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J. Biol. Chem., Vol. 275, Issue 30, 23154-23160, July 28, 2000
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From the
Received for publication, March 9, 2000, and in revised form, April 14, 2000
Phosphoglucose isomerase catalyzes the reversible
isomerization of glucose 6-phosphate to fructose 6-phosphate. In
addition, phosphoglucose isomerase has been shown to have functions
equivalent to neuroleukin, autocrine motility factor, and maturation
factor. Here we present the crystal structures of phosphoglucose
isomerase complexed with 5-phospho-D-arabinonate and
N-bromoacetylethanolamine phosphate at 2.5- and 2.3-Å
resolution, respectively. The inhibitors bind to a region within the
domains' interface and interact with a histidine residue
(His306) from the other subunit. We also demonstrated that
the inhibitors not only affect the enzymatic activity of phosphoglucose
isomerase, but can also inhibit the autocrine motility factor-induced
cell motility of CT-26 mouse colon tumor cells. These results indicate that the substrate and the receptor binding sites of phosphoglucose isomerase and autocrine motility factor are located within close proximity to each other. Based on these two complex structures, together with biological and biochemical results, we propose a possible
isomerization mechanism for phosphoglucose isomerase.
Phosphoglucose isomerase
(PGI)1 (EC 5.3.1.9), a
glycolytic enzyme, is an essential enzyme in all tissues. It
interconverts glucose 6-phosphate and fructose 6-phosphate, hence plays
a central role in both the glycolysis and the gluconeogenesis pathways. PGI deficiency in humans is an autosomal recessive genetic disorder that has the typical manifestation of nonspherocytic hemolytic anemia
of variable clinical expression (1, 2). The serum activity of human PGI
serves as a tumor marker in cancer patients (3, 4) and elevation in PGI
activity is closely correlated with metastasis (5, 6). In addition to
its essential role in carbohydrate metabolism in the cytoplasm, PGI
also functions as neuroleukin (NLK) (7-9), autocrine motility factor
(AMF) (10, 11), and maturation factor (MF) (12).
NLK is a neurotrophic growth factor that promotes the survival of
spinal and sensory neurons (13). Interestingly, PGI and NLK have
previously been reported to have differential induction capabilities
for certain neurons (9). AMF, a new class of cytokines, can stimulate
cell migration in vitro and metastasis in vivo
(10, 14-16). Amino acid sequencing and immunological cross-reactivity experiments suggest that mouse AMF is identical or closely related to
PGI/NLK (10). MF is capable of mediating the differentiation of human
myeloid leukemic HL-60 cells to terminal monocytic cells, and
apparently a high degree of homology exists between the MF of myeloid
leukemia cells and PGI or NLK (12).
It was previously suggested that PGI may recognize a sugar-containing
molecule(s) at the cell surface (7) and that NLK binds to the cell
surface in a carbohydrate-dependent manner utilizing a
PGI-like structure. Watanabe (10, 16) also pointed out that AMF
(PGI/NLK/MF) may contain a PGI-like structure and initiates signal
transduction by interacting with the carbohydrate side chains of the
extracellular domain of the AMF receptor (AMFR). Carbohydrate
phosphates can act as AMF inhibitors probably by binding and blocking
the receptor recognition site on AMF. These novel functions of PGI may
depend directly on the catabolism of phosphosugar, making an
understanding of the molecular catalytic mechanism of the enzyme
potentially significant.
We previously reported the substrate-free structure of PGI/AMF/NLK and
confirmed that PGI can function as AMF and NLK (17). Nonetheless, the
nature of the substrate-active site or the receptor binding site of
PGI/AMF/NLK remains poorly understood. In this report, we tested the
inhibitory effect of two carbohydrates containing phosphate,
5-phospho-D-arabinonate (5PA), and
N-bromoacetylethanolamine phosphate (BAP), on the PGI
enzymatic activity and the AMF-induced cell motility. 5PA, the
five-carbon homologue of 6-phosphogluconate, is the equivalent analogue
of the cis-1,2-enediolate intermediate in the reaction of
phosphoglucose isomerase (18), and BAP is characterized as an
active-site directed inhibitor to mammalian phosphoglucose isomerase in
an effort to identify active-site residues (19). We then presented the
crystal structures of PGI from Bacillus stearothermophilus
complexed with 5PA and BAP at 2.5- and 2.3-Å resolution, respectively.
