A thermodynamic study of the hydrolysis of L-glutamine to (L-glutamate + ammonia) and of L-asparagine to (L-aspartate + ammonia)

Article abstract of DOI:10.1006/jcht.1999.0496

Calorimetric enthalpies of reaction were measured for the two enzyme-catalyzed reactions, i.e., L-glutamine(aq) + H₂O(l) = L-glutamate(aq) + ammonia(aq) (1) and L-asparagine(aq) + H₂O(l) = L-aspartate(aq) + ammonia(aq) (2). The standard molar enthalpies for reference reactions involving specific species were computed using an equilibrium model that considered the multiplicity of ionic forms of the reactants and products. Using the thermodynamic quantities obtained for the reference reactions, the values of the apparent equilibrium constant for reactions 1 and 2 at 311.15 K and pH 7 were 250. In performing these calculations, it was assumed that there was no binding of Mg²⁺(aq) to any of the species involved in the reactions 1 and 2. The standard transformed Gibbs energy changes for these two reactions under physiological conditions were both -14 kJ/mole. The thermodynamic results were discussed with respect to the structural changes involved in these reactions.

Full text of DOI:10.1006/jcht.1999.0496

J. Chem. Thermodynamics 2000, 32, 1077–1090
doi:10.1006/jcht.1999.0496
Available online at http://www.idealibrary.com on
A thermodynamic study of the hydrolysis of
L-glutamine to (L-glutamate + ammonia) and of
L-asparagine to (L-aspartate + ammonia)
Nand Kishore,a Yadu B. Tewari, and Robert N. Goldbergb
Biotechnology Division, National Institute of Standards and Technology,
Gaithersburg, MD 20899, U.S.A.
Calorimetric enthalpies of reaction have been measured for the following enzyme-
catalysed reactions:
L-glutamine(aq) + H O(l) = L-glutamate(aq) + ammonia(aq),
2
L-asparagine(aq) + H O(l) = L-aspartate(aq) + ammonia(aq).
2
An equilibrium model that accounted for the multiplicity of ionic forms of the reactants and
products was used to calculate standard molar enthalpies for reference reactions involving
specific species. Values of equilibrium constants for the reference reactions were obtained
using results from the literature in thermochemical cycle calculations. The thermodynamic
results are discussed in relation to the structural changes involved in these reactions.
c
ꢀ 2000 Academic Press
KEYWORDS: ammonia; asparaginase; L-asparagine; L-aspartate; enthalpy; equilibrium
constants; glutaminase; L-glutamate; L-glutamine
1. Introduction
The aim of this study is to obtain an improved knowledge of the thermodynamic of the
reactions:
L-glutamine(aq) + H2O(l) = L-glutamate(aq) + ammonia(aq),
L-asparagine(aq) + H2O(l) = L-aspartate(aq) + ammonia(aq).
(1)
(2)
In addition to their fundamental role in the catabolism of amino acids,(1) these reactions
also serve as a thermodynamic pathway to standard molar thermodynamic quantities for
the hydrolysis of ATP† to (ADP + orthophosphate).(2–4) Also, since both of the above
a
Guest researcher from the Department of Chemistry, Indian Institute of Technology, Bombay, India.
b
To whom correspondence should be addressed (E-mail: robert.goldberg@nist.gov).
†Abbreviations used in this paper are: ATP, adenosine 5 -triphosphate; ADP, adenosine 5 -diphosphate; Tris,
tris (hydroxymethyl) aminomethane.
0
0
c
0021–9614/00/091077 + 14 $35.00/0
ꢀ 2000 Academic Press

1078
N. Kishore, Y. B. Tewari, and R. N. Goldberg
NH2
NH2
O
C
H2N
CH2 CH2 CH COOH
H2O
HOOC CH2 CH2 CH COOH NH3
L-glutamine
L-glutamic acid
NH2
O
C
NH2
H2N
CH COOH
H2O
HOOC CH2 CH COOH
NH3
CH2
L-asparagine
L-aspartic acid
FIGURE 1. Structures of the substances in reactions (1) and (2). The neutral forms of the substances
are shown.
reactions are structurally similar (see figure 1), having reliable thermodynamic values for
these reactions is important for the development of Benson-type estimation methods for
biochemical reactions in aqueous solutions.(5)
This study is part of a continuing investigation into the thermodynamics of enzyme-
catalysed reactions. Reactions (1) and (2) are catalysed, respectively, by glutami-
nase (EC 3.5.1.2) and asparaginase (EC 3.5.1.1). However, the enzyme glutamin-
(asparagin-)ase (EC 3.5.1.38) is known to catalyse both reactions as well as those of the
D-isomers,(6) and reaction (1) is also catalysed by the PabA subunit of p-aminobenzoate
synthase.(7)
2. Experimental
Relevant information on the substances used in this study is given in table 1.‡ The purities
of L-asparagine, L-aspartic acid, L-glutamic acid, and L-glutamine were checked by
using the chromatographic procedures described below {a Hewlett-Packard (HP) Hypersil
BDS C18 column was used} and found to be consistent with the purities found by
the vendor who used t.l.c. A Metrohm model 633 automatic Karl Fischer titrator, and
a model 665 dosimat were used for the determinations of the volume of water in the
various samples. The solvent used for these determinations was a mixture of methanol and
“Hydranal Composite 2” solution. “Hydranal Composite 2” was also used for the titration,
the end-point of which was determined coulometrically with an appropriate correction for
drift. The Karl Fischer apparatus was enclosed in a plexiglass box which was maintained
under a continual positive pressure of dry nitrogen gas. Calibration was achieved using a
solution consisting of 1-octanol saturated with water. The reported mass fraction moisture
contents (see table 1) are based on the solubility of water in 1-octanol at T = 298.15 K
‡Certain commercial equipment, instruments, or materials are identified in this paper to specify the experimen-
tal procedures adequately. Such identification is not intended to imply recommendation, or endorsement by the
National Institute of Standards and Technology (NIST), nor is it intended to imply that the materials, or equipment
identified are necessarily the best available for the purpose.

