From the Department of Biochemistry, Osaka
Medical College, Takatsuki 569-8686 and § Department of
Chemistry, Faculty of Science, Osaka City University, Sumiyoshi-ku,
Osaka 558-8585, Japan
Received for publication, September 9, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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Aspartate aminotransferase
has been known to undergo a significant conformational change, in which
the small domain approaches the large domain, and the residues at the
entrance of the active site pack together, on binding of substrates.
Accompanying this conformational change is a two-unit increase in the
pKa of the pyridoxal
5'-phosphate-Lys258 aldimine, which has been
proposed to enhance catalysis. To elucidate how the conformational
change is coupled to the shift in the aldimine pKa
and how these changes are involved in catalysis, we analyzed
structurally and kinetically an enzyme in which Val39
located at both the domain interface and the entrance of the active
site was replaced with a bulkier residue, Phe. The V39F mutant
enzyme showed a more open conformation, and the aldimine pKa was lowered by 0.7 unit compared with the
wild-type enzyme. When Asn194 had been replaced by Ala in
advance, the V39F mutation did not decrease the aldimine
pKa, showing that the domain rotation controls the
aldimine pKa via the
Arg386-Asn194-pyridoxal 5'-phosphate linkage
system. The maleate-bound V39F enzyme showed the aldimine
pKa 0.9 unit lower than that of the maleate-bound
wild-type enzyme. However, the positions of maleate,
Asn194, and Arg386 were superimposable between
the mutant and the wild-type enzymes; therefore, the domain rotation
was not the cause of the lowered aldimine pKa
value. The maleate-bound V39F enzyme showed an altered side-chain
packing pattern in the 37-39 region, and the lack of repulsion between
Gly38 carbonyl O and Tyr225
O Aspartate aminotransferase (aspartate, 2-oxoglutarate
aminotransferase, EC 2.6.1.1;
AspAT)1 is a pyridoxal
5'-phosphate (PLP)-dependent enzyme and catalyzes the
reversible transfer of the amino group of aspartate to 2-oxoglutarate by the following Ping Pong Bi Bi mechanism (1), in which E·PLP and
E·PMP denote the PLP form (see Equation 1) and the pyridoxamine 5'-phosphate (PMP) form (see Equation 2) of the enzyme,
respectively.
seemed to be the cause of the reduced
pKa value. Kinetic analysis suggested that the
repulsion increases the free energy level of the Michaelis complex and
promotes the catalytic reaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Eq. 1)
The homodimeric structures have been solved for cytosolic
(2), mitochondrial (3-5), and Escherichia coli (6, 7) enzymes. Each subunit is composed of large and small domains, and the
active site containing the PLP-Lys258 aldimine is located
between the two domains. The
(Eq. 2)
-carboxylate group of the
dicarboxylic substrates binds to Arg386 located at the
small domain and the
-carboxylate group to
Arg292* located at the large domain. An asterisk
indicates that the residue comes from the neighboring subunit. On the
basis of structural, steady-state (1), and transient (8, 9) kinetic
studies, the reaction mechanism of AspAT has been proposed (10, 11) and
refined (9) (see Scheme I).
The PLP-Lys258 aldimine (internal aldimine) of the
unliganded AspAT has an imine pKa value of ~6.5,
which is strikingly lower than those of the PLP-amine aldimines with
protonated pyridine N (~10; see Ref 13). We have shown that the
principle factor that decreases the aldimine pKa
value of AspAT is the imine-pyridine torsion of the
PLP-Lys258 aldimine (as represented by the torsion angle
C3-C4-C4'-N, expressed as
, in the
panel of Scheme I), rather than the historically accepted
electrostatic effect of the neighboring positive charges of
Arg292* and Arg386. The torsion of the
aldimine, the angle of which spans the range from ~35° (protonated
aldimine) (2) to ~90° (unprotonated aldimine) (7), causes a
decrease of 3 units in the aldimine pKa (14),
whereas the positive charges of the arginine residues decreases the
pKa by only 0.7 unit (15). The catalytic significance of the aldimine torsion is considered to be that it
increases the energy level of the protonated form of the aldimine in
the unliganded enzyme, thereby decreasing the free energy gap between
the starting state (the unliganded enzyme plus the free substrate) and
the transition state leading to the first irreversible step,
i.e. the release of the product oxaloacetate (see Scheme I).
This decrease in the free energy gap is estimated to be 16 kJ
mol
1 and increases the
kcat/Km value for amino acid
substrates by 103-fold (14).
