From the Department of Biology, Graduate School of Science, Osaka
University, Toyonaka, Osaka 560-0043, Japan
Aromatic amino acid aminotransferase (AroAT) and
aspartate aminotransferase (AspAT) are known as dual-substrate enzymes,
which can bind acidic and hydrophobic substrates in the same pocket (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and
Kuramitsu, S. (1997) J. Biochem. (Tokyo)
122, 55-63). In order to elucidate the mechanism of hydrophobic
substrate recognition, kinetic and thermodynamic analyses using
substrates with different hydrophobicities were performed. They
revealed that 1) amino acid substrate specificity
(kmax/Kd) depended on the
affinity for the substrate (1/Kd) and 2) binding of
the hydrophobic side chain was enthalpy-driven, suggesting that van der
Waals interactions between the substrate-binding pocket and
hydrophobic substrate predominated. Three-dimensional structures of
AspAT and AroAT bound to
-aminoheptanoic acid were built using the homology modeling method. A molecular dynamic simulation study suggested that the outward-facing position of the Arg292
side chain was the preferred state to a greater extent in AroAT than
AspAT, which would make the hydrophobic substrate bound state of the
former more stable. Furthermore, AroAT appeared to have a more
flexible conformation than AspAT. Such flexibility would be
expected to reduce the energetic cost of conformational rearrangement induced by substrate binding. These two mechanisms (positional preference of Arg and flexible conformation) may account for the high
activity of AroAT toward hydrophobic substrates.
 |
INTRODUCTION |
Many enzymes show restricted specificities for single chemical
types of substrate (1), but some have evolved binding pockets with dual
specificities for different chemical groups (aminotransferases (Refs. 2
and 3) and cysteine protease cruzain (Ref. 4)). Escherichia
coli aromatic amino acid aminotransferase
(AroAT)1 and aspartate
aminotransferase (AspAT) are unique in being active toward two entirely
different kinds of substrate (acidic and hydrophobic). These enzymes
recognize a carboxyl group of an acidic substrate with side chain of
arginine residue and recognize hydrophobic substrates in proportion to
their hydrophobicities (3). These two kinds of substrates are
accommodated in the one binding pocket of the enzyme.
Structural determination and protein engineering techniques have made
it relatively easy to define the key residues that recognize the polar
and charged groups of a substrate. However, the mechanism responsible
for hydrophobic substrate specificity is more complicated, because the
complexity of interactions within the substrate binding pocket and
multiple protein configurations must be taken into consideration. In
many cases, a number of residues contribute to hydrophobic ligand
specificity. There are also situations when the residues determining
specificity do not interact directly with the substrate. Generally,
hydrophobic side chains are well packed in the protein cores and a
hydrophobic effect is one of the major factors involved in substrate
recognition and protein folding (5). So far, three strategies designed
to estimate the extent of hydrophobic interaction have been reported.
The first involves measuring enzyme affinities for a series of
hydrophobic ligands (6-9), and detailed thermodynamic analyses of
nonpolar interactions based on ligand binding have been performed in T4 lysozyme (10), acyl-coenzyme A-binding protein (11), and cytochrome P450cam (12). The second involves measuring the effects of a series of
mutations on protein stability. Several studies have shown that
replacing a residue facing the cavity with a more hydrophobic residue
makes proteins more stable (13-19). The third involves the use of a
series of solvents with different hydrophobicities as the reaction
media (20). The results obtained suggest that the effect of a
hydrophobic interaction varies according to the environment around the
site (hydrophobicity, packing density, conformational flexibility, and
presence of water).
Aminotransferases catalyze the reversible transamination reaction
between an
-amino acid and an
-keto acid, which is accompanied by
interconversion of the cofactor between pyridoxal 5'-phosphate (PLP)
and pyridoxamine 5'-phosphate (PMP) (21, 22).
EL and EM denote the
PLP and PMP forms of the enzyme, respectively. Several kinds of
aminotransferases with different substrate specificities are known, and
they have been classified into four families on the basis of their
amino acid sequence homologies (23, 24).
E. coli AroAT shows high activity toward hydrophobic and
acidic substrates (3, 25). AroAT is quite similar in many respects to
AspAT, which is the most extensively investigated aminotransferase (26,
27). The three-dimensional structures of AspATs from various sources
have been determined by x-ray crystallography (28-30). E. coli AroAT (31) and AspAT (32) show amino acid sequence identities
of 43%, and the residues in their catalytic sites are almost fully
conserved. Both enzymes have a dimer structure consisting of identical
44-kDa subunits, and their circular dichroism spectra in the far UV
region are essentially identical (25). The absorption spectra over 300 nm and pKa values of the PLP-Lys Schiff base are
also very similar between AspAT and AroAT, which reflect the
microenvironment of the active site. Therefore, it seems reasonable to
assume that they have common three-dimensional structures and catalytic
mechanisms.
The characteristic difference between AspAT and AroAT is the their
activity toward hydrophobic substrates; in effect, AroAT shows about
10,000 times higher activity toward phenylalanine than AspAT.
Furthermore, the chimera constructed from AspAT and AroAT shows
hydrophobic substrate specificities between those of the two parent
enzymes (3, 33). Because the substrate-binding pockets of these three
enzymes are surrounded by the same set of residues (residues in
red in Fig. 1), the mechanism
of hydrophobic substrate recognition by aminotransferases has
generated considerable interest. Previous studies (3) have revealed
that the size of the substrate side chain is a major factor that
determines the enzymatic activity; the free energy stabilizations of
AroAT toward a series of hydrophobic substrates are twice those of
AspAT. In particular, AroAT shows constant activity toward the
hydrophobic unit of a substrate, irrespective of its shape, suggesting
that conformational flexibility or plasticity may be related to the hydrophobic substrate specificity of this enzyme (3).

