From the Department of Biochemistry and Biocenter Oulu, University of Oulu, P. O. Box 3000, Oulu FIN-90014, Finland
Received for publication, November 7, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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The atomic resolution structure of
Leishmania mexicana triosephosphate isomerase complexed
with 2-phosphoglycolate shows that this transition state analogue is
bound in two conformations. Also for the side chain of the catalytic
glutamate, Glu167, two conformations are observed.
In both conformations, a very short hydrogen bond exists between the
carboxylate group of the ligand and the catalytic glutamate. The
distance between O11 of PGA and O Triosephosphate isomerase
(TIM,1 EC 5.3.1.1.) is a
dimeric enzyme that catalyzes the interconversion of
D-glyceraldehyde 3-phosphate (DGAP) and dihydroxyacetone
phosphate (DHAP) during glycolysis (1) (see Fig. 1). The active site is
located at the C termini of the The catalytic residues are Glu167, His95,
Lys13, as reviewed by Knowles (1), and Asn11
(3, 12). These residues are in loops 6, 4, 1, and 1, respectively. The
catalytic mechanism of TIM has been extensively studied by means of
x-ray crystallography, NMR, infrared spectroscopy, mutagenesis, and
enzymology, including the use of labeled substrates (1). Three planar
transition state analogues (see Fig. 1) have been used for experimental
studies on the reaction mechanism. In addition, detailed quantum
mechanics/molecular mechanics calculations of the TIM reaction have
been performed recently (13). Despite the wealth of information, no
atomic resolution structures of TIM complexed with ligands have been
reported, and not all aspects of the reaction mechanism are understood.
For example, how does the enzyme environment allow for the proton
abstraction by the glutamate. Also, there are two alternative proposals
for the intermediate proton transfer steps, the relative importance of
which is not clear (12, 14).
The reaction is started with the abstraction of the pro-R
proton from the C1 carbon of the substrate, DHAP, by
Glu167, leading to a planar enediolate intermediate. In the
absence of enzyme, this is energetically a very unfavorable reaction
due to the high pKa of the C1 atom of the substrate.
In the enzyme environment, several residues contribute to facilitating this step, in particular those residues that stabilize the negative charge on the oxygen atom of the adjacent carbonyl group. These are
N2 of Glu167 is 2.61 and
2.55 Å for the major and minor conformations, respectively. In either
conformation, O
1 of Glu167 is hydrogen-bonded to a water
network connecting the side chain with bulk solvent. This network also
occurs in two mutually exclusive arrangements. Despite the structural
disorder in the active site, the C termini of the
strands that
construct the active site display the least anisotropy compared with
the rest of the protein. The loops following these
strands display
various degrees of anisotropy, with the tip of the dimer interface loop
3 having very low anisotropy and the C-terminal region of the active
site loop 6 having the highest anisotropy. The pyrrolidine ring of Pro168 at the N-terminal region of loop 6 is in a strained
planar conformation to facilitate loop opening and product release.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strands of the classic TIM barrel
fold and is shaped by the loops connecting the
strands to the
following
helices. Of special importance for substrate binding are
loops 6, 7, and 8, of which loops 6 and 7 undergo conformational
changes as the substrate is bound. In the unliganded state, loop 6 interacts with loop 5, whereas in the liganded state, it interacts with the YGGS motif (residues
Tyr210-Gly211-Gly212-Ser213)
of loop 7. O
of Tyr210 and O
of Ser213
are hydrogen-bonded to the main chain nitrogen atoms of
Ala178 and Gly175 in loop 6, respectively. N of
Gly173 is hydrogen-bonded to one of the phosphate oxygens
of the substrate. In the conformational switch from the open to the
closed state, the tip of loop 6 (near Gly175) moves 7 Å as
a rigid body (2), whereas in loop 7, the peptide planes after
Gly211 and Gly212 rotate 90° and 180°,
respectively. The rotation of these peptide planes allows for the tight
binding of the phosphate moiety and for the conformational switch of
the Glu167 side chain (3). The closure of loop 6 has also
been studied by NMR (4-6) and molecular dynamics calculations (7, 8). The calculations indicate that the loop starts to open in the region
Lys176-Val177 (7) and that this is initiated by
the loosening of the hydrogen bond between Gly175 and
Ser213 (8). The region 176-178 (Lys-Val-Ala) is also
referred to as the C-terminal hinge of loop 6 (9). The opening/closing motion of loops 6 and 7 appears to be partially rate-limiting for the
catalysis of the reaction in both directions (5, 6). It is unclear
which structural features are associated with the energy barrier of
this conformational switch (8, 10, 11).