Preparation of the Wild Type and N-Bromoacetylethanolamine
Phosphate-linked Phosphoglucose Isomerase--
The isolation and
purification of PGI have been previously reported (20). The complete
inactivation of PGI was achieved by incubating the enzyme (0.6 mg/ml)
at 30 °C overnight with 4 mM
N-bromoacetylethanolamine phosphate (BrAcNHEtOP) in a
phosphate buffer (20 mM, pH 7.0). BrAcNHEtOP was
synthesized according to the protocol described by Hartman et
al. (21). Free inhibitors were removed by passing the reaction
mixture through a desalting column.
Cell Motility Assay--
The cell motility assay of mouse CT-26
cells was measured by the method of Lin et al. (22) with
minor modifications. Polyvinylpyrrolidone-free polycarbonate filters
(Nuclepore, 8-µm pore size) were soaked in 0.5 M acetic
acid overnight then washed with distilled water and coated with 50 µl
of Matrigel for 2 h. Cells were placed on the top part of the
Boyden chamber at a cell dose of 5 × 104 per 200 µl
in Dulbecco's modified Eagle's medium (DMEM) with 10%
phosphate-buffered saline. The bottom part of the chamber was filled
with or without PGI in DMEM for positive and control experiments. For
the BAP inhibition assay, BAP-linked PGI was used instead of PGI in the
bottom chamber. For the 5PA inhibition assay, the PGI and 5PA were
preincubated for 2 h before filling the bottom chamber together
with DMEM. Incubation was carried out at 37 °C for 16 h. The
filters were removed and fixed in 4% paraformaldehyde for 15 min at
room temperature. Cells on the upper filter surface were removed with a
cotton swab. The filters were stained with hematoxylin for 40 min, and
the cells on the lower filter surface were counted under a light microscope.
Enzymatic Assay--
The phosphoglucose isomerase activity was
determined at 30 °C by the coupled glucose-6-phosphate dehydrogenase
method (23). The standard assay mixture (1 ml) contains 20 mM potassium phosphate (pH 7.0), 10 units of
glucose-6-phosphate dehydrogenase, 2 mM Crystallization and Data Collection--
The PGI·BAP crystals
were grown at room temperature by hanging-drop vapor diffusion of a
2-µl protein solution (15 mg/ml in 50 mM potassium
phosphate buffer, pH 7.0) mixed with 2-µl reservoir solution
containing 0.8 M potassium sodium tartrate in 0.1 M HEPES buffer (pH 7.5). The PGI·BAP crystals belong to
space group I222 with cell dimensions of a = 74.94 Å, b = 93.64 Å, and c = 171.99 Å and a diffraction to a 2.3-Å
resolution, with one molecule per asymmetric unit. For the PGI·5PA
complex, crystals were also grown by the hanging-drop vapor diffusion
method at room temperature from 2 µl of protein solution (15 mg/ml
containing 0.05 mM 5PA in 50 mM phosphate, pH
7.0) mixed with 2 µl of reservoir solution containing 0.4 M ammonium dihydrogen phosphate. Crystals measuring 0.3 x 0.56 x 0.1 mm grew in about 4 days and had a diffraction resolution of 2.5 Å. The PGI·5PA crystal also belongs to space group
I222 with cell dimensions of a = 74.80 Å, b = 94.71 Å, and c = 171.74 Å and contains one molecule per asymmetric unit.