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Fig. 1.
Structure of the active site of E. coli AspAT complexed with -methylaspartate (29). The
direction of view is from the C atom of the substrate
analog to the side chain terminus. a, -methylaspartate is
shown in cyan. b, the analog has been omitted
from a. Red, side chains conserved in AspAT and
AroAT; blue, non-conserved side chains; green,
backbone.
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Model structures for AroAT have been proposed (34, 35), and
subsequently, the structure of an AspAT hexamutant (V39L, K41Y, T47I,
N69L, T109S, and N297S)2 with
higher aromatic activity was determined by x-ray crystallography (36).
The hexamutant structure is thought to be a better template for AroAT
model building than those used previously. In this study, we re-built
an AroAT model in its complexed form with
-aminoheptanoic acid that
we expected to be helpful for understanding the hydrophobic substrate
specificity of AroAT. Recently, the dynamic natures of proteins have
been evaluated using a variety of methods. Increasing instances of
multiple conformations of substrates and proteins, even in their
crystallographic structures, have been found as the result of advances
in the various techniques. Protein dynamics, as well as protein
structures, are thought to be hierarchical (37). Not only large scale
conformational changes (movements of domains and/or segments), but also
relatively small fluctuations, must be biologically important. In this
paper, we discuss protein fluctuation, that may be related to van der
Waals interactions within the AroAT enzyme and between this enzyme and
its substrates. Molecular dynamic (MD) simulation has become a powerful
tool for analyzing the dynamic natures of macromolecules. We used this technique to investigate the flexibility of AroAT and performed thermodynamic analysis to examine further the effects of hydrophobicity on substrate binding.
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EXPERIMENTAL PROCEDURES |
Enzyme Preparation--
The plasmid pUC19GpY (3) overproduces
E. coli AroAT coded by the tyrB gene in the
E. coli TY103 strain (38), which was derived from the JM103
strain (39), by disrupting the aspC, tyrB, and
recA genes. Cells carrying this plasmid were grown overnight at 37 °C, harvested by centrifugation, frozen, and stored at
20 °C. The enzyme was purified and its concentration determined
using the protocols described previously (33).
Pre-steady-state Kinetic Analysis--
After the addition of an
amino acid substrate to the PLP form of AroAT, the 360-nm absorption
band shifts to 330 nm, which means the PLP form has converted to the
PMP form. The converse spectral change is observed with the reaction
between a keto acid and the PMP form of AroAT. The substrate
concentrations used were high enough to convert the enzyme between the
PLP and PMP forms. Under single-turnover conditions, the absorption
changes at 360 nm were monitored using a stopped-flow spectrophotometer
(SX-17MV, Applied Photophysics). The dead time was 2.0 ms under the
conditions used (7 kg·cm
2). About 10 progress curves
for each substrate concentration were recorded and curve-fitting to a
single exponential time course was performed using the program provided
with SX-17MV. The kinetic parameters were determined using the
following model (Reaction 2) and Equation 1.
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(Eq. 1)
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kapp is the apparent rate constant at a
given substrate concentration, kmax is the
maximum rate constant, and Kd is the dissociation
constant.
For slow kinetic experiments, a Hitachi U-3000 spectrophotometer was
employed. When the kapp value was directly
proportional to the substrate concentration, Equation 2, instead of
Equation 1, was used to determine the catalytic efficiency,
kmax/Kd(2).
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(Eq. 2)
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The reaction conditions were 10 µM AroAT, 1-50
mM substrate, 100 mM KCl, 50 mM
HEPES-KOH, pH 8.0, and 15-35 °C.
Binding of Inhibitors--
Aliphatic acid competitive inhibitors
bind noncovalently to AroAT and form Michaelis complexes. Upon complex
formation, the pKa of the internal aldimine, which
forms between N
of Lys258 and C4' of PLP,
increases from 6.8 to over 8.0 (40, 41). Therefore, the conversion from
the unprotonated (360 nm) and protonated (430 nm) species can be
followed spectrophotometrically. The spectra of the PLP form of AroAT
with various concentration of aliphatic acids were recorded using a
spectrophotometer (Hitachi U-3000). The reaction conditions were 100 mM KCl, 50 mM HEPES-KOH, pH 8.0, 25 °C, and
the protein concentration was about 10 µM. The binding of
aliphatic acid inhibitors to the PMP form of AroAT was monitored using
a spectropolarimeter (Jasco J-720). The Kd values for the inhibitors were obtained by fitting the absorbances at 430 (PLP
form) or 330 nm (PMP form) to theoretical curves using the Igor Pro
version 3.01 application (WaveMetrics, Inc.).
Thermodynamic Analysis--
The free energy change for the fast
binding step (
Gs) was obtained using the
equation
RTln(1/Kd), where R
is the gas constant (1.99 cal/K·mol) and T is the absolute
temperature, and the enthalpy change (
Hs) was
derived from a van't Hoff equation.
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(Eq. 3)
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This equation is often integrated under the assumption that
Cp = 0.
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(Eq. 4)
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Hs in Equation 4 is the so-called
van't Hoff enthalpy. The reactions between the PMP form of the enzyme
and keto acid substrates were analyzed using Equation 4. When it is
supposed that the enthalpy depends linearly on temperature, the
enthalpy change is expressed as shown in Equation 5.
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(Eq. 5)
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Hr is the enthalpy change at
temperature Tr, and
Cp is the
non-zero heat capacity change. Therefore, integration of the van't
Hoff equation (Equation 3) yields Equation 6.
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(Eq. 6)
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Kr is the dissociation constant at
Tr. The reactions between the PLP form of the
enzyme and amino acid substrates were analyzed using Equation 6.
The entropy change was calculated using the equation
Gs =
Hs
T
Ss, and the activation free energy for the
rate-determining step (
G
) was calculated
using Equation 7.
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(Eq. 7)
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kB is the Boltzmann constant (1.38 × 10
34 J·K
1), and h is the
Planck constant (6.63 × 10
34 J·s). The activation
energy (EA) was obtained from an Arrhenius plot
of the rate constant versus temperature
(ln(kmax) versus 1/T),
and the slope of this plot yields
EA/R. Thus, the enthalpy change
(
H
) is calculated using the equation
H
= EA
RT, and the activation entropy change
(
S
) was calculated using the equation
G
=
H
T
S
.
Homology Modeling and Molecular Dynamic Simulation--
The
x-ray crystallographic structure of the hexamutant of AspAT (Protein
Data Bank code 1AHG; see Ref. 36) complexed with the cofactor-substrate
analog N,5'-phosphopyridoxyl-L-tyrosine (PPY)
was used as the model for the hydrophobic substrate ligand-enzyme complex. As the crystallographic structure of AspAT complexed with
hydrophobic substrate analog has not been resolved, it was built by
reconverting the six mutated residues of the AspAT hexamutant. Modeling
of E. coli AspAT and AroAT was carried out using the Homology program (Biosym Technologies). AspAT and its hexamutant have
the same number of residues, whereas AroAT is one residue longer.
Pro64 was interpreted as an insertion residue, when
performing homology modeling of AroAT. The three-dimensional structure
data base (Protein Data Bank, Release 76) was searched for the
structure of residues 58-70, and the most probable loop structure was
chosen as an initial structure and spliced into the AroAT structure in
order to fill the insertion gap. The tyrosine residue of PPY in the
hexamutant was replaced by
-aminoheptanoic acid (sC7), a double bond
between N of sC7 and C4' of PLP and partial double bonds in the
aromatic ring of PLP were introduced and a 5-Å shell of water was
added to both the AspAT and AroAT structures. The AspAT and AroAT
systems contained water molecules found in the template structure (36) and a total of 24,279 and 24,391 atoms, respectively.
MD simulation was carried out using the Discover version 2.97 program
(Biosym Technologies) with the consistent valence force-field. The
dielectric constant was fixed at 1.0, and a 14-Å cutoff was used for
the nonbonded interactions with a switching function which smoothly
turns off the interaction over a range of 1.5 Å. Minimization was
performed according to the strategy used by Kasper et al.
(42). Briefly, both the AspAT and AroAT systems were subjected first to
500 energy minimization steps with all the heavy atoms fixed and then
to 500 steps with all the atoms free to move, using the
steepest-descents algorithm. Subsequently, they were subjected to 2000 steps using the conjugate-gradient algorithm. After minimization, the
total energies in AspAT and AroAT were
57 Mcal·mol
1
and
53 Mcal·mol
1, respectively, and their respective
root mean square derivatives were 0.066 and 0.062 kcal·mol
1·Å
1. The temperatures of both
systems reached 300 K during the initial 2 ps of MD simulation. The
Discover program assigned initial velocities to the atoms in order to
maintain a Maxwell-Boltzmann distribution at a given temperature. The
rest of the simulation protocol was run for about 280 and 370 ps for
AspAT and AroAT, respectively, at 300 K with a time step of 1 fs, and
the molecular coordinates of the molecules were saved every 1 ps.
In order to remove the overall translation and rotation of the systems,
the backbone N, C
, and C atoms of 100 conformers (last
100-ps interval of simulation) were superimposed on those of the first
conformer within this time interval and each atom position was
averaged. The mean square fluctuation was calculated for each
C
atom using Equation 8.
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(Eq. 8)
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Xi, Yi, and
Zi are the C
coordinates of a given
conformer in the 100-ps trajectory; Xav, Yav, and Zav are the
C
coordinates of the time-averaged structure.
 |
RESULTS |
The Effect of Temperature on Hydrophobic Substrate Specificity
A series of aliphatic substrates of uniform chemical nature was
used in this study. The enzymatic activities toward substrates of
various hydrophobicities were studied by analyzing the single-turnover reactions (half-reactions) using a stopped-flow apparatus. Table I shows the values of
kmax/Kd at 15-35 °C. A
temperature increase of 5 °C increased the
kmax/Kd value for each substrate 2-fold, and the catalytic efficiency increased as the side
chain length increased. The energetic contribution of each hydrophobic
substrate side chain to the enzymatic activity was evaluated by
calculating the
GT
value using
Equation 9 (Scheme 1) (43).
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(Eq. 9)
|
The
GT
value
decreased as the carbon number of the substrate increased and,
consistent with previous studies (3, 44), the relationship was linear
over the 4- to 7-carbon range (Fig. 2).
The slopes of the straight lines represented the dependence of the
enzymatic activity on the hydrophobic unit of the substrate side chain
and were referred to as the hydrophobic "substrate specificities."
Over the temperature range tested (15-35 °C), these slopes were
almost identical,
1.6 kcal·mol
1. Contrary to
expectation, these results suggest that entropy was not a major
determinant of the substrate specificity of AroAT, and they may be
attributable to strong enthalpy-entropy compensation.
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Table I
The kmax/Kd (s 1 · M 1) values for E. coli AroAT with a series of
aliphatic substrates at various temperatures
Conditions: 50 mM HEPES, 100 mM KCl, pH 8.0. sCn,
CH3-(CH2)n 3-CH(NH2)-COOH.
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Fig. 2.
Correlation between
GT and the substrate carbon
numbers. , 15 °C; , 20 °C; , 25 °C; ,
30 °C; , 35 °C. The substrates are a series of aliphatic amino
acids with straight side chains. The free energy differences
( GT ) between the unbound
enzyme and substrate (E+S) and the transition state
(ES ) was calculated using the equation
GT = Gs + G = RT(ln(kBT/h) ln(kmax/Kd)). The reaction
conditions were 50 mM HEPES buffer containing 100 mM KCl, pH 8.0, at the given temperatures.
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From this analysis, it was not clear how the temperature and substrate
hydrophobicity affect the bound and transition states of the reaction.
Therefore, separate determinations of kmax and Kd values were required.
Kinetic Analysis of Half-reactions
Two directions of the half-transamination reaction were defined;
the reaction between an amino acid substrate and the PLP form of the
enzyme was referred to as the "forward reaction," and that between
a keto acid substrate and the PMP form of the enzyme was referred to as
the "reverse reaction." Table II
shows the kinetic parameters of the forward and reverse reactions at 25 °C.
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Table II
Kinetic parameters of the half-reactions and dissociation constants
for E. coli AroAT
Conditions: 50 mM HEPES, 100 mM KCl, pH 8.0, 25 °C. The abbreviations: sCn,
CH3-(CH2)n 3-CH(NH2)-COOH;
ksCn,
CH3-(CH2)n 3-CO-COOH;
sCnI,
CH3-(CH2)n 3-CH2-COOH.
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Forward Reaction--
It was not possible to determine the kinetic
parameters for the alanine (sC3),
-aminobutyric acid (sC4), or
norvaline (sC5) substrate due to their large dissociation constants.
The Kd values for norleucine (sC6) and
-aminoheptanoic acid (sC7) at 25 °C were 58 and 5.1 mM, respectively. The affinity (1/Kd) of
AroAT for the sC7 substrate was 10 times that for the sC6 substrate. In
contrast, the kmax values, the first order rate
constants, for these two substrates were almost equal. These results
suggest that the hydrophobicity of the substrate side chain affected
only the bound state and did not change the transition state and that the affinity for the substrate was the major factor that determined the
catalytic efficiency in the forward reaction. Consequently, the
difference between the free energy changes from the unbound to
substrate-bound state (
Gs(7-6),
1.4
kcal·mol
1 between sC6 and sC7) was nearly same as the
difference between the free energy changes from the unbound to
transition state (
GT
(7-6),
1.5 kcal·mol
1).
The aliphatic acid AroAT inhibitors we used can bind noncovalently to
the enzyme and form Michaelis complexes. The dissociation constant for
the reaction of heptanoic acid (sC7I) with the PLP form of AroAT was 6 times higher than that of its cognate amino acid substrate, sC7 (Table
II). The lower affinity for sC7I was probably attributable to the lack
of covalent binding to the cofactor PLP. The energetic contribution of
each hydrophobic unit to the affinity,