The intrinsic pKa of the external aldimine,
i.e. the aldimine of PLP and the substrate aspartate, has
been estimated to be >11 (14). The elevated pKa is
considered to be caused by the aldimine being fixed to a near
planar conformation ( =
25°) (7) and the deprotonated
form being destabilized (14). Accordingly, the external aldimine
reduces the fraction of the dead end species
H+EL=S. The shift from
H+EL=S to ELH+=S
is favorable for the progress of the 1,3-prototropic shift (ELH+=S
K in Scheme I), the first
rate-determining step of the entire catalytic reaction (16).
In comparison to the case of the external aldimine, less is understood
about the mechanism of controlling the aldimine pKa and its role in catalysis in the Michaelis complex, where PLP still
forms the aldimine with Lys258 (6). The intrinsic
pKa of the aldimine has been estimated to be 8.8, by
spectroscopic analysis of the maleate-bound enzyme, and it has been
explained that the increase in the pKa is because of
the electrostatic effect of the anionic ligand (17, 18). However,
taking into account the relatively weak electrostatic effect (0.7 pH
unit) of Arg292* and Arg386 on the aldimine
pKa value as described above, we must consider other
mechanisms for the upward pKa shift in this complex,
such as alterations in the torsion angle of the aldimine and the
hydrogen bond network surrounding it. Furthermore, although the
increase in the aldimine pKa in the Michaelis complex has been claimed to accelerate the transaldimination step (ELH+·S ELH+=S in
Scheme I) by shifting the equilibrium from
EL·SH+ to ELH+·S in
the Michaelis complex (10), its exact role in catalysis is obscure,
because the transaldimination step is not rate-determining at all in
the catalytic reaction (16).
To solve these problems associated with the Michaelis complex, we
set out to carry out structural and mechanical analysis of the complex.
Upon formation of the Michaelis complex, AspAT undergoes a
conformational change (2-7), in which the small domain approaches the
large domain. Together with the domain rotation, the side chains of the
residues aligned at the entrance of the active site are reorganized and
correctly packed to form a lid over the active site. These
changes are expected to cause alterations in the conformation of the
aldimine and the hydrogen-bonding pattern around the aldimine in the
Michaelis complex. Therefore, we have chosen the mutation of
Val39 (19), which exists at the interface of the large and
small domains, to a more bulky residue Phe, with the anticipation of hampering the domain rotation and side-chain packing. This mutant enzyme was analyzed together with the wild-type enzyme, to explore the
effect of the substrate-induced conformational change on the important
catalytic groups.
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EXPERIMENTAL PROCEDURES |
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Materials-- The medium used for bacterial growth contained 0.5% yeast extract, 1% peptone, and 0.5% NaCl, pH 7.0. All other chemicals were of the highest grade commercially available.
Site-directed Mutagenesis-- The V39F (19) and N194A (18) mutants of pUC19 containing the E. coli AspAT gene (aspC) were prepared as described. The double mutant V39F/N194A was constructed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The V39F plasmid was used as a template. The primers for mutagenesis used were 5'-CAT GGC TGC TGC CAT GCC CCA ACC GGT ATC-3' and 5'-GAT ACC GGT TGG GGC ATG GCA GCA GCC ATG-3'. The obtained mutant plasmids were expressed as described previously (20).
Spectroscopic Measurements--
Absorption spectra were
measured using a Hitachi U-3300 spectrophotometer (Tokyo, Japan) at 298 K. The buffer solution contained 50 mM buffer component(s)
and 0.1 M KCl. The buffer components used were MES-NaOH,
HEPES-NaOH, and TAPS-NaOH. The concentration of the AspAT subunit in
solution was determined spectrophotometrically using the apparent molar
extinction coefficient of app = 4.7 × 104 M
1cm
1 for the
PLP form of the enzyme at 280 nm (20). The
pKa value of the aldimine N of the
PLP-Lys258 Schiff base was calculated by fitting the data
as follows in Equation 3.
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(Eq. 3) |
Crystallization of the Mutant Enzymes--
Crystallization
of V39F AspAT was performed by the hanging drop vapor diffusion method.