Hydrolysis of L-glutamine and L-asparagine
1079
determined by Leo and Hansch.(8) These mass fraction moisture contents w are judged to
be reliable to within approximately ±0.03 · w. The values of w were applied as corrections
in all subsequent calculations.
An HP 1050 h.p.l.c. and an HP model 1100 h.p.l.c. were used to determine the
residual amounts of L-glutamine and L-asparagine in solution following the reaction in the
microcalorimeters (see below). The column, mobile phase, and method of detection were
the same for both instruments. The column was an HP Hypersil BDS C18 column (4 mm
i.d., 250 mm long). The mobile phase was {tetrabutyl ammonium hydroxide (concentration
c = 0.01 mol · dm−3) + ammonium phosphate (c = 0.025 mol · dm−3)}; it ran at a
constant flow rate of 0.00667 cm3 · s−1. The u.v. detectors were set at the wavelength
λ = 215 nm. With this procedure, the retention times of L-glutamine and L-glutamate were
5.4 min, and 10.3 min, respectively; the retention time of L-asparagine and L-aspartate
were 5.7 min and 11.0 min, respectively. 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 have been given by Steckler et
al.(9, 10) The data acquisition system has recently undergone significant modernization.
The voltages of the thermopiles of the microcalorimeters are now measured with HP
model 34420A nanovolt meters. These voltages are then recorded on a microcomputer
with a data acquisition program written in HP-VEE. The integration of the areas of the
thermograms is performed by using a code written in C++.
The calorimetric sample vessels were fabricated from high density polyethylene. Each
vessel had two compartments that held a volume V ≈ (0.55 and 0.40) cm3 of solution,
respectively. The substrate solutions were placed in the 0.55 cm3 compartment and the
enzyme solutions were placed in the 0.40 cm3 compartment. The substrate solutions
for reactions (1) and (2) initially contained, respectively, L-glutamine and L-asparagine
dissolved in a phosphate buffer. The enzyme solutions were prepared by the addition
of either glutaminase or asparaginase to the respective stock buffer solutions that were
used for the preparation of the substrate solutions. The purpose of this procedure was to
minimize the “blank” enthalpies (see below).
The vessels and their contents were allowed to equilibrate in the microcalorimeters
for ≈60 min before the enzyme and substrate solutions were mixed. After this mixing,
approximately 75 min were allowed to elapse for reaction (1) and 42 min for reaction (2).
Following the reaction, the vessels were removed from the microcalorimeters, and h.p.l.c.
was promptly used to determine the amounts of L-glutamine and L-asparagine remaining
in solution. Thus, the mole fraction of unreacted L-glutamine {reaction (1)} ranged
from 0.0037 to 0.0101; the mole fraction of unreacted L-asparagine {reaction (2)} was
0.0063. The “blank” enthalpy changes for mixing of the L-glutamine and L-asparagine
solutions (the substrate solutions) with the buffer were, respectively, (0.02 ± 0.69) mJ
and (0.60 ± 0.96) mJ. On mixing the glutaminase and asparaginase solutions (the enzyme