Gs(8-7), was calculated to be
1.2
kcal·mol
1 (between sC7I and sC8I). This value was
similar to the 
GT
(8-7) of
1.3 kcal·mol
1 between the sC7 and sC8 substrates,
indicating that the hydrophobicities of the inhibitors and amino acid
substrates had the same effects on the affinities of the PLP form of
AroAT.
The kinetic parameters of the half-reactions were determined at various
temperatures, and Table III shows the
thermodynamic data for each reaction. The linear relationships between
ln(kmax) and 1/T shown by the
Arrhenius plots (Fig. 3a)
indicate that the reaction mechanisms for sC6 and sC7 substrates did
not alter over the temperature range studied and that their
H
values were similar. Furthermore, their
similar kmax values mean that the
G
values are almost equal. Therefore, it
is indicated that the
S
values of sC6 and
sC7 are also similar and that the extra methylene group of the latter
did not affect the conformation of the transition state. The van't
Hoff plot for the forward reaction yielded a convex curve (Fig.
3c), indicating large heat capacity changes (
Cp) had occurred. Negative
Cp values may,
at least in part, mean that during enzyme-substrate binding,
hydrophobic areas of the substrate and enzyme were removed from contact
with water. Similarly, a negative
Cp value was observed
during the binding of octanoic acid (sC8I) to the PLP form of AroAT
(data not shown). Murphy and Freire (45) proposed that the
contributions of polar and nonpolar interactions to
Cp
are proportional to the changes in their accessible surface areas (the
polar and nonpolar parameters were 0.26 and
0.45
cal·mol
1·K
1·Å
2,
respectively, when those areas were removed from contact with water).
However, the
Cp value for the sC6 substrate was more negative than that for the sC7 substrate, even though the interacting hydrophobic area of sC7 was greater than that of sC6. Therefore, contributions of other factors, i.e. van der Waals
interactions, must be considered.
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Table III
Thermodynamic parameters of half-reactions for E. coli AroAT at
25 °C
Conditions: 50 mM HEPES, 100 mM KCl, pH 8.0. Abbreviations: sCn,
CH3-(CH2)n 3-CH(NH2)-COOH;
ksCn,
CH3-(CH2)n 3-CO-COOH. The data
shown are means ± S.D.
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Fig. 3.
Arrhenius (a and b) and van't Hoff (c and d)
plots of half-reactions. a and c,
-Aminohexanoic acid (sC6) ( and solid
line); -aminoheptanoic acid (sC7) ( and broken
line). b and d, -ketovaleric acid (ksC5)
( and solid line); -ketocaproic acid (ksC6) ( and
broken line). Straight and curved
lines were fitted under the assumption that Cp = 0 and a constant Cp 0, respectively. The
reaction conditions were 50 mM HEPES buffer containing 100 mM KCl, pH 8.0, at the given temperatures.
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Reverse Reaction--
The catalytic efficiencies of many
aminotransferases are greater with keto acid substrates than their
cognate amino acid substrates, due to the higher affinities for keto
acids. The affinity has a greater effect on the catalytic efficiency
than the rate constant (2, 25). The kinetic parameters for
-ketovaleric (ksC5) and
-ketocaproic (ksC6) acids were determined
(Table II). The affinity of AroAT for the ksC6 substrate was 6 times
that for the ksC5 substrate and the kmax value
for the former was 4 times higher than that for the latter. Both
kmax and Kd of the reverse
reaction were affected by side chain hydrophobicity, whereas the
kmax values for the amino acid substrates we
used were not. Consequently, 
Gs(6-5)
(
1.1 kcal·mol
1 between ksC5 and ksC6) was
significantly smaller than the cognate 
GT
(6-5) (
1.7
kcal·mol
1) for the reverse reaction. With the PMP form
of the enzyme, the difference between the Kd values
of sC7I and sC6I was responsible for the