All data sets were taken at room temperature on a Rigaku R-AXIS II
imaging plate system using double-mirror-focused CuK Structure Determination and Refinement--
The AMoRe program
package (25) was used in the rotation and translation search. The
previous PGI crystal structure (17) was used as the search model
(Protein Data Bank (PDB) accession code: 2PGI). The search model was
placed in a P1 cell with a = b = c = 90 Å and
Structure refinement and model building were carried out using the
XPLOR (26) and XTALVIEW programs (27). The rigid body refinement was
used first, and the model was treated as one group to optimize the
orientational and translational parameters. Simulated annealing omit
maps were then used to reduce the model bias. A bulk solvent mask was
calculated to improve the reflection data. The final models contain
3661 non-hydrogen atoms, including 134 oxygen atoms from water
molecules for PGI·BAP (PDB accession code: 1C7Q); and 3,539 non-hydrogen atoms, including 94 oxygen atoms from water molecules for
PGI·5PA (PDB accession code: 1C7R). The first and the last two amino
acids are presumably disordered in both crystal structures and residues
96 to 105 of PGI·5PA were not visible in the electron density map.
The final refinement statistics are summarized in Table I. The
2|Fo| Cell Motility and Enzyme Inactivation--
Previously, we
demonstrated by cell migratory stimulation assay that PGI from B. stearothermophilus exhibits AMF cell motility activity (17). To
directly address the question whether the isomerase and the cell
motility functions of the protein share the same substrate binding
site, we tested the effect of two carbohydrates containing phosphates,
5PA and BAP, on these functions. The PGI activities were monitored by
the rate of isomerizing fructose 6-phosphate (28). In the presence of
0.1 mM 5PA, the isomerase activity of the enzyme was
decreased by 86%. PGI that has been conjugated with BAP was
essentially inactive. A similar result with the BAP-linked PGI has been
reported by Gibson et al. (19).
Results from the motility assay and the stimulatory effect on CT-26
cells in the absence (Fig. 2A)
or presence (Fig. 2B) of PGI are presented. In the presence
of 5PA (C) and BAP (D), the inhibitory effect on
cell motility can be readily observed. The current data indicate that
5PA and BAP not only inhibit PGI enzymatic activity but also constrain
the induced cell motility of PGI with no effect on basal migration. The
results confirm that the substrate and the receptor binding site of
PGI/AMF are overlapped.
General Structure--
The orthorhombic crystals of PGI·BAP and
PGI·5PA are isomorphous to the substrate-free PGI (17) crystals. The
folding topology of these two inhibitor complexes is structurally
identical to the substrate-free form as shown in Fig. 3a.
Briefly, the structure is composed of two globular domains (designated
as the large and small domain) and an "arm-like" C-terminal tail.
Each domain has a
Even though there is one subunit in the crystallographic asymmetric
unit, the active form of the enzyme is a dimer. The monomer-monomer associate in an arm-to-arm hug fashion with intimate contacts and form
a hydrophilic channel that coincides with the crystallographic 2-fold
axis, which runs through the dimer as indicated by the arrow
in Fig. 3b. The
solvent-accessible surface (29) of the subunit is 19505 Å2. The intermolecular contact area is 10,751 Å2, with a summation of 3192 and 7559 Å2 of
the hydrophilic and hydrophobic surface areas, respectively. Dimer
formation causes a burial of 55% of the monomer surface area.
Fig. 4 shows the structural comparisons
of substrate-free PGI with those of the PGI·5PA and PGI·BAP
complexes. Both inhibitors gave similar, but not identical, results.
Their binding sites lie in the slight cleft located within the large
domain, the small domain, and the C-terminal tail of the monomer as
predicted (Sun et al., 1999). The cleft is close to the
subunit interface, which is formed by the association of the two
subunits. Least-squares superposition (C Inhibitor Binding Site of Transition State Analogue
5PA--
5-Phosphoarabinonate (5PA) is a stable analogue of the
cis-1,2-enediolate intermediate that is believed to occur
transiently in the phosphoglucose isomerase reaction (18). 5PA is also
one of the strongest known competitive inhibitors of PGI. Therefore, it
possibly represents a transition state analogue and the 5PA-bound form
of the protein may resemble a catalytic intermediate or a transition state.