A 5-µl drop containing 40 mg of protein/ml, 10 mM
potassium phosphate, pH 7.0, 10 µM PLP, and 0.3 mM NaN3 was added to 5 µl reservoir solution
containing 10 mM potassium phosphate, pH 7.0, and 40%
saturated ammonium sulfate and was equilibrated against 400 µl of
reservoir solution at 293 K. After 2 days, the drop was seeded with a
small crystal of the wild-type (WT) AspAT obtained previously. Crystals
with a size (about 0.8 × 0.5 × 0.4 mm) suitable for x-ray
experiments were grown for 7 days. Crystals of V39F/N194A AspAT were
obtained using the same procedure for V39F AspAT except that the
reservoir solution contained 45% saturated ammonium sulfate. Crystals
of maleate-bound V39F AspAT were obtained by a co-crystallization
method, using the same procedure as that for V39F AspAT except that 1 µl of 1 M maleate was mixed with 5 µl of enzyme
solution, and a small crystal of V39F AspAT was used for seeding. The
diffraction data for V39F AspAT were collected on the BL6A station at
the Photon Factory, High Energy Accelerator Research
Organization, Tsukuba, Japan using an x-ray beam of wavelength
1.00 Å at 293 K and Fuji imaging plates with a screenless Weissenberg
camera for macromolecular crystallography (21). Data collection was
performed using two crystals. The diffraction data for V39F/N194A AspAT
and maleate-bounded V39F AspAT were collected with a Rigaku R-AXIS IIc
image plate detector mounted on a Rigaku RU-200 rotating anode
generator operated at 40 kV and 100 mA with monochromatized CuK
radiation at room temperature. Each data collection was performed using
one crystal. All data were processed and scaled using the programs
DENZO and SCALEPACK (22) (see Table I). The diffraction data for V39F and V39F/N194A AspATs showed good isomorphism with those of WT AspAT;
therefore, the coordinates of 1ARS (7) were used as the first model for
refinement. The crystals of maleate-bounded V39F AspAT were isomorphous
with those of WT AspAT and the 2-methylaspartate complex; thus, the
coordinates of 1ART (7) were used as the first model for refinement.
Simulated annealing and several subsequent rounds of least squares
refinement using X-PLOR (23) were carried out. After each round of
refinement, the model obtained was refitted to an electron density map
using program O (24). The mutated residues and solvent molecules were
modeled on the basis of 2Fo
Fc and Fo
Fc electron density maps. The solvent molecules whose thermal factors were above 100.0 Å2 after refinement
were removed from the model. The quality of the model for each
structure was evaluated using PROCHECK (25). Table
I contains a summary of the refinement
statistics. The mean positional error of the atoms from the Luzzati
plot was 0.32 Å for both V39F and WT AspATs.
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RESULTS |
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Aldimine pKa Values--
The absorption spectra of the
V39F mutant AspAT (V39F) show an absorption maximum at 358 nm in the
alkaline pH region and at 430 nm in the acidic pH region (data not
shown), which are identical to those of the WT AspAT and correspond to
the unprotonated (EL; see Scheme
I) and protonated
(ELH+; see Scheme I) structures of the
PLP-Lys258 aldimine, respectively (13, 17). The pH
dependence of the apparent molar absorptivity at 430 nm is shown in
Fig. 1. The aldimine
pKa value of V39F is obtained by fitting the data to
Equation 3 to be 6.1, which is 0.7 unit lower than that of WT (6.8; see
Refs. 9 and 27). Because the aldimine pKa value is
closely related to the presence of the hydrogen bond between PLP O3'
and Asn194 (14, 15), the effect of the V39F mutation was
studied on the N194A mutant AspAT (Fig. 1). The aldimine
pKa value of the V39F/N194A double mutant AspAT is
8.7, essentially identical to that of the N194A mutant AspAT
(pKa = 8.6; see Ref. 18).
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The binding of maleate to WT causes an increase in the 430-nm
absorption band with a concomitant decrease in the 360-nm band, reflecting the increase in the aldimine pKa value
(17). Similar spectral changes are observed for the binding of maleate to V39F (data not shown). The aldimine pKa value of
the maleate-bound V39F (V39F·maleate) is 7.9 (Fig. 1), which is 0.9 unit lower than the value 8.8 (18) of the maleate-bound WT
(WT·maleate). The aldimine pKa values of WT and
mutant AspATs in the presence and absence of maleate are summarized in
Table II, together with the aldimine
torsion angle () obtained from the crystallographic analysis.
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Structure of V39F--
The structure of V39F is shown in Fig.