Gs(7-6) of
1.7
kcal·mol
1, which was equal to the

GT
(7-6) (
1.7
kcal·mol
1) value estimated from the substrate
specificities for keto acid substrates (see Fig. 1a in Ref.
3). These results suggest that the energy required to stabilize the
Michaelis complex was used to stabilize the transition state of the
reverse reaction, as well as that of the forward reaction.
Thermodynamic analysis revealed large positive enthalpy changes (3-6
kcal·mol
1) during the substrate-binding step (Fig.
3d). These large
Hs value may mean
that the solvation energies are higher for keto acids than amino acids.
Another explanation is that more water molecules are associated with
the PMP form of AroAT than with the PLP form, although fewer water
molecules were observed in the crystallographic structure of the PMP
form of E. coli AspAT than in that of the PLP form (46). The
activation free energies (
G
) were 14-15
kcal·mol
1 for all the substrates we used (Table III)
and were higher than that for the reaction of aspartate with AspAT by
only 1 kcal·mol
1 (2). Therefore, these substrates
appear to be almost good substrates for AroAT.
Unlike the forward reaction, the activation free energy of the reverse
reaction was altered by the side chain length (Fig. 3b).
Furthermore, the contribution of a methylene group in a keto acid
substrate to the bound state differed from that of one in an inhibitor
(Table II); 
Gs and