of Lys13 and N
2 of His95 if the
substrate is DHAP and N
2 of His95 and N
2 of
Asn11 if the substrate is DGAP. Alternative mechanisms have
been discussed for the steps following the initial proton abstraction
(reviewed in Ref. 12), in particular the "classic" and the
"criss-cross" mechanism. In the classic mechanism, the proton
abstracted by Glu167 is delivered to the adjacent carbon,
unless, in the mean time, exchanged with solvent (15). In the
criss-cross mechanism, this proton is transferred to O2 (Fig.
1) of the substrate. NMR experiments have
indicated that in wild-type TIM, both mechanisms contribute to the
reaction (14).
View larger version (16K):
[in a new window]
Fig. 1.
A, the proton shuttling events in the
reaction catalyzed by TIM according to the criss-cross mechanism.
B, covalent structures of the transition state analogues
PGH, PGA, and IPP. The numbering scheme used for PGA is
indicated.
The mode of binding of 2-phosphoglycolate (PGA) to TIM has been studied
ever since this compound was proposed to be a transition state analogue
of the TIM reaction (16). 31P NMR studies indicate that PGA
binds to TIM as a trianion (17). It is assumed that the tight binding
of PGA is largely due to electrostatic interactions between PGA and the
protonated Glu167 and neutral His95 side chains
(18). Here, we report on an atomic resolution structure of
Leishmania mexicana TIM complexed with PGA and discuss its implications for the reaction mechanism.
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EXPERIMENTAL PROCEDURES |
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Crystallization and Data Collection-- For these experiments, the superstable E65Q variant of L. mexicana TIM was used, in which Glu65 has been mutated to Gln. The kinetic properties of this variant and the wild type enzyme are the same (19, 20). The enzyme was purified and crystallized as described before (3, 19, 20). The crystals grew at room temperature in less than a week from a mother liquor containing 20% polyethylene glycol 6000 in 0.1 M acetic acid/NaOH, pH 4.5, to a size of ~0.8 × 0.4 × 0.2 mm. The crystals belong to the space group C2, and there is one subunit per asymmetric unit. Two complete sets of diffraction data were collected at beamlines I711, MAX-Lab, Lund, Sweden (to 0.90-Å resolution) and BW7B, EMBL/DESY, Hamburg, Germany (to 0.83-Å resolution) on a MAR345 image plate at 100 K. The crystals were flash-frozen in the cryo stream after a brief soak in a cryoprotectant consisting of the mother liquor supplemented with 23% glycerol and additional 2% polyethylene glycol 6000 for cryo protection. The wavelengths used were 0.9600 and 0.8453 Å on I711 and BW7B, respectively. The data collection was carried out, respectively, in two and three passes for high, medium, and low resolution data from a single crystal. The data were processed and scaled with Denzo and Scalepack (21). The data processing statistics for the 0.83-Å structure are summarized in Table I. Truncate (22) was used to calculate the Wilson statistics for the 0.83-Å structure, indicating a Wilson B-factor of 6.9 Å2 (Table I).
