A thermodynamic investigation of some reactions involving prephenic acid

Article abstract of DOI:10.1006/jcht.1998.0444

Calorimetric enthalpies of reaction have been measured for the following enzyme-catalysed reactions at the temperature 298.15 K: prephenate(aq) = phenylpyruvate(aq) + carbon dioxide(aq), prephenate(aq) + NAD_ox(aq) + H₂O(1) = 4-hydroxyphenylpyruvate(aq) + NAD_red(aq) + carbon dioxide (aq). Here, NAD_ox and NAD_red are, respectively, the oxidized and reduced forms of β-nicotinamide adenine dinucleotide. The enzymes that catalyse these respective reactions, prephenate dehydratase and prephenate dehydrogenase, were prepared by expression of the appropriate plasmids using the techniques of molecular biology. The calorimetric measurements together with the equilibrium modeling calculations lead to a standard molar enthalpy change Δ_rH^o_m = -(126 ± 5) kJ·mol^-1 for the reference reaction: prephenate^2-(aq) = phenylpyruvate^-(aq) + HCO^-₃(aq). Similarly, Δ_rH^o_m = -(74 ± 3) kJ·mol^-1 for the reference reaction: prephenate^2-(aq) + NAD^-_ox(aq) + H₂O(1) = 4-hydroxyphenylpyruvate^- (aq) + NAD²_red(aq) + HCO^-₃(aq) + H⁺aq). Both results pertain to T = 298.15 K and ionic strength I = 0. Benson estimates for the entropies lead to approximate values of the equilibrium constants K ≈ 1·10²⁶ and K ≈ 1·10¹², respectively, for the above two reference reactions.

Full text of DOI:10.1006/jcht.1998.0444

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J. Chem. Thermodynamics 1999, 31, 211–227
Article No. jcht.1998.0444
Available online at http://www.idealibrary.com on
A thermodynamic investigation of some reactions
involving prephenic acid
Nand Kishore,a Marcia J. Holden, Yadu B. Tewari, and
Robert N. Goldberg b
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 at the temperature 298.15 K:
prephenate(aq) = phenylpyruvate(aq) + carbon dioxide(aq),
prephenate(aq) + NAD (aq) + H O(l) = 4-hydroxyphenylpyruvate(aq) +
ox
2
NAD (aq) + carbon dioxide(aq).
red
red
Here, NAD and NAD
are, respectively, the oxidized and reduced forms of β-
ox
nicotinamide adenine dinucleotide. The enzymes that catalyse these respective reactions,
prephenate dehydratase and prephenate dehydrogenase, were prepared by expression of the
appropriate plasmids using the techniques of molecular biology. The calorimetric measure-
ments together with the equilibrium modeling calculations lead to a standard molar enthalpy
o
−1
change 1 H = −(126 ± 5) kJ·mol for the reference reaction:
r
m
−
2−
−
prephenate (aq) = phenylpyruvate (aq) + HCO (aq).
3
o
−1
Similarly, 1 H = −(74 ± 3) kJ·mol for the reference reaction:
r
m
2−
−
prephenate (aq) + NAD (aq) + H O(l) =
2
ox
2−
−
−
+
4-hydroxyphenylpyruvate (aq) + NAD (aq) + HCO (aq) + H (aq).
red
3
Both results pertain to T = 298.15 K and ionic strength I = 0. Benson estimates for
26
the entropies lead to approximate values of the equilibrium constants K ≈ 1·10 and
12
c
K ≈ 1·10 , respectively, for the above two reference reactions. ꢀ 1999 Academic Press
KEYWORDS: enthalpy; 4-hydroxyphenylpyruvic acid; β-nicotinamide adenine dinucl-
eotide; phenylpyruvic acid; prephenate dehydratase; prephenate dehydrogenase; prephenic
acid
a
b
Guest researcher from the Department of Chemistry, Indian Institute of Technology, Bombay, India.
To whom correspondence should be addressed (E-mail: robert.goldberg@nist.gov).
c
0021–9614/99/020211 + 17 $30.00/0
ꢀ 1999 Academic Press