GT
were both
1.7
kcal·mol
1 for a set of inhibitors, whereas their
respective values were
1.1 and
1.7 kcal·mol
1,
substantially different, for a set of keto acid substrates. These
results suggest that there were some differences between the kinetic
bound states in both directions. A possible explanation for this is
that an intermediate (E·I), the energy level of
which depends on the side chain length, forms and is in rapid
equilibrium (Scheme 2), as shown by
Reaction 3.
EM is the PMP form of AroAT, KA a keto
acid,
the transition state of the reaction,
K1 the dissociation constant
([EM][KA]/[EM·KA]), and
K2 the equilibrium constant
([EM·KA]/[E·I]).
In this model, the apparent Kd value for a substrate
is expressed by K1·K2/(1+K2).
More detailed analysis of the reverse reaction will be reported
elsewhere.

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Scheme 2.
Energy profiles of the transamination
reactions with substrates with n and n+1
carbons. EL, pyridoxal form of enzyme;
AA, amino acid substrate; E·I,
intermediate form; EM, pyridoxamine form of enzyme;
KA, keto acid substrate.
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Energy Profile of the Transamination Reaction
Fig. 4 shows the reaction profiles
for the transamination reactions of AroAT with sC6 and ksC6. The free
energy level of the unbound PLP form of AroAT plus an amino acid
substrate (EL + sC6 in Fig. 4) was a
little lower by 1.3 kcal·mol
1, than that of the unbound
PMP form of AroAT plus a keto acid substrate (EM + ksC6 in Fig. 4). This value agreed well with the value
estimated from the equilibrium constant of the half-reaction, which was
determined spectrophotometrically (data not shown).

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Fig. 4.
Profiles of the thermodynamic parameters for
the half-transamination reactions between AroAT and -aminohexanoic
and -ketocaproic acids. G (solid
line), H (broken line), and
T S (dotted line) are shown.
EL, pyridoxal form; EM,
pyridoxamine form; sC6, -aminohexanoic acid;
ksC6, -ketocaproic acid.
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In order to evaluate the hydrophobic effects, the energy difference
profiles were evaluated (Table IV).
Although, overall, the binding steps were entropy-driven (Fig.
5, a and b),
binding of a single methylene group was enthalpy-driven (Fig.
5c). We thought this was because the van der Waals
interactions in the substrate-binding pocket were strong and because
the substrate side chain was packed well by the surrounding residues of
AroAT.
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Table IV
Energetic contribution of a single methylene group in the substrate
side chain
The data shown are means ± S.D.
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Fig. 5.
Profiles of the thermodynamic parameters for
the forward transamination reaction. a, reaction between
-aminohexanoic acid (sC6) and the pyridoxal form of the enzyme.
b, reaction between -aminoheptanoic acid (sC7) and the
pyridoxal form of the enzyme. c, subtraction of a
from b.
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Homology Modeling and Molecular Dynamic Simulation
The hexamutant structure (Protein Data Bank code 1AHG) was chosen
as a template for model building, because it is the only high
resolution structure complexed with a hydrophobic substrate analog,
PPY. In this study, the tyrosine residue of this analog was replaced by
-aminoheptanoic acid (sC7), because AroAT showed high activity
toward not only aromatic substrates, but also aliphatic substrates.
Furthermore, sC7 can bind to both AspAT and AroAT without substantial
energy cost, and it has been suggested that the substrates larger than
sC7 are subject to steric hindrance in the binding pocket of AspAT
(3).
After minimizing the initial AroAT and AspAT structures complexed with
sC7 molecules, MD simulation for 280 and 370 ps for AspAT and AroAT,
respectively, was performed (Fig. 6). The
temperature of both systems was 300 ± 2 K, and the kinetic
energies were maintained at about 21.5-22.0 Mcal during simulation.
The potential energies declined gradually, and that of the AspAT system
almost reached equilibrium in 200 ps. Therefore, the 181-280-ps
trajectory was analyzed, as described below. Only one water molecule
(B128 HOH in 1AHG), which was originally defined in the crystal
structure, was apart from the AspAT molecule. A longer simulation time
was required for the AroAT system to reach equilibrium, probably
because the initial structure of AroAT differed more from that yielded by MD simulation than was the case with AspAT simulation. Therefore, the 271-370-ps trajectory of the AroAT system was analyzed. First, the
overall translation and rotation of the molecule were removed by
superimposition of the first conformer at the 100-ps interval. The root
mean square deviations of the aligned positions were about 0.6 and 0.9 Å for AspAT and AroAT, respectively. Subsequently, the positionally
averaged structures were obtained from the 100 aligned conformers.

