. B. Tewari et al.
reference reactions and with the equilibrium model, the following values of the
Ž . Ž .
Ž .
apparent equilibrium constant KЈ for reactions 1 , 2 , and 3 , respectively, are
calculated for these conditions: 0.77, 1.09, and 1.28. In performing this calculation,
Ž .
we assumed, 1 that the neutral species and those having charge numbers of y1
2qŽ 2qŽ
Ž .
.
.
do not bind to Mg aq , and 2 that the binding constant for Mg aq with
2yŽ
.
.
2qŽ
.
Ž22.
2-oxoglutarate aq is equal to the known
binding constant for Ca aq with
2yŽ
Ž
.
y3
.
2-oxoglutarate aq , namely K T s 298.15, and Ic s 0.1 mol dm
s 19.5. Thus,
2y
Ž
.
only a small fraction f0.02 of the total amount of 2-oxoglutarate
will be
complexed with Mg2q. The calculated value KЈ s 0.77 for biochemical reaction 1
under physiological conditions is not too different from the value KЈ s 0.68
obtained by Brosnan et al.Ž9. under these actual conditions. The standard molar
Ž .
transformed Gibbs energy changes ⌬r GmXT for biochemical reactions 1 , 2 , and 3
Ž . Ž .
Ž .
.
y1
.
Ž
.
under physiological condition are, respectively: 0.68, y0.22, and y0.64 kJ mol
Inspection of figure 1 shows that the nearest neighbor interactions are unchanged
in these three reactions. Therefore, any estimation method that considers only
nearest neighbor interactions must necessarily lead to the prediction that the
thermodynamic reaction quantities ⌬r GTm , ⌬r HTm , ⌬r STm , and ⌬r CTp, m are zero for
these transamination reactions. This prediction is consistent with the results
obtained in the present study, i.e. the thermodynamic reaction quantities are all
small. Additionally, examination of the valuesŽ10. of the apparent equilibrium
constants for other transamination reactions shows that, with only two exceptions,
the apparent equilibrium constant falls within a very limited range: 0.016 F KЈ F
100. These two exceptions are for the following biochemical reactions catalysed by
glutamine-pyruvate transaminase
L-glutamine aq q glyoxylate aq s2-oxoglutaramate aq q glycine aq , 16
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
.
L-glutamine aq q pyruvate aq s2-oxoglutaramate aq q L-alanine aq .
Ž
.
Ž
.
Ž
.
Ž
.
17
Ž
Ž
.
Ž
Respective values of KЈ T s 310.15 K, pH s 7.4 s 607 and KЈ T s 310.15 K,
Ž29.
.
pH s 8.4 s 340 were reported by Cooper and Meister
A likely explanation for these relatively high values is that the 2-oxoglutaramate
for these two reactions.
undergoes a cyclization reaction that is greater than 99 per cent complete under
the reaction conditions used by Cooper and Meister.Ž29. Thus, by Le Chatelier’s
ˆ
Ž
.
Ž
.
principle, very little of the reactants on the left hand side of reactions 16 and 17
will remain at equilibrium. Since the concentrations of both forms of 2-
Ž
.
oxoglutaramate cyclized and uncyclized were used in the calculation of the
apparent equilibrium constants reported by Cooper and Meister,Ž29. large values of
KЈ were obtained. However, when corrections are appliedŽ29. for the concentration
of the uncyclized form of 2-oxoglutaramate, values of KЈ s 1.8 and KЈ s 1.0 are
Ž
.
Ž .
obtained for reactions 16 and 17 , respectively. These corrected values are in
accord with the prediction that thermodynamic reaction quantities are small for
transamination reactions.

Thermodynamics of transamination reactions
793
We thank Drs Jack F. Kirsch and Vincent L. Vilker for their encouragement and
support of this research, and Dr Arthur J. L. Cooper for his helpful discussion on
the reactions catalysed by glutamine-pyruvate transaminase. The work of Tinh N.
Luong was supported by National Institutes of Health Grant GM35393 to Jack F.
Kirsch.
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Recei¨ed 15 September 1997; in final form 9 January 1998
WA97r066