2A, in comparison with the
structure of WT. Most of the small domain and a part of the large
domain are shown. The large domains of the two enzymes are almost
superimposable. However, the position of the small domain of V39F
deviates from that of WT. The deviations are most pronounced at the
N-terminal part (Met5-Pro48)2
of the small domain and the two helices at the C terminus. The remaining part of the small domain, containing the last part of the
long helix and the following helix, shows little deviation.
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Val39 is one of the residues that locate at the entrance of the active site (6) (Fig. 2A). In V39F, the space between the Tyr70* and Asn69* side chains cannot accommodate the bulky side chain of Phe39, and the phenyl ring is excluded from the space. The phenyl ring has a direct steric effect on the side chain of Ile37 and pushes it outward from the active site. This displacement causes the movement of the main chain of the N-terminal part of the small domain outward of the active site, providing a more open conformation of V39F as described above (Fig. 2A). Together with the rotation of the small domain induced by the V39F mutation, the side chain of Arg386 is moved by 0.63 Å outward from the active site (see Fig. 2B and Fig. 4A). This movement caused a small shift (0.33 Å) in the position of the side chain of Asn194, which is hydrogen-bonded to the guanidinium group of Arg386. The entire structure of the V39F/N194A double mutant AspAT is essentially identical to that of V39F (data not shown; see PDB 1IX8).
Structure of the Maleate-bound V39F--
As in the case of WT,
maleate forms a complex with V39F with its two carboxylate groups bound
to Arg292* and Arg386, causing a significant
rotation of the small domain (Fig. 3). Although the improper packing of the side chains around
Phe39 hampers the complete rotation of the small domain as
observed for WT, the difference in the structure around
Arg386 is not significant between V39F·maleate and
WT·maleate. As a result, the guanidinium groups of Arg386
and Arg292*, maleate, and the side chain of
Asn194 of V39F·maleate are closely superimposable to
those of WT·maleate (see Fig. 3 and Fig.
4B). A great difference
between the two structures is observed in the region of residues 37-39
(Fig. 4B). On maleate binding to WT, Ile37 moves
3.1 Å to cover the active site with a rotation of 120° (Fig. 4,
comparison of A and B). This movement drives the
carbonyl O of Gly38 to approach Tyr225
O, causing the formation of a weak hydrogen bond (3.6 Å)
between the two atoms. In V39F, on the other hand, the presence of the bulky phenyl ring of Phe39 hampers the movement of the side
chain of Ile37 (Fig. 4, comparison of A and
B). The position and conformation of residues
Ile37-Gly38-Phe39 in V39F·maleate
remain like those of the unliganded enzymes.
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Reaction of V39F with 2-Methylaspartate-- To determine the effect of the V39F mutation on the first two steps of the catalytic reaction, the substrate binding and the transaldimination, we analyzed the reaction of V39F with the substrate analogue 2-methylaspartate. The spectral transitions are essentially the same as in the case of WT (14), and the results are analyzed in the same way (data not shown). Briefly, a single exponential process of the spectral change is observed, and extrapolation to t = 0 yielded a spectrum apparently different from that of the unliganded enzyme, indicating the presence of an enzyme-substrate complex rapidly formed from the unliganded enzyme and 2-methylaspartate. The observed single exponential spectral transition is considered to reflect the equilibration process between the rapidly formed species and another species. By combining the results and the chemical properties of 2-methylaspartate, which stops the catalytic reaction at the external aldimine (8), we assigned the two species to be the Michaelis complex (MC) and the external aldimine (EA), as shown in Equation 4 (14).
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(Eq. 4) |
Reaction of V39F with Aspartate and the Kinetic Isotope
Effect--
V39F has 2.5-fold reduced kcat
values and 5-fold increased Km values for its amino
acid substrates, aspartate and glutamate (19). To analyze the effect of
the V39F mutation on the elementary steps, we studied the kinetic
isotope effect of the substitution of -1H
with 2H on the kinetic parameters. The ratio of
kcat for aspartate to kcat for [2-2H]aspartate was 4.6 in V39F, compared with the value 2.0 of WT (20). Essentially no kinetic
isotope effect was observed for Km (ratio is 1.0) in
either enzyme (20) (this study).
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DISCUSSION |
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Protein Conformation and the Aldimine pKa--
The
pKa values of dissociation groups at the active site
of enzymes are generally considered to be regulated mainly by the
polarity of the microenvironment and the electrostatic effects
(including that of hydrogen bonds) of the surrounding residues. In this
study on AspAT, we observed that the conformational change in the
enzyme protein significantly affects the pKa of the
PLP-Lys258 aldimine at the active site. The V39F mutation
causes movement of the small domain outward from the active site (Fig.