1080
N. Kishore, Y. B. Tewari, and R. N. Goldberg

Hydrolysis of L-glutamine and L-asparagine
1081
solutions) with their respective buffers, these “blank” enthalpy changes were, respectively,
(0.32±1.4) mJ and (0.79±1.1) mJ. These uncertainties are equal to two estimated standard
deviations of the mean. These “blank” enthalpies of mixing were applied as corrections
to the measured calorimetric enthalpies which were ≈ − 239 mJ for reaction (1) and
≈ − 247 mJ for reaction (2).
Measurement of pH was performed by using an Orion Model 811 pH meter and
a Radiometer combination glass micro-electrode. The pH meter was calibrated with
Radiometer standard buffers and with a standard phosphate buffer (pH = 7.42 at T =
298.15 K). This latter buffer was prepared from KH2PO4 and Na2HPO4, standard reference
materials 186-Id and 186-IId, respectively, from NIST.
3. Results and discussion
The apparent equilibrium constants(11) for reactions (1) and (2), respectively, are:
Km0 = m(L-glutamate) · m(ammonia)/{m(L-glutamine) · mo},
(3)
(4)
Km0 = m(L-aspartate) · m(ammonia)/{m(L-asparagine) · mo}.
The molalities m in the above equations are the total molalities of the various charged
and uncharged species that are formed from the dissociation of the various substances in
solution. For example, ammonia refers to a mixture of (NH+ + NH3) and L-glutamate
4
refers to a mixture of (L-glutamic acid0 + L-glutamate− + L-glutamate2−). In the
discussion to follow, we shall also find it useful to refer to the following reference reactions
that pertain to specific species:
L-glutamine±(aq) + H2O(l) = L-glutamate−(aq) + NH+4 (aq),
(5)
(6)
L-asparagine±(aq) + H2O(l) = L-aspartate−(aq) + NH+4 (aq).
The equilibrium constants for reactions (5) and (6), respectively, are:
Km = m(L-glutamate−) · m(NH4+)/{m(L-glutamine±) · mo},
(7)
(8)
Km = m(L-aspartate−) · m(NH4+)/{m(L-asparagine±) · mo}.
The standard state used in this study is the hypothetical ideal solution of unit molality
(mo = 1 mol · kg−1). The subscript m has been appended to both K and K0 to indicate
a molality basis.(12) Also, mo has been used in equations (3), (4), (7), and (8) to keep
the equilibrium constants dimensionless. While the choice of the reference reactions is
arbitrary, we have selected those species that are predominant for the pH values used in
this study.
The results of the calorimetric measurements are given in table 2. The uncertainties in
the measured values of h1r Hm(cal)i that are given in this table represent only the ran-
dom errors inherent in the measurements and do not reflect possible systematic errors
which are now considered. We judge that reasonable estimates of the standard uncertain-
ties(13) due to possible systematic errors in the values of 1r Hm(cal) are: 0.003 · 1r Hm(cal)
and 0.007 · 1r Hm(cal) due to impurities in the respective samples of L-glutamine and L-
arginine; 0.005 · 1r Hm(cal) due to uncertainties in the calorimetric measurements (this

1082
N. Kishore, Y. B. Tewari, and R. N. Goldberg
includes uncertainties in the “blank” enthalpies); and 0.001 · 1r Hm(cal) due to possible un-
certainties in the determination of the amounts of unreacted L-glutamine and L-asparagine.
These estimates of possible systematic error are combined in quadrature together with
the statistical uncertainties in the measured values of 1r Hm(cal) expressed as one es-
timated standard deviation of the mean, to obtain combined standard uncertainties.(13)
These combined standard uncertainties are then multiplied by two to arrive at the final
set of results: 1r Hm(cal) (T = 298.15 K, pH = 5.14, and Im = 0.11 mol · kg−1) =
−(24.39 ± 0.31) kJ · mol−1 for reaction (1), and 1r Hm(cal) (T = 298.15 K, pH = 7.03,
and Im = 0.27 mol · kg−1) = −(25.63 ± 0.45) kJ · mol−1 for reaction (2).
The pK values and standard molar enthalpies for the proton dissociation reactions
of the reactants and the other solutes are needed to relate the experimental results for
reactions (1) and (2) to thermodynamic quantities for the respective reference reactions (5)
and (6). These pK values and standard molar enthalpies 1r Hmo are given in table 3. The
pK and 1r Hmo values for the ionization of L-asparagine, L-aspartic acid, L-glutamine,
and L-glutamic acid are from Martell and Smith.(14) Where necessary, the pK and 1r Hmo
values were adjusted to Im = 0 by using an “ion-size” parameter of 1.6 kg1/2 · mol−1/2 in
the extended Debye–Hu¨ckel equation to estimate the activity coefficients of the aqueous
species in solution. The thermodynamic quantities for the ionizations of NH+4 and H2PO4−
are calculated, respectively, from the NBS tables of chemical thermodynamic properties(15)
and from the CODATA tables.(16) The pK values of all of the reactants, with the exception
of the first ionization of L-glutamic acid, are well removed (i.e. |pK − pH| > 1.9) from
the pH values at which the measurements were performed. Fortunately, the ionization of L-
glutamic acid (pK = 4.30) has been well studied (Martell and Smith(14) give 77 references
to pertinent studies) and we shall rely upon their(14) selected values for pK and 1r Hmo for
this ionization reaction.
The equilibrium model used for the calculation of the equilibrium constants Km and
standard molar enthalpies 1r Hmo for the reference reactions from the measured values of
Km0 and 1r Hm(cal) has been described previously.(17) This model has been modified so that
it utilizes the Mathematica(18) computer code of Alberty and Krambeck(19) to solve the
simultaneous non-linear equations that describe the various equilibria. The calculations
also include corrections for non-ideality and are made self-consistent(17) with regard to
the ionic strength. The non-ideality corrections are based on the extended Debye–Hu¨ckel
equation,(17) in which the “ion-size” parameter has been set at 1.6 kg1/2 · mol−1/2. Thus,
by applying this model, and by using both the experimental values of 1r Hm(cal), and the
thermodynamic quantities given in table 3, we obtain 1r Ho = −(25.16±0.31) kJ · mol−1
for reaction (5) and 1r Hmo = −(26.17 ± 0.45) kJ · mol−m1 for reaction (6). These values
pertain to T = 298.15 K and I = 0. The uncertainties given here are based on the
uncertainties given in the experimentally determined values of 1r H(cal).
The uncertainties in the values of 1r Hmo for the reference reactions (5) and (6) have
two components: the experimental uncertainties in the measured values of 1r Hm(cal) and
possible errors in the quantities used in the equilibrium model. This latter component of
uncertainty was examined by perturbing each of the pertinent quantities in the model by an
assumed possible error. Specifically, the appropriate pK values (see table 4) were perturbed