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Structure Solution and Refinement-- The structure was solved by molecular replacement using the program AMoRe (23). For the 0.90-Å structure, the previously solved 2-Å structure of the E65Q variant of L. mexicana TIM (1IF2 (3)) was used as the search model. The structure was refined first with Refmac (24) using isotropic B-factors and in the later stages anisotropically using SHELX97 (25) to R and Rfree values of 0.1205 and 0.1385, respectively. The resulting model was used as the initial model for the refinement of the 0.83-Å structure.
The refinement of the 0.83-Å structure was carried out using SHELX97
(25) adding the high resolution data gradually as the refinement
proceeded. At this stage, the default geometric restraints and
weighting scheme were used. Model building was done with O (26). A test
set of 2% of all reflections was used to calculate Rfree. During the first six cycles, the model
was refined isotropically. At this stage, water molecules were added to
the structure, but other solvent molecules or disordered residues were
not modeled. The following cycles of refinement were done
anisotropically, and a detailed description of the model with
disordered side chains and solvent was built. Most hydrogens were
clearly visible in the Fo Fc
maps after moving to anisotropic refinement, as can be seen for example
in Fig. 8, and were added to the model at the 16th cycle of refinement.
For the last cycles, the geometric restraints were released for the
protein part and removed for PGA. The refinement was continued until
the highest peaks in the Fo
Fc
map were below 0.5 e
/Å3; the r.m.s.d. of the
final Fo
Fc map is 0.07 e
/Å3. At this stage, the highest peaks were
due to disordered solvent or side-chain atoms not located close to the
active site. The final crystallographic R and
Rfree values were 0.0949 and 0.1085, respectively. The final statistics of the resulting model are presented
in Table II. The programs O (26),
XtalView (27), PARVATI (28), Ortep (29), Raster3D (30),
AnisPlot,2 Molray (31),
and DINO3 were used
for analyzing the structure and generating the figures. The coordinates
and structure factors have been deposited at the Protein Data Bank at
the Research Collaboratory for Structural Bioinformatics.
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RESULTS |
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Structural Heterogeneity--
Due to the high resolution of the
diffraction data, many of the disordered residues could be modeled with
confidence. Altogether, 42 of the 250 residues have been built in more
than one conformation. Nevertheless, some of the solvent-exposed side
chains had to be considered as highly disordered with no preferred
conformations. Those side chains have been included in the model as one
or two conformations, depending on the electron density, but sometimes there was residual electron density in the surroundings that could not
be explained by the model. Mostly, the alternative conformations occur
in the side chains, but in a few cases, also the main chain is
disordered. For example, the tip of the flexible loop 6 (residue Gly175) has two distinct conformations. In the major
conformation (60% occupancy), the peptide proton of
N(Gly175) points to O of Ser213, providing a
reasonable hydrogen-bonding interaction
(O
(Ser213)-N(Gly175) distance is 3.29 Å).
In the minor conformation, this interaction is weakened and
Gly175 is unfavorably close to O
of Ser213;
for instance, the distance
C
(Gly175)-O
(Ser213) is 3.21 Å. This
disorder seems to reflect the intrinsic loop-opening properties of loop
6, which is also visualized by the anisotropic B-factors of
residues adjacent to Gly175 (Thr174 and
Lys176) (Fig. 2).
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In the active site, PGA is bound in two conformations (Fig.
3). In both of them, the O11 atom of PGA
is hydrogen-bonded to O2 of Glu167. In the predominant
conformation (70% occupancy), O12 of PGA is at a hydrogen-bonding
distance to N
of Lys13 and N
2 of His95,
whereas in the minor conformation (30% occupancy), it points further
down and is weakly hydrogen-bonded to N
2 of Asn11 (Fig.