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Fig. 6.
Time courses of potential energies
(a), kinetic energies (b), and temperatures
(c) of the AroAT (solid line) and AspAT
(dotted line) systems. All data were saved every 1 ps.
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The many secondary structural segments defined by the DSSP method (47)
were shorter than those in the crystallographic structure (hexamutant,
Protein Data Bank code 1AHG; or wild type AspAT with
-methylaspartate, Protein Data Bank code 1ART) by one or two
residues, particularly for the
-helices, although their positions in
the primary structure remained constant, and this was probably related
to the high mobilities of the atoms at both ends of the secondary
structures. The averaged structures were compared with the structures
determined experimentally by superimposing their main chain (N,
C
, and C) atoms, and the root mean square deviations of
the two structures in their aligned positions are shown in Table
V. The averaged structure of AroAT had
higher values than that of AspAT, which may be due to their different loop structures. The structures of both AspAT and AroAT were closer to
that of the closed conformation complexed with a substrate analog (1AHG
or 1ART) than to that of an open one (Protein Data Bank code 1ARS)
(Fig. 7). When wild type AspAT bound to
the substrate analog, the small domain (5-52 and 364-409 residues) is
observed to move 5° toward the large domain (53-363 residues) and
the dihedral angles of three residues (36, 37, and 38) changed (29).
The dihedral angles of these residues in the averaged structures were
similar to those of wild type AspAT in the closed conformation, except
that Gly36 in subunit A of the averaged AspAT adopted a
dihedral angle similar to that of the open conformation. These results
suggest that even wild type AspAT in its closed conformation complexed
with a non-dicarboxylate substrate (hydrophobic substrate) was
sufficiently stable to catalyze the transamination reaction. Two Pro
residues (138 and 195) maintained their cis-conformations during
simulation, and these were the same as the conformations observed
in the crystallographic structures.

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Fig. 7.
Stereo drawings of the backbone
structures. The averaged structure of AroAT (magenta),
crystallographic structure of the open form of wild type AspAT
(yellow), and crystallographic structure of the closed form
of wild type AspAT (cyan) are superimposed on the averaged
structure of AspAT (white).
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The structures of the substrate binding sites of AspAT and AroAT were
fairly similar with the main differences around the Arg292
residue. In subunit A of AspAT, the side chain of Arg292
had turned toward the interior of the active site and its guanidino group had formed hydrogen bonds with two water molecules, a position like that of the
-carboxyl group of the natural substrate for AspAT
(Fig. 8a). The side chains of
Arg292 in the B subunit of AspAT (Fig. 8b) and
both AroAT subunits (Fig. 8, c and d) were at the
edge of the binding site, where all four Arg292 residues
existed when simulation was started. The movement of the
Arg292 side chain from the edge to the interior of subunit
A occurred 4-7 ps after starting the MD simulation. At the beginning
of simulation, the total energy of the system was higher than that
during the equilibrium phase, and such high energy would enable
Arg292 to change its orientation. However, the inner
orientation of Arg292 was maintained during the following
simulation period, suggesting that this orientation was relatively
stable in AspAT. The Arg292 side chain of AroAT formed a
hydrogen bond with one water molecule and a salt bridge with the
-carboxyl group of Glu141 in a side-by-side geometric
fashion. Furthermore, this water molecule was fixed by the side chain
of Asp15. We consider these interactions were the major
forces that kept the Arg292 side chain at the edge of the
substrate binding site.

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Fig. 8.
Structures of the active sites of simulated
AspAT and AroAT. a, AspAT subunit A. b, AspAT
subunit B. c, AroAT subunit A. d, AroAT subunit
B. -Aminoheptanoic acid is shown in orange, water
molecules are shown as cyan spheres, and the others are
colored by their atoms.
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A difference distance matrix (48) has often been used to compare two
structures. As superimposition of two structures is not required, this
analytical technique is more likely to detect subtle differences than
direct comparison of structural images. In order to compare the
substrate binding sites of AspAT and AroAT,
rij and 
rij,
which are the atomic displacement within one structure and difference
between the displacements in two enzymes, respectively, were
calculated. The 
rij values obtained were
normalized by the average displacement values, and the matrix element
was expressed by the following equations.
|
(Eq. 10)
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(Eq. 11)
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rijAsp and
rijAro are the atomic
displacements within the simulated AspAT and AroAT structures,
respectively. The difference distance matrices calculated for subunits
A and B are shown in the upper and lower
triangles, respectively, of Fig. 9.
A red spot indicates that the distance between two atoms was
greater in AroAT than AspAT, and a blue one means it was
shorter in AroAT than AspAT. There were many red and blue colonies in the matrix for subunit A, and they were thought to originate and derive
from the different conformations of the Arg292 side chains
of AspAT and AroAT. In subunit B, where the two side chains of
Arg292 adopted similar conformations, substantial
differences were detected between Asn142 and
Arg292 as red colonies. The average distances between two
residues in AspAT and AroAT were 6.2 and 9.5 Å, respectively. Detailed
examination of the region around Asn142 revealed that the
positions of the backbone structures had changed slightly and these
changes may originate from the difference of residue 149, which is Ser
in AspAT and Gly in AroAT.

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Fig. 9.
Difference distance matrices of atomic
displacement in the active sites of simulated AspAT and AroAT. The
matrix element (Rij) is calculated using the
equation, Rij = ( ijAsp ijAro)/(( ijAsp + ijAro)/2)2. The calculations for
subunits A and B are shown in upper and lower
triangles, respectively, and the color scale is shown at the
bottom.
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Van der Waals contact and dense packing of the substrate binding site
are very important for hydrophobic substrate binding. Fig.
10 shows the numbers of all the atoms
within 3-5 Å of the five carbon atoms of the sC7 side chain. No
differences between the packing densities of AspAT and AroAT were
detected. Both structures had about 13 non-hydrogen and 21 hydrogen
atoms within the 5 Å layer around each carbon atom of their sC7 side
chains, indicating that the densities of their interior were relatively
high, as would be expected for typical globular proteins. These results suggest that both AspAT and AroAT had similar levels of van der Waals
contact between the substrate and protein in their hydrophobic substrate-binding pockets and that the packing density was not a major
determinant of the hydrophobic substrate specificities of these
enzymes.