Hydrolysis of L-glutamine and L-asparagine
1083
TABLE 2. Results of the calorimetric measurements at T = 298.15 K. The molalities
m are those obtained after mixing of the enzyme and substrate solutions and prior to any
reaction. All molalities are equal to the sums of the molalities of the indicated substances
in their various ionic forms. L-Glutamine and L-asparagine are abbreviated as Gln and Asn,
respectively. 1 H (cal) is the calorimetrically determined enthalpy of reaction. The values
r
m
of the ionic strength I are calculated. Glutaminase and asparaginase were used to catalyse
m
reactions (1) and (2), respectively. The respective mass fractions w of these enzymes in
−5
−5
solution were ≈4 · 10
and ≈2 · 10 . The uncertainties given for 1 H (cal) are equal
r
m
to two estimated standard deviations of the mean. Estimates of total error for 1 H (cal)
r
m
are given in the text (see Results and discussion)
Reaction (1): L-glutamine(aq) + H O(l) = L-glutamate(aq) + ammonia(aq)
2
m(KH PO )
m(Gln)
I
1 H (cal)
r m
m
2
4
Experiment
pH
−1
−1
−1
−1
mol · kg
mol · kg
mol · kg
kJ · mol
1
2
3
4
5
6
7
5.14
5.14
5.14
5.14
5.14
5.14
5.14
0.1004
0.1004
0.1004
0.1004
0.1004
0.1004
0.1004
0.01194
0.01185
0.01188
0.01184
0.01200
0.01196
0.01197
0.11
0.11
0.11
0.11
0.11
0.11
−24.38
−24.46
−24.12
−24.31
−24.61
−24.52
−24.29
0.11
−1
h1 H (cal)i = −(24.39 ± 0.12) kJ · mol
r
m
Reaction (2): L-asparagine(aq) + H O(l) = L-aspartate(aq) + ammonia(aq)
2
m(Na HPO )
m(H PO )
m(Asn)
I
1 H (cal)
r m
m
2
4
3
4
Experiment
pH
−1
−1
−1
−1
−1
mol · kg
mol · kg
mol · kg
mol · kg
kJ · mol
1
2
3
4
5
6
7.03
7.03
7.03
7.03
7.03
7.03
0.09874
0.09876
0.09874
0.09874
0.09876
0.09876
0.00386
0.00386
0.00386
0.00386
0.00386
0.00386
0.01135
0.01148
0.01137
0.01136
0.01148
0.01148
0.27
0.27
0.27
0.27
0.27
0.27
−25.72
−25.59
−25.57
−25.68
−25.68
−25.55
−1
h1 H (cal)i = −(25.63 ± 0.06) kJ · mol
r
m
by ±0.05, the value of 1r Hmo for the ionization of H2PO−4 by ±0.2 kJ · mol−1, and the
“ion-size” parameter used in the activity coefficient model by ±0.3 kg1/2 · mol−1/2. The
combined effect of these perturbations in the calculated values of 1r Hmo were relatively
small (<0.04 · 1r Hmo ). Therefore, the final uncertainties in the calculated values of 1r Hmo
for reactions (5) and (6) are the same as the uncertainties given above.
The equilibrium model was also used to calculate the change in binding of the hydrogen
ion 1r N(H+). Thus, 1r N(H+) = 0.099 for reaction (1) at T = 298.15 K, pH = 5.14,