3). In the predominant conformation, the
N
2(Asn11)-O12(PGA) distance is 4.17 Å, whereas in the
minor conformation this distance is 3.22 Å. The difference between the
two conformations concerns a rotation about the C1-C2 bond of PGA. The
torsion angle O1P-C2-C1-O12 is
35° for the major conformation
and 0° for the minor conformation. The Glu167 side chain
is also 2-fold-disordered. The disorder concerns the position of O
1,
whereas O
2 adopts always the same position, being hydrogen-bonded to
O11 of PGA. The structural disorder of the carboxylate moieties of PGA
and the Glu167 side chain appears not to be a concerted
motion, instead, the active-site geometry (Fig. 3) suggests that these
two groups move independently of each other, while keeping the
O11(PGA)-O
2(Glu167) hydrogen bond intact. In the major
conformation of Glu167 (80% occupancy,
1 =
52,
2 =
169,
3 =
66), the O
1 atom points to S
of
Cys126 at a distance of 4.12 Å, whereas in the minor
conformation (
1 =
50,
2 =
161,
3 =
53),
O
1 is rotated away from S
of Cys126 at a distance of
4.69 Å. This structural disorder appears to be correlated with
heterogeneity of S
of Cys126, because there exists a
minor conformation in which this atom is rotated into the space created
by the rotation of O
1 of Glu167. In both
Glu167 side-chain conformations, the O
1 atom is within a
hydrogen-bonding distance from O11 of PGA as well as Wat28
(Fig. 3). This water is the first water of a network between Glu167 and bulk solvent. It consists of waters 28, 114, 38A, and 58. Wat58 is also hydrogen-bonded to O
of
Ser96 (Fig. 3). There also exists an alternative
arrangement for this network, consisting of waters 28, 114, 38B, 107, and 58.
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Anisotropy--
The large amount of data at this high resolution
allowed for expanding the description of the model to anisotropic
displacement parameters. The anisotropic description was applied to all
atoms, including solvent molecules, which caused a significant drop in both R and Rfree. As seen in other
structures at atomic resolution, none of the atoms agree with the
traditional isotropic description. As examined by PARVATI (28) and
Rastep (30), the mean anisotropy of all protein atoms in the structure
is 0.53 (with a of 0.1) deviating significantly from the value 1, which means perfect isotropy. The anisotropy value refers to the ratio
of the shortest and longest principal axis of the thermal ellipsoid
describing the anisotropic scattering properties of an atom (28). The
mean isotropic B of the protein atoms is 11.8 Å2.
In this crystal form, the dimer 2-fold axis coincides with a
crystallographic axis, and consequently, the molecular center is on
this 2-fold axis. The analysis of the mean anisotropy of the protein
atoms of the dimer as a function of the distance from the dimer center
shows the least anisotropy at the center (with a mean anisotropy of
0.9), which increases smoothly to a mean anisotropy of 0.3 at the
extremities (25 Å away from the center). A more detailed analysis of
the variation of the anisotropy along the polypeptide chain is shown in
Fig. 4. Consistently, very low anisotropy
is seen at the C termini of the strands. This correlates with the
low isotropic B-factors of these regions. The regions with
the highest anisotropy are in the TIM-barrel framework
helices, in
particular at their C termini.
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In the active site, the side chain of His95 shows
significant anisotropic behavior with an anisotropy value of 0.47 for
N2. This anisotropic movement occurs in the plane of the imidazole ring (Fig. 5). The side chains of
Lys13 and Asn11 are less anisotropic with
anisotropy values of 0.63 for N
of Lys13 and 0.62 for
N
2 of Asn11. Glu167, which is present in two
conformations, displays significant anisotropy in the minor
conformation with anisotropy values of 0.59, 0.41, 0.37, and 0.54 for
C
, C
, O
1, and O
2, respectively. For the major conformation,
the corresponding anisotropy values are 0.66 (C
), 0.87 (C
), 0.50 (O
1), and 0.43 (O
2). The mean anisotropy of PGA is 0.47 and 0.43 for the major and minor conformation, respectively. It is interesting
to note the relatively high anisotropy of PGA and the side chain of the
catalytic glutamate, despite the fact that the main chain regions
defining the active site have relatively low anisotropy as well as low
isotropic B-factors (Fig. 4). For example, the anisotropy of
the C
atoms of Lys13, His95,
Asn11, and Glu167 are 0.86, 0.77, 0.84, and
0.69, respectively.