As shown in Fig. 5A, the
specific binding of 5PA involves a network of polar interactions
between 5PA and Ile80, Gly82,
Gly201, Arg202, Gln281,
Glu285, His306, Gln413, and
Lys420. The phosphate group on 5PA is essential for binding
and is stabilized by interacting with residues Gly201,
Arg202, Glu285, and Gln413. At the
other end of 5PA, the O1 carboxylate oxygen of 5PA form hydrogen bonds with the N
In the PGI·5PA complex, a total of 16 hydrogen bonds, 7 from the
large domain, 6 from the small domain, and 2 from the C-terminal region, are observed between the protein and the ligand. Residues participate in 5PA binding are shown in Table
II and include charged, polar, and
nonpolar side chains, as well as atoms from the peptide backbone. It is
also interesting to point out that the His306 positioned in
the binding site is contributed by the other subunit of the dimer. This
positioning may explain why the active form of the isomerase is a
dimer.
Inhibitor Binding Site of BAP--
Even though BAP may not
represent a transition state analogue, it can modify PGI
stoichiometrically, resulting in complete and rapid inactivation of the
enzyme (19). In addition, data from mutagenesis (30) suggest that
His306 is an active-site residue of PGI. The imidazole of
His306 could be the nucleophile that attacks BrAcNHEtOP. As
shown in Fig. 1b, BAP formed a covalent bond with
His306. Therefore, the complex structure of PGI·BAP can
confirm that BAP is indeed as an active site inhibitor and react with
His306.
In contrast to the fairly deep binding location of 5PA (Fig.
5A), BAP is situated near the entrance of the binding pocket (Fig. 5B). The noteworthy difference between 5PA and BAP is
the orientation of the phosphate moieties on these inhibitors. The phosphate group on 5PA is pointed toward the bottom of the pocket. Conversely, the phosphate moiety on BAP swings about 180° and orients
in the opposite direction. As shown in Fig. 5B, the binding pocket of BAP is lined with Ser140, Thr142,
Thr143, Glu145, His306,
Glu417, and Lys420. The His306
residue was previously suggested to act as a general base to initiate
the isomerization reaction (31). As expected, the PGI enzyme is
alkylated by BAP specifically at position N Inhibitor-induced Local Conformational Changes--
Achari
et al. (32, 33) have proposed an occurrence of local
conformational changes upon inhibitor binding. We compared the
structures of the inhibitor-bound complexes with that of the uncomplexed PGI (17) and observed significant conformational changes in
the active site of either PGI·5PA or PGI·BAP. The comparison of the
PGI structures with and without 5PA bound is shown in Fig. 5A. The most significant difference appears at the region
between residues Gly200 and Val205. This
variation may be due partially to the enhanced interaction between
Arg202 and the phosphate group on 5PA. The quanidino group
of Arg202 in the PGI·5PA complex structure moved 3-4 Å toward the inhibitor from its original position in the unbound protein.
Because the phosphate moiety of BAP points to the entrance of the
binding pocket, a local conformational change upon BAP binding is found
at the region between residues Lys139 and
Thr144 as shown in Fig. 5B. Ser140
is the key residue responsible for this structural variation. Upon
inhibitor binding, the side-chain hydroxyl group of Ser140
moves into proximity with BAP and forms two hydrogen bonds with an
oxygen atom of the phosphate group and the backbone oxygen O4.
Proposed Catalytic Mechanism of Phosphoglucose
Isomerase--
Previous results suggest that the availability of the
active site on PGI is required for stimulating the migration of tumor cells. Therefore, the understanding of the molecular catalytic mechanism of the enzyme should be physiologically significant.
It has long been proposed that the catalytic mechanism of
phosphoglucose isomerase should include the following steps: 1) binding
of the cyclic form of the substrate to the enzyme, 2) ring opening of
the substrate, 3) base-catalyzed isomerization via a
cis-endiol intermediate, 4) ring closure of the product, and
5) release of product (31, 34-35). Isotope effect studies suggest that
the isomerization step is rate-limiting (34). The transition state
should have a structure similar to cis-enediolate, because
both 5PA (18) and 5-phosphoarabinohydroxamate (36) behave as transition
state inhibitors. The isomerization activity is
pH-dependent and follows a bell-shaped curve and, together with the temperature dependence of the pKa values,
suggests that histidine and lysine residues participate in the
catalysis (31). Mutagenic studies indicate that one of the conserved
lysine (Lys420 in PGI) and arginine (Arg202 in
PGI) residues play indispensable roles in catalysis (30). Affinity
labeling of the conserved histidine (His306 in PGI) by
BrAcNHEtOP, an active site-directed inhibitor of phosphoglucose isomerase, suggests that the conserved histidine functions as a general
base in the isomerization step (30).