1084
N. Kishore, Y. B. Tewari, and R. N. Goldberg
TABLE 3. The pK values and standard molar enthalpy changes
o
1 H for the aqueous proton dissociation reactions of substances
r
m
pertinent to this study at T = 298.15 K and I = 0. See Results and
discussion for the basis of these values
o
1 H
r
m
Reaction
L-asparagine = L-asparagine + H
pK
−1
kJ · mol
+
±
±
−
+
+
2.16
8.94
3.0
L-asparagine = L-asparagine + H
40
0
−
2−
+
+
L-aspartic acid = L-aspartic acid + H
3.90
4.1
38
−
L-aspartic acid = L-aspartic acid + H
10.00
+
±
±
−
+
+
L-glutamine = L-glutamine + H
2.19
9.21
3.0
40
L-glutamine = L-glutamine + H
0
−
+
L-glutamine acid = L-glutamate + H
4.30
9.96
3.0
40
−
2−
+
L-glutamate = L-glutamate + H
+
+
2−
NH = NH + H
9.25
52.2
3.6
3
4
−
+
H PO = HPO + H
7.212
2
4
4
and Im = 0.11 mol · kg−1; and 1r N(H+) = 0.0102 for reaction (2) at T = 298.15 K,
pH = 7.03, and Im = 0.27 mol · kg−1. These values of 1r N(H+) were used in the
buffer protonation corrections(20) to calculate the values of 1r Hmo for the reference
reactions (5) and (6) from the experimentally determined values of 1r Hm(cal) for the
overall biochemical reactions (1) and (2). Thus, the buffer protonation correction is small
(<0.04 kJ · mol−1).
Results from the literature for the apparent equilibrium constant (concentration basis) Kc
and 1r H(cal) for biochemical reactions (1) and (2) are given in table 4. The comparison
of these results requires that they all be brought to a common basis. Therefore, we have
utilized the same equilibrium model as was used for the treatment of our results to calculate
values of Kc and 1r Hmo for the reference reactions (5) and (6) at T = 298.15 K and I = 0.
This calculation required a knowledge of the pK and 1r Hmo values for the ionizations of
acetic acid and boric acid:
C2H4O2(aq) = H+(aq) + C2H3O−2 (aq),
(9)
H3BO3(aq) = H+(aq) + H2BO−3 (aq).
(10)
Accordingly, the following values for these quantities were taken from Martell and
Smith:(14) pK = 9.24 and 1r Hmo = 13.0 kJ · mol−1 for reaction (10); and pK = 4.76 and
1r Hmo = −0.41 kJ · mol−1 for reaction (9). These quantities pertain to T = 298.15 K and
I = 0. The values of 1r Hmo determined in this study for reactions (5) and (6) were used,
where necessary, to adjust results to T = 298.15 K. Thus, by using the equilibrium model,
we have calculated the values of Kc and 1r Hmo given in table 4. The only equilibrium data

Hydrolysis of L-glutamine and L-asparagine
1085
TABLE 4. Results from the literature for biochemical reactions (1) and (2). The thermodynamic
temperatures T , the pH values, the buffers used, and either the apparent equilibrium constants
0
(concentration basis) K , or the calorimetrically determined enthalpies of reaction 1 H(cal) are
r
c
given for each study. Approximate ionic strengths (concentration basis) I were calculated from the
c
information given on the compositions of the solutions used in each study. We have also calculated the
values of the equilibrium constants (concentration basis) K and standard molar enthalpies of reaction
c
o
1 H for the respective reference reactions (5) and (6) at I = 0 and at T = 298.15 K. The uncertainties
r
m
o
in the values of 1 H(cal) and 1 H are equal to two estimated standard deviations of the mean
r
r
m
Biochemical reaction (1): L-glutamine(aq) + H O(l) = L-glutamate(aq) + ammonia (aq)
2
T
I
c
0
pH
K
K
Reference
c
c
−3
K
mol · dm
a,b
a,b
a,b
(2)
(2)
(2)
298.0
0.23
0.45
0.89
0.32
0.54
0.98
773
826
385
374
389
370
387
420
Benzinger and Hems
Benzinger and Hems
Benzinger and Hems
298.0
297.4
950
b
(3)
298.15
298.15
298.15
5.5
5.5
5.5
770
Benzinger et al.
b
b
(3)
877
Benzinger et al.
(3)
1040
Benzinger et al.
Biochemical reaction (1): L-glutamine(aq) + H O(l) = L-glutamate(aq) + ammonia (aq)
2
o
T
I
1 H(cal)
1 H
c
r
r
m
pH
4.5
Buffer
acetate
Reference
−3
−1
−1
K
mol · dm
0.004
kJ · mol
kJ · mol
−(21.0 ± 0.2)
(21)
295.0
−(22.0 ± 0.2)
Kitzinger et al.
Biochemical reaction (2): L-asparagine(aq) + H O(l) = L-aspartate(aq) + ammonia (aq)
2
o
T
I
1 H(cal)
1 H
c
r
r
m
pH
Buffer
Reference
−3
−1
−1
K
mol · dm
kJ · mol
kJ · mol
(21)
295.0
8.5
borate
≈0.022
0.13
− (21.5 ± 0.3)
−(24.56 ± 0.2)
−(20.6 ± 0.3)
−(25.1 ± 0.2)
Kitzinger and Hems
(22)
298.15
6.86
phosphate
Rekharsky et al.
a
(2)
Benzinger and Hems did not report the pH at which their measurements were performed. We have assumed
(3)
b
that the pH was the same as that used by Benzinger et al., namely pH = 5.5. It does not appear that a buffer was
(2)
(3)
used in the equilibrium studies of Benzinger and Hems or of Benzinger et al.
in the literature are those of Benzinger et al.(2, 3) from which we obtain hKci = 387 for
reaction (5) at T = 298.15 K and I = 0. The adjustment of the value of Kc to Km requires
the density values of the solutions. Thus, with the densities reported by Benzinger et al.,(3)
we obtain the value Km = 378 for reaction (5).
Examination of the calorimetric results shows that the value of 1r Hmo for reaction (5) ob-
tained from the study of Kitzinger and Hems(21) differs from the result of the present study
by 4.2 kJ · mol−1. Also, the value of 1r Hmo for reaction (6) obtained by them(21) is in dis-