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The anisotropy of the flexible loop 6 has a direction parallel to the
loop opening/closure event. The anisotropy is more pronounced at the
C-terminal end of the loop, concerning residues
Gly173-Ala178 (Fig. 2). The most anisotropic
part concerns residues Thr174 and Gly175 when
only the main chain atoms are considered, whereas the side chain of
Lys176 has one of the highest anisotropy values in the
whole structure (0.10 for the C atom).
Active-site Geometry--
Fig. 6
highlights important hydrogen bonding interactions in the active site,
in particular the hydrogen bonds of the major conformations of PGA and
the Glu167 side chain. The phosphate moiety is
hydrogen-bonded to main chain N atoms from loops 6, 7, and 8 (Fig. 3),
whereas the triose moiety forms hydrogen bonds to side-chain atoms, in
particular Lys13, His95, and Glu167
(Fig. 3). The phosphate moiety is also hydrogen-bonded via four water
molecules to the protein: via Wat44 to Lys13
and via Wat186, Wat8, and Wat1 to
loops 7 and 8. Wat1 is a buried water molecule, not part of
a hydrogen bonding network to bulk solvent. Wat1 is buried
between the ligand and protein and hydrogen-bonded to N of
Gly212, O2P of PGA, and O of Leu232. It is at
3.69 Å distance from C2 of PGA.
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The side chains of Lys13 and His95 are well
hydrogen-bonded to the bulk of the protein. N of Lys13
is tightly fixed to its position by three hydrogen bonds to O
1 of
Glu97, O2 of PGA, and Wat45. N
2 of
His95 is hydrogen-bonded to O12 of PGA (2.74 Å), whereas
the side-chain position of His95 is stabilized by a
hydrogen bond between N
1 and N of Glu97 (2.96 Å). The
Glu167 side chain is only weakly interacting with the rest
of the protein. The Glu167 side-chain carboxylate oxygen
atoms span the region between C
of Gly211 (at 3.39 Å from O
2), O of Leu232 (at 3.60 Å from O
2) and S
of Cys126 (at 4.12 Å from O
1). The hydrogen atom on
S
of Cys126 points toward the main-chain oxygens of 124 and 93 (Figs. 6 and 7), as is also
confirmed by the residual positive density in the Fo
Fc map; therefore, the
O
2(Glu167)-S
(Cys126) is a van der Waals
contact. O
1 is at a distance of 3.29 Å from N
2 of
His95, but the geometry does not allow for a good hydrogen
bond (Fig. 3). Two other fully conserved side chains
(Ile172 in loop 6 and Leu232 at the beginning
of loop 8) also contact the Glu167 carboxylate group (Fig.
7). None of these contacting protein atoms allow for good hydrogen
bonding. A good hydrogen bond is observed with Wat28 (Figs.
3 and 7). This hydrogen bond exists in both the major and the minor
conformation of the Glu167 side chain with distances of
2.71 and 2.80 Å. In both the major and the minor conformation, there
exists a short (2.61/2.55 Å) hydrogen bond between O
2 of
Glu167 and O11 of PGA. The C
-O
2 and C
-O
1 bond
distances in Glu167 are 1.26 and 1.19 Å, respectively,
whereas the C1-O11 and C1-O12 bond distances are 1.22 and 1.19 Å,
respectively. These distances are in the range of the C-O bond
distances of the carboxyl moiety of the fully protonated crystal
structure of PGA (33), which are 1.23 Å for the C=O double bond and
1.31 Å for the C-OH single bond. In the trianionic species of PGA,
the C-O distances of the carboxylate group are 1.24 and 1.26 Å (33).