Considering all of the above data from the literature in combination
with the active-site structure of phosphoglucose isomerase unveiled in
the current study (Fig. 5), we propose the following molecular
catalytic mechanism for PGI: (i) The reaction is initiated by binding
the substrate to the enzyme. A network of hydrogen bonds between the
substrate and the active site amino acids stabilizes this binding. It
is worth noting that the amino acids comprising the active site,
especially those directly contacting the substrate, are conserved
throughout the PGI family (17). (ii) Lys420 is involved in
the opening of the phosphoglucopyranose ring by functioning as a
general base. The side-chain amino group of Lys420 is
within 3.0 Å of the C1 and C2 hydroxyl groups of the inhibitor (Fig.
6a) and the importance of
Lys420 in catalysis has been confirmed by mutagenic studies
(30). (iii) His306, acting as a general base, extracts the
proton from C2. Simultaneously, Glu285 acts as a general
acid and donates a proton to the C1 carbonyl oxygen (Fig.
6b). The concerted action of these two residues transforms the substrate to the cis-enediol intermediate and
inaugurates the isomerization step. By reversing their roles in the
subsequent step, the carboxylate of Glu285, acting as the
base, and the imidazolium of His306, acting as the acid,
work on the intermediate to complete the isomerization reaction. The
substrate at the transition state of isomerization presumably has a
structure similar to the cis-enediolate (Fig. 6). Through
electrostatic interaction, Glu145 stabilizes the developing
partial positive charge on the side chain of His306, while
Arg202 stabilizes the partial developing negative charge on
Glu285. Arg202 also contributes to the
interaction of the phosphate group and may lock the substrate in an
optimum position for isomerization. The ammonium of Lys420
provides charged hydrogen bonds to the C1 and/or C2 oxygen atoms of the
cis-enediolate. (iv) After isomerization, Lys420
performs an acid-catalyzed ring closure reaction, and the product is
released from the enzyme.
The crystal structure of rabbit skeletal muscle PGI (rPGI) with a
competitive inhibitor D-gluconate 6-phosphate was published by Jeffery et al. (37) during the preparation of the present manuscript. The topology of rPGI is very similar to that of PGI from
B. stearothermophilus, but the orientation of the
bound D-gluconate 6-phosphate is different from that of 5PA
in the present study. Consequently, the catalytic mechanism proposed by
Jeffery et al. (37) has features that differ from that
proposed in this study. In rPGI, a
His388-Glu216 dyad and Lys518
(corresponding to His306, Glu145, and
Lys420 of the B. stearothermophilus PGI) were
proposed as the general base-acid pair to initiate the isomerization
step. The function of the His-Glu dyad is in close agreement with our
proposed mechanism. However, a function of providing charged hydrogen
bonds to the C1 and C2 oxygen atoms of the transition sate is suggested
for the active-site Lys in the present study.
A conserved Glu (Glu285 in this study) is thought to be the
residue that donates a proton to the substrate in the initial
isomerization step (Fig. 6). The proposed role of Glu285 is
supported by the following reasons: 1) the closeness of the O1 carboxylate oxygen of 5PA and the
O
In summary, we have elucidated the crystal structures of PGI complexed
with the transition state analogue 5-phospho-D-arabinonate (5PA) and N-bromoacetylethanolamine phosphate (BAP) at 2.5- and 2.3-Å resolution, respectively. We also demonstrate that the
inhibitors, 5PA and BAP, not only inhibit the enzymatic activity of PGI
but also inhibit the AMF-induced cell motility of CT-26 mouse colon tumor cells. Thereby, the present study provides the first view that
the locations of the substrate binding site for phosphoglucose isomerase and the receptor binding site for autocrine motility factor
are overlapped. We also propose a possible isomerization mechanism for
PGI.