1086
N. Kishore, Y. B. Tewari, and R. N. Goldberg
agreement by 5.6 kJ · mol−1. In both cases, the results of the earlier studies are less exother-
mic than the results of the present study. This is consistent with either the presence of non-
reactive impurities in the sample, or less than complete reaction in their microcalorimeter.
The result of Rekharsky et al.,(22) 1r Hmo (T = 298.15 K, I = 0) = −(25.1 ±
0.2) kJ · mol−1 for reaction (6), differs from the present result, 1r Hmo (T = 298.15 K,
I = 0) = −(26.2 ± 0.5) kJ · mol−1, by 1.1 kJ · mol−1. However, while their(22) result is
very precise, no allowance was made for possible systematic errors in the measurements
(see above). Thus, if we assume that a more reasonable assignment of error is an uncer-
tainty comparable to that assigned to 1r Hmo for reaction (6) in this study (±0.5 kJ · mol−1),
the result of Rekharsky et al.(22) and of the present study are seen to be in near agreement.
A thermodynamic cycle that can be used to calculate Km for reaction (5) is based
upon values of the standard molar enthalpy of formation 1f Hmo , standard molar entropy
Smo , and the saturation molality m(sat) of L-glutamine(s). In the calculations that are
now described, and which will be referred to as thermochemical cycle “A”, T
=
298.15 K and p = 0.1 MPa. Thus, we use the following values for L-glutamine(s):
1f Hmo = −826.4 kJ · mol−1 from Pedley et al.(23) based on the standard molar enthalpy of
combustion 1c Hmo determined by Tsuzuki and Hunt;(24) Smo = 195.06 J · K−1 · mol−1 from
Domalski et al.(25) based on the heat capacity measurements of Hutchens et al.;(26) and
m(sat) = 0.291 mol · kg−1 given by Hutchens.(27) Use of these values with Smo for C(s),
H2(g), N2(g), and O2(g) from the CODATA tables(16) leads to 1fGmo = −532.3 kJ · mol−1
for L-glutamine(s) and 1fGom = −529.3 kJ · mol−1 for L-glutamine±(aq). Here, we
have assumed that the activity coefficient γ of L-glutamine±(aq) is 1.0. To complete
the calculation, we use 1fGom for L-glutamine±(aq) together with values of 1fGom for
NH+(aq) and H2O(l) from the CODATA tables,(16) and 1fGom for L-glutamate−(aq) from
4
Miller and Smith-Magowan(28) {see table 10 (succinate + acetate pathway)}. This gives
1rGom = −10.4 kJ · mol−1 and Km = 66 for reaction (5) at T = 298.15 K and I = 0.
A similar calculation (this will be referred to as thermochemical cycle “B”) can
be performed that leads to Km for reaction (6). Here, we have the following for L-
asparagine · H2O(s): 1f Hmo = −1086.6 kJ · mol−1 given by Pedley et al.(23) based on
the value of 1c Hmo determined by Huffman et al.;(29) Smo = 213.4 J · K−1 · mol−1
given by Domalski et al.(25) based on the heat capacity measurements of Hutchens
and Borsook;(30) and m(sat) = 0.19 mol · kg−1 given by Hutchens.(27) This leads to
1fGmo = −769.1 kJ · mol−1 for L-asparagine · H2O(s), and 1fGom = −527.9 kJ · mol−1
for L-asparagine±(aq). In performing this calculation, it was assumed that γ = 1 for
L-asparagine±(aq) and that the activity of water aw = 1. We then use 1fGom for L-
asparagine±(aq) together with 1fGom for NH+4 (aq) and H2O(l) from CODATA,(16) and
1fGom for L-aspartate−(aq) from Miller and Smith-Magowan(28) to calculate 1rGmo
−13.3 kJ · mol−1 and Km = 213 for reaction (6) at T = 298.15 K and I = 0.
=
A final thermodynamic pathway (thermochemical cycle “C”) uses the reaction catalysed
by glutamate-ammonia ligase (EC 6.3.1.2):
ATP(aq) + L-glutamate(aq) + ammonia(aq) = ADP(aq) +
orthophosphate(aq) + L-glutamine(aq).
(11)