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Fig. 10.
Numbers of all the atoms within a given
distance from each carbon atom of the -aminoheptanoic acid side
chain. a, AspAT; b, AroAT.
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The mean square fluctuations about the positions of the
C
atoms among the superimposed conformations were
calculated on the basis of the averaged structures (Fig.
11). The averaged values over the whole
subunit were 0.16 and 0.34 Å2 for AspAT and AroAT,
respectively. Generally, the parts between the secondary structures
showed higher than average values. Both large and small domains showed
fluctuations of similar magnitudes, suggesting that both enzymes had
adopted the closed conformation in the 100-ps time range. AroAT showed
substantially greater fluctuations than AspAT, suggesting that AroAT is
more flexible than AspAT, which has been suggested experimentally (3).
Another interesting observation was that some of the dihedral angles of
the sC7 side chain changed during simulation (each sC7 side chain has
three dihedral angles). During a simulation of sC7 with water
molecules, no such changes were observed within 1 ns.

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Fig. 11.
Mean square fluctuations of C
atoms. a, AspAT; b, AroAT. The helical and sheet
regions are also shown. The coordinates from the trajectories during
the last 100 ps were used for this analysis. Differences in position
were calculated after every conformer during this 100-ps interval had
been superimposed on the first structure.
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 |
DISCUSSION |
Substrate Binding Energy--
It is possible to estimate the
effect of one carbon in a substrate side chain by comparing the binding
energies of substrates with different chain lengths. The energy
contribution of one methylene group was about 1.4 kcal·mol
1, and the total contribution of the aliphatic
side chain of sC7 was estimated to be 6 kcal·mol
1. From
the difference between the affinity of a substrate and its cognate
aliphatic acid inhibitor, the contribution of an amino group (Schiff
base) was estimated to be 1 kcal·mol
1. The contribution
of the
-carboxyl group of an amino acid substrate to binding was
estimated by analyzing the Arg386 mutant, in which the
residue recognizing the
-COO
group of the substrate
was replaced by Lys, and found to be 1-2 kcal·mol
1
(49, 50). Therefore, the sum of the energy of sC7 is 8-9 kcal·mol
1, whereas the net binding energy of sC7 was
only 3 kcal·mol
1. The binding energy is decreased by
mixing free energy (2.3 kcal·mol
1). Furthermore,
conformational change upon substrate binding costs energy 2-3
kcal·mol
1, estimated in studies on AspAT (51, 52). The
total energy of substrate binding, 7.3-8.3 kcal·mol
1,
is roughly consistent with the above sum.
Thermodynamic Cycle--
The solvent-transfer free energy is often
used for analyzing hydrophobic interactions, and it accounts for some
of the hydrophobic effect, i.e. the transfer of a substrate
from water to a nonaqueous solvent. In terms of an enzyme-substrate
interaction, not only the substrate, but also the enzyme, loses the
unfavorable contact with the aqueous solvent in the pocket when the
complex is formed. Therefore, the maximal effect is restricted to
2-fold, as much as can be derived from the solvent-transfer free energy
in this model, which indicates that the hydrophobic binding pocket is also filled with aqueous solvent. In this study, the contributions of
one methylene group to 
Gs,

Hs, and
T
Ss in the forward reaction
were calculated to be
1.4,
2.3, and
0.9
kcal·mol
1, respectively (Table IV). This

Gs value is 1.5-fold the transfer free
energy of one methylene group from water to pure hydrocarbon liquid
(
0.9 kcal·mol
1) (53), suggesting that the
interactions between AroAT and the aliphatic substrate side chains we
studied were relatively strong. However, the thermodynamic parameters

Gtrans,

Htrans, and
T
Strans for the transfer of one
methylene group have been estimated to be
1.0,
0.6, and 0.4 kcal·mol
1, respectively (53). From our thermodynamic
cycle data, the thermodynamic parameters for hydrophobic substrate
binding, 
GE-SBind,

HE-SBind, and
T
SE-SBind,
were calculated to be
0.4,
1.7, and
1.3
kcal·mol
1, respectively, when

GEStrans was assumed to
be zero (Scheme 3). The large negative

HESBind value also
suggests strong van der Waals interactions exist in the
substrate-binding pocket of AroAT.