We thank Dr. Ming F. Tam for comments on the manuscript.
*
This work was supported by the National Science Council
(Grant NSC89-2311-B-001-092 to C. D. H.) and by Academia Sinica,
Republic of China.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.
**
To whom correspondence should be addressed: Institute of Molecular
Biology, Academia Sinica, Taipei, Taiwan, 11529. Tel.: 886-2-2788-2743;
Fax: 886-2-2782-6085; E-mail: mbhsiao@ccvax.sinica.edu.tw.
Published, JBC Papers in Press, April 17, 2000, DOI 10.1074/jbc.M002017200
The abbreviations used are:
PGI, phosphoglucose
isomerase;
NLK, neuroleukin;
AMF, autocrine motility factor;
MF, maturation factor;
5PA, 5-phosphoarabinonate;
BrAcNHEtOP, N-bromoacetylethanolamine phosphate;
DMEM, Dulbecco's
modified Eagle's medium;
rPGI, rabbit phosphoglucose isomerase;
AMFR, AMF receptor.
The Crystal Structure of Phosphoglucose Isomerase/Autocrine
Motility Factor/Neuroleukin Complexed with Its Carbohydrate Phosphate
Inhibitors Suggests Its Substrate/Receptor Recognition*
§,
**, and Graduate Institute of Life Sciences,
National Defense Medical Center, Taipei, Taiwan, Republic of China, the
§ Institute of Molecular Biology, Academia Sinica, Taipei,
Taiwan 11529, Republic of China; the ¶ Institute of Cellular & Molecular Biology,Taipei Medical College, Taipei, Republic of China;
and the Graduate Institute of Agricultural Biotechnology,
National Chung Hsing University, Taichung, Taiwan 40227, Republic of
China
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-NAD+, 4 mM fructose 6-phosphate, and 0.5 µg of PGI or PGI covalently linked with BAP. To assess the inhibitory
effect, 5-phosphoarabinoate was included in the assay mixture to a
final concentration of 0.1 mM.
x-ray radiation generated from a RigakuRU-300 rotating anode operating at 50 kV and 80 mA. Data were indexed, integrated, and scaled using
DENZO and SCALEPACK software packages (24) (Table
I).
Summary of diffraction and refinement data
= = 
= 90°. Data between 8.0 and 4.5 Å and a
Patterson radius of 20 Å were used for all rotation and translation
function calculations. The data sets from PGI·5PA and PGI·BAP both
gave significant solutions. After rigid body refinement, the optimal
solution was determined for PGI·BAP data at Euler angle = 71.32°, = 76.29°, and = 305.05°;
x = 0.482, y = 0.139, and
z = 0.150. When compare with the non-normalized structure factors between search model and target crystal in the definition region, the correlation coefficient and R-factor are 84.0 and 23.5%, respectively. The solution of PGI·5PA was at = 71.71°, = 76.34° and = 305.77°, x = 0.483, y = 0.137, z = 0.151 with a correlation coefficient of 77.6 and an R-factor of 28.3%.
|Fc|-omitted density maps around the
inhibitors, with the final refined model superimposed over them,
are shown in Fig. 1.
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Fig. 1.
The omitted
2|Fo| |Fc|
electron density maps contoured at the 1.0
level (39) of
PGI at the inhibitor binding sites. The maps were calculated
by omitting (a) 5PA and (b) BAP from the x-ray
model.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 2.
The inhibitory effects of carbohydrate
phosphates on motility stimulation of CT-26 mouse colon cancer cells by
purified PGI from B. stearothermophilus. CT-26 cells were
plated in culture medium in the (A) absence or
(B) presence of PGI and in the (C) presence of
BAP-linked PGI and (D) presence of PGI with 5PA.
-sheet core surrounded by -helices that link
the -strands. The active site of substrate/inhibitor binding is
located within the two globular domains, the C-terminal tail, and the
interface between the two subunits. The large domain also contains a
protruding loop region opposite to the C-terminal tail. The structure
can be represented by a prolate ellipsoid with an "arm-like"
structural feature on each side. The dimensions of the monomer are
about 71 x 75 x 33 Å.