Hydrolysis of L-glutamine and L-asparagine
1087
Apparent equilibrium constants for this reaction have been determined by Levintow and
Meister,(31) Varner and Webster,(32) and Rosing and Slater.(4) We have used the equilibrium
model (see above) to calculate values of K for the reference reaction:
ATP4−(aq) + L-glutamate−(aq) + NH4+(aq) = ADP3−(aq) + HPO24−(aq) +
L-glutamine±(aq) + H+(aq).
(12)
In performing these calculations, we used the pK and 1r Hmo values for the L-glutamate, L-
glutamine, and ammonia species given in table 4 as well as the pK and 1r Hmo values given
in Alberty and Goldberg’s(33) table 1 for the ATP, ADP, and orthophosphate species. To
adjust results to T = 298.15 K, it is necessary to have a value of 1r Hmo for reaction (12).
This required a value of 1r Hmo for the reaction:
ATP4−(aq) + H2O(l) = ADP3−(aq) + HPO24−(aq) + H+(aq).
(13)
Thus, with 1r Ho (13) = −20.5 kJ · mol−1 from Alberty and Goldberg(33) and 1r Hmo (5) =
−25.2 kJ · mol−m1 from the present study, we obtain 1r Hmo (12) = 1r Hmo (13)−1r Hmo (5) =
4.7 kJ · mol−1. Also required in these calculations are the values pK = 8.075 and 1r Hmo
=
47.48 kJ · mol−1 for the ionization of Tris · H+(aq). These were taken, respectively, from
(35)
the studies of Bates and Hetzer(34) and Ojelund and Wadso¨.
All of the aforementioned
¨
thermodynamic quantities pertain to T = 298.15 K and I = 0. The “ion-size” parameter
in the extended Debye–Hu¨ckel equation was, as above, kept fixed at 1.6 kg1/2 · mol−1/2
.
The results of these calculations of K(T = 298.15 K, I = 0) for reaction (12) are
summarized in table 5. Averaging the results of the respective investigations, we obtain
hKi = 4.4 · 10−3 given by Levintow and Meister,(31) hKi = 0.0025 given by Varner
and Webster,(32) and hKi = 8.3 · 10−4 given by Rosing and Slater.(4) The corresponding
values of 1rGom are 13.5 kJ · mol−1, 14.9 kJ · mol−1, and 17.6 kJ · mol−1. In the discussion
to follow, we shall adopt a final grand average h1rGomi = (15.3 ± 2.3) kJ · mol−1 for
reaction (12). The estimated uncertainty is large enough to encompass the results of
all three investigations. We then use 1rGom(5) = 1rGmo (13) − 1rGmo (12) to calculate
1rGom = −12.3 kJ · mol−1 and Km = 143 for reaction (5) at T = 298.15 K and I = 0.
The estimated uncertainties in Km are large enough that its value could be as low as 50 and
as large as 360. The value 1rGom(13) = 3.03 kJ · mol−1 used in the above calculation is
that used by Alberty and Goldberg,(33) and is based upon a recalculation(36) of the results
of Guynn and Veech(37) for reaction (13).
Thus, for reaction (5) we have the experimental result K = 378 from Benzinger et
al.,(31) K = 66 from thermochemical cycle “A”, and K = 143 from thermochemical
cycle “C”. The average is K = 196 for reaction (5). For reaction (6) the only result
(thermochemical cycle “B”) is K = 213. We simply adopt K = 200 for both reactions (5)
and (6) at T = 298.15 K and I = 0. We consider both values to be approximate. Values
for 1rGom, Km, 1r Hmo , and the standard molar entropy change 1rSmo for reactions (5)
and (6) at T = 298.15 K and I = 0 are given in table 6. Since the values of the Kms
were approximate, the values for 1rGom and 1rSmo in table 6 must also be considered
approximate.
That the values of 1r Hmo for reactions (5) and (6) should be very nearly the same