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N. Kishore et al.
COOH
O
C
C
O
HOOC
HO
CH2
COOH
CH2
=
+
H2CO3
H
prephenic acid
phenylpyruvic acid
O
OH OH
NH2
O
O
P
O
P
HOOC
HO
CH2 C COOH
N
N
N
H2C
CH2
O
O
O
N+
O–
OH
N
+
+
O
H2O
=
NH2
H
C
O
OH OH
prephenic acid
COOH
ꢀ-nicotinamide adenine dinucleotide (oxidized form)
O
OH OH
NH2
C
O
O
P
O
CH2
N
N
N
H2C
P
CH2
O
O
O
N+
+
O
OH
N
+
O
H2CO3
NH2
C
O
OH
OH OH
ꢀ-nicotinamide adenine dinucleotide (reduced form)
4-hydroxyphenylpyruvic acid
FIGURE 1. Structures of the substances in reactions (1) and (2). The neutral forms of the substances
are shown.
1. Introduction
The enzymes prephenate dehydratase (EC 4.2.1.51) and prephenate dehydrogenase (EC
1.3.1.12) catalyse, respectively, the reactions:
prephenate(aq) = phenylpyruvate(aq) + carbon dioxide(aq),
prephenate(aq) + NADox(aq) + H2O(l) = 4-hydroxyphenylpyruvate(aq) +
NADred(aq) + carbon dioxide(aq).
(1)
(2)
Here, NADox and NADred are, respectively, the oxidized and reduced forms of β-nicotina-
mide adenine dinucleotide. The structures of these substances are shown in figure 1.
These two reactions, which are the first steps in two branches of the chorismate metabolic
pathway,(1) arefollowedinvivobytransaminationreactionsinwhichthephenylpyruvateand

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Prephenic acid reactions
213
4-hydroxyphenylpyruvate are converted, respectively, to L-phenylalanine and L-tyrosine. In
thissamemetabolicpathway, prephenateisformedfromchorismatebyanenzyme-catalysed
(chorismate mutase, EC 5.4.99.5) Claisen rearrangement. The transamination reactions and
the conversion of chorismate to prephenate have been the subject of recent thermodynamic
studies.(2, 3) Accordingly, the aim in the present study was to perform calorimetric and
equilibrium measurements on reactions (1) and (2) and thus obtain a complete picture of the
thermodynamics of a major portion of the chorismate metabolic pathway. This necessitated
the preparation of prephenate dehydratase and prephenate dehydrogenase by starting with
the expression of the appropriate plasmids and by using the techniques of molecular biology.
In addition to its inherent biochemical interest, this pathway is also of importance because
of its potential use for the large-scale production of aromatic amino acids and other valuable
products.(4, 5)
2. Experimental
Relevant information on the substances used in this study is given in table 1.† In the ab-
sence of a sample of prephenic acid having sufficient purity, it was decided to prepare this
substance in situ by using a well-characterized sample of chorismic acid(2) and the enzyme
chorismate mutase (EC 5.4.99.5). This conversion of chorismate to prephenate was found
to be essentially complete under the conditions used for the calorimetric experiments (see
below). Specifically, the mole fraction of chorismate remaining was <8·10−4 following a
reaction time of 1 h. Previous kinetic results(6, 7) showed that the half life for the decom-
position of prephenate via reaction (1) is 130 h at the temperature 298.15 K and pH = 7.0.
Thus, for the calorimetric experiments, where the prephenate was in solution for ≈1 h
prior to initiation of the reaction(s) of interest, the mole fraction of prephenate converted to
(phenylpyruvate + carbon dioxide + H2O) was ≈0.0053.
Prephenate dehydrogenase was produced by expression of the plasmid pJX3 of Xia et
al.(8) This plasmid is a recombinant of pUC19 with a 1600 base DNA insert from Er-
winia herbicola. This insert contains a defective pheA gene and a tyrA gene that is missing
the coding for 37 amino acids at its amine terminus (replaced by 18 amino acids from
the polycloning site of the lacZ α-peptide of pUC19). The resulting DNA encodes for a
monofunctional prephenate dehydrogenase with no chorismate mutase activity. The pro-
tein was expressed in E. coli KA12 cells that are deficient in pheA and tyrA resulting
in no endogenous chorismate mutase, or prephenate dehydratase, or prephenate dehy-
drogenase activity.(9) The cells were grown in a Terrific Broth medium batch culture(10)
at T = 310 K for ≈15 h. The cells were harvested by centrifugation at the accelera-
tion a ≈ 3000·gn (gn = 9.80665 m·s−2), washed in the buffer {tris (hydroxymethyl)
aminomethane (Tris) (concentration c = 0.01 mol·dm−3) + ethylenediaminetetraacetic
acid (EDTA) (c = 0.0001 mol·dm−3) + dithiothreitol (DTT) (c = 0.001 mol·dm−3),
adjusted with HCl to pH = 7.4}. These cells were then re-harvested by centrifugation
†Certain commercial equipment, instruments, or materials are identified in this paper to specify the experimental
proceduresadequately. SuchidentificationisnotintendedtoimplyrecommendationorendorsementbytheNational
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.

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