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Scheme 3.
Thermodynamic cycle. a, binding
of substrate to enzyme. b, binding of one methylene group to
enzyme. The dark shaded box indicates a protein, and the
light shaded circle denotes a carbon of a hydrophobic
substrate.
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Morton et al. (10) performed a thorough analysis of
hydrophobic ligand binding in the cavity of T4 lysozyme created by
site-directed mutation. According to them, the entropic cost of one
methylene group in the sC7 substrate for binding is
0.9
kcal·mol
1, if it is assumed that the substrate loses
its translational, rotational and internal degrees of freedom (dihedral
angle). The effect of the latter term of freedom must be viewed with
caution, because Wynn et al. (54) found that the terminal
carbon atoms in the flexible straight side chain of the structure
(unnatural amino acid with hydrocarbon side chain) were disordered in
their x-ray crystallographic study. However, this estimated value
(
0.9 kcal·mol
1) may contribute to our observed value
(
1.2 kcal·mol
1), the rest of which may be accounted
for by fixations of the protein main and side chains. Such fixation
(disorder-to-order transition) may be required for modulating the
affinity and specificity of the enzyme (55).
The energetic contribution of one methylene group in the alkyl chain to
the binding of AroAT is relatively large (
1.4
kcal·mol
1) and quite high in comparison with those
reported in the literature:
0.4 kcal·mol
1 for T4
lysozyme (from the class III compounds in Ref. 10),
0.4
kcal·mol
1 for acyl-coenzyme A-binding protein (11),
0.8 kcal·mol
1 for Streptomyces griseus
proteases (9, 56, 57),
1.0 kcal·mol
1 for
Paracoccus denitrificans AroAT (58), and
1.2
kcal·mol
1 for
-chymotrypsin (56, 59). Even when
compared with those for more closely related enzymes, the values were
0.73 and
1.2 kcal·mol
1 for AspAT and the chimeric
enzyme constructed from AspAT and AroAT, respectively (3), it is quite
high. Although more detailed comparisons are required for accuracy,
AroAT may be one of the enzymes with very hydrophobic substrate-binding
pockets. The above findings lead to the interpretation that the
hydrophobic interactions are more efficient and/or the entropic costs
of such interactions are lower with AroAT than other enzymes. Previous
work has suggested that the energetic cost of conformational
rearrangement of AroAT is relatively small, because this enzyme is more
flexible than AspAT (3). Therefore, the latter interpretation seems
feasible. Interesting phenomena have been reported with
-lytic
protease and chymotrypsin; regions that are conformationally plastic
and distinct from their substrate-binding sites play important roles in
the substrate specificities of these enzymes (60-63). Recent studies
by Rader and Agard (64) revealed that such plasticity is inherent and
related to accommodating substrates, and they called it "dynamic
close packing."
Averaged Structure of AroAT Complexed with sC7 Substrate--
In
this study, the structures of AspAT and AroAT were re-built, using
model building and MD simulation, as their bound forms with sC7
molecules. AroAT showed higher activities than AspAT toward not only
aromatic substrates, but also aliphatic substrates. The activities of
these two enzymes per hydrophobic unit of the substrate side chain
differed about 2-fold. Therefore, their activities toward the sC7
substrate differed about 10,000-fold. Furthermore, it has been
suggested that there is no striking steric hindrance, not even in the
substrate binding site of AspAT, of sC7. For these reasons, we chose
sC7 as the model substrate for the study to investigate the differences
between the hydrophobic substrate specificities of AspAT and AroAT.
The orientation of the Arg292 side chain has been one of
the main topics of the structural analysis carried out during the last two decades, because it is a most important and direct determinant of
the recognition of dicarboxylic substrates (Table
VI). In the presence of a dicarboxylic
substrate, the guanidino group of Arg292 forms a salt
bridge with the
-carboxylic group of the dicarboxylate. When a
substrate is not present, the side chain of Arg292 is
generally oriented toward the outside of the substrate-binding pocket.
Asp15 assists the orientations of Arg292 in
both the open and closed forms of E. coli AspAT (29) and mitochondrial AspAT (mAspAT). However, cytosolic AspAT (cAspAT) has no
aspartate residue in this position, it has been replaced by valine.
Instead of Asp15, Glu141 interacts with
Arg292 in the closed, but not the open, form of cAspAT
(30). In the MD structure of AspAT, one of two Arg292
residues, which belong to each subunit, was directed toward the inside
(Fig. 8a) and the other toward the outside of the
substrate-binding pocket (Fig. 8b). Two conformations of
Arg292 have been observed in the same subunit (occupancy is
0.5 for each conformer) of porcine cAspAT in the absence of a substrate analog (30). Arg292 has also been observed to direct its
side chain toward the inner of the substrate-binding pocket of the PMP
form of E. coli AspAT in the substrate-free state (46).
These observations suggest that the outside-facing position of the
Arg292 side chain in AspAT is not quite stable.
The hexamutant of E. coli AspAT (V39L, K41Y, T47I, N69L,
T109S, and N297S), which was constructed by Onuffer and Kirsch (65), is
the nearest mutant to AroAT of those constructed so far. This hexamutant shows about 1,000 times higher activity than its parent AspAT toward aromatic substrates. Interestingly, the hexamutant adopts
the closed conformation and its Arg292 side chain faces the
inside, even in the absence a substrate (36), which indicates that the
orientation of the Arg292 side chain is not always coupled
to the open-closed conformational change. Seville et al.
(34) proposed that, when a hydrophobic substrate is bound,
Arg292 in AroAT is directed toward the outer side of the
substrate-binding pocket in order to make room for large hydrophobic
substrates, as has been observed in the complex structures formed
between the AspAT hexamutant and aromatic substrate analogs. AroAT
possesses both Asp15 and Glu141 residues, as
discussed above, and there is interest in exactly how these residues
interact with the Arg292 side chain in AroAT.
The MD structure of AroAT described above was relatively stable during
380-ps simulation at 300 K and the Arg292 side chain
interacted with Asp15, Glu141 and a water
molecule (some interactions are mediated by water molecules). These
results suggest that the outward-facing position of the
Arg292 side chain was the preferred state to a greater
extent in AroAT than AspAT, which would make the hydrophobic substrate
bound state of the former more stable. The major difference is the
presence of Glu141 in AroAT and Pro at the same position in
AspAT. Previous research showed that Glu141 only has slight
effect on the enzymatic activity of cAspAT (66). However, the
importance of Glu141 in AroAT is still unclear, because
Glu141 of AroAT can interact with Arg292
outside the substrate-binding pocket, whereas that of cAspAT may be not
be able to do so. Therefore, if Glu141 really is important
for the activity of AroAT, the positions of Arg292 and
Glu141 in the substrate-free state will attract
considerable interest.
Enzyme Flexibility--
In the enzyme-substrate complex, the
hydrophobic interaction is entirely due to van der Waals interactions.
Two mechanisms are thought to strengthen a hydrophobic interaction. One
is to construct a substrate binding site from the hydrophobic residues, a notable example being retinal-binding protein (67), and the other is
to increase the density of the substrate-binding pocket packing. In the
cases of AspAT and AroAT, the former does not explain the different
hydrophobic substrate specificities of the two enzymes, because their
residues that interact directly with the substrate are almost fully
conserved. The latter mechanism was explored using homology modeling
and MD simulation. The results suggest that the substrate binding sites
of both enzymes are densely packed and this is supported by the large
enthalpy effects on substrate binding to AroAT we observed. Therefore,
the latter mechanism is not directly responsible for hydrophobic
substrate specificity either. However, an interesting observation was
that the fluctuations of the main chain C
atoms of AroAT
were larger than those of AspAT and large fluctuations would be
expected to facilitate subtle rearrangements of the substrate-binding
pocket (3). Therefore, the energetic cost of dense packing in AroAT may
be lower than that in AspAT. Furthermore, large fluctuations of the
entire AroAT molecule, or a large part of it, may enable rearrangement,
not only in the vicinity of the pocket, but also of the majority of
AroAT. Such global rearrangement may be a particularly effective
strategy for reducing the steric hindrance and/or distortion of the
molecular conformation induced by substrate binding.
We thank the Research Center for Protein
Engineering, Institute for Protein Research, Osaka University, for
performing the computer calculations.