1088
N. Kishore, Y. B. Tewari, and R. N. Goldberg
TABLE 5. Results from the literature for biochemical reaction (11): ATP(aq)
+ L-glutamate(aq) + ammonia (aq) = ADP(aq) + orthophosphate(aq) + L-
glutamine(aq). The thermodynamic temperatures T , the pHs, the buffers used, and
0
the apparent equilibrium constants K are given for each study. Approximate ionic
strengths (concentration basis) I were calculated from the information given on
c
the compositions of the solutions used in each study. The values of the equilibrium
constant K for the reference reaction (12) at I = 0 and at T = 298.15 K were
calculated by using the equilibrium model (see Results and discussion)
T
I
c
0
3
pH Buffer
K
10 · K
Reference
−3
K
mol · dm
a
(31)
(31)
(31)
(31)
295.15
310.15
310.15
310.15
308.15
310.15
310.15
310.15
310.15
310.15
7.0
0.23
0.23
0.24
0.16
0.16
0.25
0.30
0.20
0.23
0.16
1800
400
6.3
Levintow and Meister
Levintow and Meister
Levintow and Meister
Levintow and Meister
a
6.0
5.2
a
7.0
1233
3000
1700
162
4.3
a
7.9
1.6
(32)
7.4
Tris
Tris
Tris
Tris
Tris
Tris
2.5
Varner and Webster
(4)
6.6
1.13
0.98
0.56
0.84
0.63
Rosing and Slater
(4)
7.0
270
Rosing and Slater
(4)
7.1
280
Rosing and Slater
(4)
7.50
7.58
668
Rosing and Slater
(4)
831
Rosing and Slater
a
(31)
Levintow and Meister
stated that they used either imidazole or Tris buffers.
However, they did not state which buffer was used for the individual results given in their
table 1. For purposes of our calculations, we have assumed that Tris buffer was used in all
cases.
o
TABLE 6. Standard molar Gibbs energy change 1 G , the equilibrium constant
r
m
o
K , the standard molar enthalpy change 1 H , and the standard molar entropy
change 1 S for the reference reactions in aqueous solution at T = 298.15 K
m
r
m
o
r
m
and I = 0
o
o
o
1 G
r
1 H
r
1 S
r
m
m
m
Reaction
K
m
−1
−1
−1
kJ · mol
kJ · mol
kJ · mol
±
L-glutamine + H O(l) =
2
+
−
L-glutamate + NH
≈ 200
≈ 200
−13
−13
−(25.2 ± 0.3)
−(26.2 ± 0.5)
−40
−44
4
±
L-asparagine + H O(l) =
2
+
−
L-aspartate + NH
4
is to be expected on the basis of their structural similarity and simple group additivity
principles. Additional insight into the correlation of the thermodynamic results with
the structures of these substances involves a calculation of the standard partial molar
entropies S2o,m for L-glutamine±(aq) and L-asparagine±(aq). Thus, we use the values
of 1rSmo for the reference reactions (5) and (6) together with the values of S2o,m for

Hydrolysis of L-glutamine and L-asparagine
1089
L-glutamate−(aq), L-aspartate−(aq), NH+4 (aq), and H2O(l) tabulated by Wilhoit(38) to cal-
culate S2o,m = 258 J · K−1 · mol−1 for L-glutamine±(aq), and S2o,m = 243 J · K−1 · mol−1
for L-asparagine±(aq). These results pertain to T = 298.15 K and I = 0. The difference
is {S2o,m (L-glutamine±) − S2o,m (L-asparagine±)} = 15 J · K−1 · mol−1. A positive
difference in these entropies is expected since L-glutamine± has one more methylene
group than L-asparagine±. Indeed, Domalski and Hearing(39) give group-contribution
values (T = 298.15 K and p = 0.1 MPa) to Smo = 39 J · K−1 · mol−1 for the gas and
Smo = 32 J · K−1 · mol−1 for the liquid for a methylene group in the Benson estimation
method. A similar pair of amino acids is L-leucine±(aq) and L-valine±(aq) with the former
substance having an additional methylene group. Again, with data given by Wilhoit,(38)
the difference is {S2o,m (L-leucine±) − S2o,m(L-valine±)} = 31 J · K−1 · mol−1. Thus, both
sets of differences in the standard partial molar entropies are approximately consistent.
Alberty(5) has recently discussed the application of the Benson method to biochemical
reactions. Every indication is that this approach can be made to work well, given reliable
thermodynamic data for reactions that encompass the chemical groups and structural
changes needed to generate Benson group parameters.
It is also desirable to have values of the apparent equilibrium constants K0 for these
reactions under approximately physiological conditions, which are taken to be:(40) T =
311.15 K, pH = 7.0, pMg = 3.0, and Im = 0.25 mol · kg−1. With the thermodynamic
quantities obtained in this study for the reference reactions and with the equilibrium
model, the values of the apparent equilibrium constant K0 for reactions (1) and (2)
under these conditions are calculated to be ≈ 250. In performing these calculations,
it was assumed that there was no binding of Mg2+(aq) to any of the species involved
in these two biochemical reactions. The standard transformed Gibbs energy changes
1rG0mo for biochemical reactions (1) and (2) under physiological conditions are both
≈ −14 kJ · mol−1
.
We thank Dr Sam Margolis (National Institute of Standards and Technology) for his helpful
advice regarding the chromatographic methods.
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(Received 15 October 1998; in final form 26 January 1999)
WA98/058