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The active site is at the dimer interface, and the well-defined
conformations of Asn11 and Lys13 are stabilized
due to the tight interactions with loop 3 of the second subunit; for
example, Thr75 at the tip of this loop is hydrogen-bonded
to N2 of Asn11 and O
2 of Glu97, which
forms a salt bridge to N
of Lys13 (Figs. 6 and 7).
Analysis of the B-factors (Fig. 4) shows that the tip of
this loop is very rigid (BC
Gly76 = 5.4 Å2) and the anisotropy is very low
(AC
Gly76 = 0.82).
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DISCUSSION |
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The reaction catalyzed by TIM is conceptually rather simple,
because it only concerns shuttling of protons (Fig. 1). The active site
can be seen to consist of two parts: a binding pocket for the phosphate
moiety and a catalytic site for the binding of the triose moiety.
During the catalytic cycle, the phosphate group of the substrate has to
be held in plane with respect to the rest of the molecule to avoid
phosphate elimination (18). Therefore, mechanistically, the reaction
becomes complicated, also because C-H bonds are very stable (34, 35).
For example, the pKa value of methane is estimated
to be 40 (36). Consequently, under physiological conditions, a
deprotonated carbon atom is very unstable. If the resulting carbanion
is stabilized, for example in the presence of an adjacent carbonyl
function, then the pKa is much lower, near 20 (37).
This is still considerably different from the pKa of
a glutamate side chain, which is known to extract the proton from DHAP
in the first step of the TIM reaction (1, 15). Nevertheless, the
active-site geometry of TIM allows for efficient proton shuttling. The
active site is located at the C termini of the TIM-barrel strands
and in fact, residues from each of the strands (or subsequent loops)
contribute to the active site (Fig. 7). Thr75 from loop 3 of the opposing monomer is hydrogen-bonded to the side chains of
Asn11 and Glu97, thus providing essential
stabilizing interactions.
In the atomic resolution structure of the current study, the active site of TIM is complexed with the transition state analogue PGA. It is observed that the triose moiety of PGA occurs in two conformations, whereas the phosphate moiety is bound in a well-defined single conformation. The P-O1P (O1P is bonded to C2) distance is 1.62 Å, whereas the other P-O distances are 1.49, 1.50, and 1.55 Å. The corresponding distances in the crystallized trianionic species of PGA are 1.64, 1.51, 1.50, and 1.53 Å, respectively (33). Thus, the phosphate is hydrogen-bonded to main-chain nitrogen atoms from loops 6, 7, and 8 (Fig. 3). The triose moiety is hydrogen-bonded to side chains only (Asn11, Lys13, His95, and Glu167). In addition, it is in van der Waals contact with the side chains of Ile172 in loop 6 and Leu232 in loop 8.
Loop 6 is a loop with conformational flexibility, which is known to be
in an open conformation in the unliganded state and in a closed
conformation in the liganded state. The conformational differences
between the open and closed conformation have been described previously
(7), and the loop can be divided into an N-terminal hinge
(Pro168, Val169, and Trp170), a
rigid tip (Ala171, Ile172, Gly173,
Thr174, and Gly175), and a C-terminal hinge
(Lys176, Val177, and Ala178) (9).
After this, helix 6 starts with Thr179 as the N-cap
residue, followed by Pro180 in the N-cap+1 position. The
tip of the loop moves as a rigid body, while small changes in the and
angles of the N- and C-terminal hinge cause loop opening and
closure. The largest changes occur for
(Lys176) and
(Val177), which are +57 and
38°, respectively (2).
In the closed conformation, as seen in this structure, the
Ile172 side chain interacts with the triose moiety of the
substrate, as well as with the Glu167 side chain (Fig. 7),
whereas N of Gly173 is part of the phosphate binding pocket
(Fig. 3). Ile172 is in a very well-defined conformation,
and the position of most of its hydrogen atoms can be seen in an
Fo
Fc omit map (Fig.