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Fig. 3.
a, a ribbon drawing (40, 41) of the PGI
monomer with the small domain on the top, the large domain
on the bottom, and the C-terminal domain on the
left. -Helices are red and labeled
1-18; -strands are in
gold and labeled
1-12. The inhibitors are
cyan and dark blue for 5PA and BAP, respectively.
b, ribbon drawing (40, 41) of the PGI dimer. One molecule is
red and gold while the other molecule is
green and purple. The crystallographic 2-fold
axis runs through the dimer as shown by the arrow.
) of the PGI·BAP and
PGI·5PA complexes with the substrate-free structure gave root mean
square deviations of 0.25 and 0.46 Å, respectively. This small
difference indicates that the overall conformations of these three
structures are very similar, except for local conformational changes in
the loop and substrate binding regions. We will discuss some important
local structural variations later to shed light on the action of the
inhibitor binding.
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Fig. 4.
Superposition of the C
atoms of the substrate-free (black), 5PA complex
(green), and BAP complex (red)
structures.
1 atom of
His306 and the O1 carboxylate
oxygen of Glu285. The oxygens of the hydroxyl groups
(O2 and O4) on 5PA interact with
His306, Lys420, Ile80, and
Gly82 via hydrogen bonds. In addition, the backbone oxygen
(O5) is hydrogen-bonded with N
2
of Gln281. It should be noted that one water molecule is
H-bonded to the O3 oxygen atoms and that the interaction of
5PA and Thr143 is mediated via this water molecule.
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Fig. 5.
Stereo drawings showing the local
conformational change between substrate-free PGI in black
and the inhibitor-bound structures as well as the observed
inhibitor-binding pockets (A), the PGI·5PA complex in
green (B), and the PGI·BAP complex
colored in red. The 5PA and BAP are represented by
spheres and lines.
Hydrogen bonds (
3.5 Å) between inhibitors and PGI
1
of His306 contributed from the other subunit of the dimer.
In addition to a water molecule, the phosphate moiety of BAP
constitutes hydrogen bonds with the hydroxyl group of
Ser140 and Thr143, whereas the side chain of
Lys420 neutralizes the opposite charges and forms one salt
bridge with the phosphate group.
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Fig. 6.
A schematic drawing of the proposed mechanism
for the conversion of glucose 6-phosphate to fructose 6-phosphate as
catalyzed by phosphoglucose isomerase. The mechanism includes the
ring opening (a and b), isomerization
(b through e), and ring closure steps
(e and f). The putative residues involved in
isomerization are shown. TS stands for the
cis-enediol intermediate transition state.
1 carboxylate oxygen of Glu285;
2) the indispensable role of Glu285 in catalysis suggested
by our unpublished mutational data; and 3) the involvement of glutamate
in catalysis of PGI suggested by affinity labeling by
1,2-anhydrohexitol 6-phosphate (38). No glutamate was assigned in the
proposed mechanism of rPGI. In our catalytic model, all three residues
(His306, Glu285, and Lys420) play
significant roles in the isomerization step; mutation of any one of
them would drastically impair the catalytic ability of PGI. The
function of Arg272 in rPGI (corresponding to
Arg202 in this study) was proposed to make the overall
electrostatic potential at the substrate positive and provide
stabilization to the developing negative charge on the enediolate-like
transition state. A similar function is fulfilled by Lys420
by providing charged hydrogen bonds to the transition state in our
model. Upon binding of 5PA, the quanidino group of Arg202
moved toward the carboxylate group of Glu285 by 0.6 Å, and
as a result, the pKa of Glu285 could be
lowered and the acidic role of Glu285 in the isomerization
step could be enhanced. Due to the close contact of Arg202
and the phosphate group of 5PA, we believe that Arg202 also
plays a key role for the recognition of phosphosugar during isomerization. It should be noted that our crystal structure dose not
rule out the possibility of Lys420 being the role of a
general acid during isomerization as suggested by the previous rPGI
study. The actual function of each active-site residue may be defined
in more precise fashion in the future after more biochemical data are available.
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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