2A). Together with this domain rotation, Arg386,
the small domain residue located near the PLP-Lys258
aldimine, moves away from the aldimine. Contrary to the expectation that the electrostatic effect of the positive charges of
Arg386 on the aldimine would be attenuated in V39F, the
aldimine pKa of V39F is 0.7 unit lower than that of
WT (see Fig. 1 and Table II). If Asn194, which bridges PLP
O3' and Arg386, is removed in advance, the V39F mutation
does not decrease the aldimine pKa (see Fig. 1 and
Table II), although V39F/N194A shows an essentially identical
conformation as V39F (comparison of 1IX6 and 1IX8). These results
indicate that the effect of the displacement of Arg386 on
the V39F mutation is transmitted through Asn194 to the
PLP-Lys258 aldimine and affects its pKa.
A possible mechanism for the pKa shift is
that the torsion angle of
PLP-Lys258, which is the main factor controlling the
aldimine pKa (14), is altered. However, because the
resolution of V39F AspAT is not high (2.2 Å), the change in
is not assessed structurally. The modeled structure of
V39F AspAT (1IX6) has a
value only 10° larger than
that of WT AspAT (1ARS); the difference is not significant. However, it
should be noted that what determines the internal aldimine
pKa value is the
value of the
protonated PLP aldimine (14). This structure is not obtained under the crystallization conditions used for 1ARS and 1IX6 (pH 7). A model study
indicates that the direction of the movement of the residues Arg386 and Asn194 increases the
value of the protonated internal aldimine, by pulling the O3' atom to
the re face of the
aldimine.3 This is consistent
with the decreased pKa value of the aldimine.
Maleate Binding and the Conformational Change--
The above
discussion shows the importance of the
PLP-Asn194-Arg386 linkage system that controls
the aldimine pKa. The structure of V39F·maleate
shows that Arg292*, Arg386, Asn194,
and maleate are almost superimposable on those of WT·maleate. Therefore, the contribution of the
PLP-Asn194-Arg386 system to the control of the
aldimine pKa is the same between V39F·maleate and
WT·maleate. Accordingly, the 0.9-unit decrease in the
pKa of V39F·maleate should be ascribed to
alterations in the other part of the structure. The most remarkable change is seen for residues 37-39. In V39F·maleate, because of the
steric hindrance of the Phe39 side chain, Gly38
carbonyl O does not approach Tyr225 O (Fig.
4B). Assuming that this change between V39F·maleate and
WT·maleate is the cause of the lowered aldimine
pKa value of V39F·maleate, we can interpret the
results as follows. In the unprotonated aldimine, Tyr225
O
forms a hydrogen bond with PLP O3' (2.90 Å for 1ARS
and 2.86 Å for 1IX6), in which Tyr225 is the hydrogen
donor. The lone pair electrons of Tyr225 O
are pointed toward the Gly38 main chain. On the other hand,
if the aldimine is protonated, the proton is expected to be shared by
PLP O3', imine N, and Tyr225 O
, and the
hydrogen atom of the Tyr225 OH is pointed toward the
Gly38 main chain. Therefore, if the Gly38 main
chain approaches Tyr225 on the binding of maleate, the lone
pair electrons of Gly38 carbonyl O cause repulsion with
Tyr225 O
in the unprotonated aldimine
(EL·SH+; see Fig.
5A), whereas a hydrogen bond
is formed between Tyr225 O
and
Gly38 carbonyl O in the protonated aldimine
(ELH+·S; see Fig. 5A). In
ELH+·S, the hydrogen bond lifts the
Tyr225 side chain and allows rotation of the PLP pyridine
ring, reducing the aldimine torsion angle
and thereby
stabilizing the structure. As a consequence, the approach of
Gly38 to Tyr225 on binding of maleate to WT is
considered to destabilize EL·SH+ relative to
ELH+·S and increases the aldimine
pKa in the Michaelis complex. The binding
of maleate to V39F will not increase the aldimine pKa as much, because the movement of
Gly38 main chain on the maleate binding is
restricted.
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Effect of V39F Mutation on the Elementary Steps-- For the analysis of the reaction of AspAT with aspartate at high pH, we consider the information shown in Equation 5 below (9), where MC, EA, and K are the Michaelis complex of the PLP form of AspAT and aspartate, the external aldimine, and the ketimine, respectively (for the structures, see Scheme I).
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(Eq. 5) |
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(Eq. 6) |
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(Eq. 7) |
Energetic Interpretation of the Acid-Base Chemistry and Kinetic Parameters-- The results of the acid-base chemistry and kinetics of WT and V39F are interpreted in the three-dimensional free energy profile shown in Fig. 5B. In this free energy profile, the energy levels of all the reaction intermediates are arranged two-dimensionally; one coordinate displays the catalytic step, and the other shows the chemical species with different protonation states. The advantage of this expression is that we can understand energetically how the pKa of a dissociation group is involved in catalysis (14). For comparison of the WT- and V39F-catalyzed reactions, the energy level of the transition state is aligned in Fig. 5B.
In the Michaelis complex of WT, EL·SH+ and
ELH+·S have similar energy levels (14). On
the V39F mutation, the free energy level of
EL·SH+ is decreased by 6.9 kJ·mol1 whereas that of
ELH+·S is decreased by only 1.7 kJ·mol
1. The energy level of the external aldimine is
unchanged. The obtained free energy diagram can explain 1) the 0.9-unit
decrease in the aldimine pKa in the Michaelis
complex, 2) the 9-fold equilibrium shift from the external aldimine to
the Michaelis complex, and 3) the increase in the free energy gap
between the ground state (the rapid-equilibrium mixture of the
Michaelis complex and the external aldimine) and the transition state
of the 1,3-prototropic shift, which results in the 7-fold decrease in
k
is distant from the carbonyl O of
Gly38 (2, 7) (1ART and 1AJS). We consider that the lack of
the Gly38-Tyr225 interaction in the external
aldimine is the reason why the V39F mutation does not affect the energy
levels of the external aldimine species.
Strain in the Michaelis Complex--
Structural and kinetic
analyses of the V39F mutant AspAT described as above provide important
findings as follows. First, the conformation of AspAT, i.e.
the position of the small domain relative to that of the large domain,
affects the aldimine pKa value, and the effect of
the conformational change on the aldimine is mediated through the
PLP-Asn194-Arg386 linkage system. Second,
despite the incorporation of the bulky Phe side chain to the domain
interface, which causes outward rotation of the small domain, the
position and conformation of the bound maleate and the residues that
interact with it, i.e. Arg292*,
Asn194, and Arg386, are the same as those in
the WT·maleate, showing the flexibility of the small domain.
Therefore, the lowered aldimine pKa of
V39F·maleate as compared with that of WT·maleate is not because of
the outward domain rotation but with the reduction in the
Gly38-Tyr225 interaction provided by the
ligand-induced packing of the residues at the active site. Together
with the kinetic analysis of V39F, it was proposed that the
Gly38-Tyr225 interaction destabilizes the
unprotonated aldimine species in the Michaelis complex (i.e.
ground-state destabilization), and contributes to the increase in
kcat. The role of the substrate-induced strain
shown in this study is in contrast to that of the torsional strain of
the PLP-Lys258 aldimine in the unliganded enzyme, which
reduces the energy gap between the starting state (unliganded enzyme
plus unbound substrate) and the transition state
ES by elevating the free energy level of the
starting state and contributes to the increase in
kcat/Km (14).
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FOOTNOTES |
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* This work was supported in part by Scientific Research on Priority Areas 13125101 (to H. H. and K. H.) and by Grant-in-aid for Scientific Research 13680697 (to H. H.) from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1IX6, 1IX7, and 1IX8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be sent: Dept. of Biochemistry, Osaka Medical College, 2-7 Daigakumachi, Takatsuki 569-8686, Japan. Tel: 81-72-683-1221 (ext. 2645); Fax: 81-72-684-6516; E-mail: med001@ art.osaka-med.ac.jp.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M209235200
2 The amino acid residues are numbered according to the sequence of pig cytosolic aspartate aminotransferase (12). According to this numbering system, the N-terminal residue is Met-5.
3 H. Hayashi and H. Kagamiyama, unpublished results.
4 H. Hayashi and H. Kagamiyama, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: AspAT, aspartate aminotransferase (aspartate, 2-oxoglutarate aminotransferase, EC 2.6.1.1); PLP, pyridoxal 5'-phosphate; PMP, pyridoxamine 5'-phosphate; V39F AspAT, mutant AspAT in which the residue Val39 has been replaced with a phenylalanine residue (other mutant AspATs are expressed in the same way); WT, wild-type; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid.
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