8). In the closed conformation, loop 6 tightly interacts via O of Ala171 with O
of
Ser213 (in loop 7) and via N of Gly173 with the
phosphate of PGA (Fig. 7). Weaker hydrogen bonding is seen for the
C-terminal part, between N of Gly175 and O
of
Ser213 and between N of Ala178 and O
of
Tyr210; also the main chain of this part of the loop shows
one of the highest anisotropy properties of the entire structure. It
seems, therefore, that the C-terminal part of loop 6 has evolved not to
interact too tightly with loop 7, allowing for efficient loop opening,
which is a requirement for efficient catalysis (5, 6). These structural
features are in good agreement with NMR and computational studies. For
example, solid-state NMR studies (4) have shown that the opening of
loop 6 is not ligand-gated; instead, also in the liganded state, the
loop opens at a rate in the millisecond timescale. Computational
studies have also indicated that loop opening is initiated by the
loosening of the hydrogen-bonding interactions between the C-terminal
region of loop 6 and the YGGS motif of loop 7 (7, 8). The observed interactions between loop 6 and the YGGS motif of loop 7 require small
structural rearrangements of the Tyr210 and
Ser213 side chains as compared with the open conformation
(10). These rearrangements are correlated with peptide rotations of the
peptide bonds after Gly211 and Gly212, which
generate the phosphate binding pocket as well as are required for the
Glu167 side chain to move into the "swing-in"
position.
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As seen in Fig. 7, the pyrrolidine ring of Pro168 in the
N-terminal region of loop 6 is planar. This is unusual, because the proline ring is known to occur in either of two different
conformational states; "up" (1 =
25°) or "down"
(
1 = +25°) (38, 39), which differ in the position of the C
atom with respect to the other members of the five-atom ring. The
properties of the pyrrolidine ring of proline confer a unique
structural role for this residue as reviewed previously (40). In this
TIM·PGA structure, both puckerings of proline rings are
observed, except for Pro168, which has
1 of
2°. This
conformation is enforced by close van der Waals contacts of the C
atom with the side chains of Tyr166 and Ala171
(Fig. 7). This strained conformation is undoubtedly an important structural feature of the closed form of loop 6. For example, in
natural TIM sequences, this proline is fully conserved and random
mutagenesis studies have shown that active variants require a proline
at this position (9). This strained conformation, not seen in the open
loop 6, will destabilize the closed loop, thus facilitating loop
opening and product release at the end of the reaction cycle.
In the closed form of loop 6, the side chain of Ile172
folds over the substrate, shielding it and the catalytic glutamate from bulk solvent. Glu167 and the substrate are also contacted
by Leu232 (Fig. 7). In this way, the Glu167
side chain in the closed conformation is buried in a pocket formed by
the side chains of Ile172 and Leu232 but also
lined by C of Gly211, O of Leu232, S
of
Cys126, and Wat28. This pocket does not anchor
the glutamate side chain via specific hydrogen bonds to the rest of the
protein, and the width of the pocket (the distance from C
1 of
Ile172 to C
of Leu232 is 8.35 Å) allows for
some conformational flexibility for this side chain. This
conformational flexibility correlates with the proton shuttling
function of Glu167. The importance of this hydrophobic
pocket is highlighted by the full sequence conservation of
Leu232 and Ile172. Moreover, a disease-related
point mutation Ile172
Val has been characterized (41).
The patients display severe symptoms due to TIM deficiency (42).
The only (non-ligand) hydrogen-bonding partner for the Glu167 side chain is Wat28. This water is part of a water network, which extends to the bulk solvent (Figs. 3 and 6). The importance of these waters for catalysis has been discussed by Zhang et al. (37). Proton exchange between the catalytic glutamate and bulk solvent via this network might also explain the low yield of tritium transfer from the labeled C1 of DHAP to C2 of DGAP during catalytic turnover (15).
Phosphoglycolohydroxamate (PGH) and
2-(N-formyl-N-hydroxy)aminoethyl phosphonate
(IPP) (Fig. 1) are transition state analogues mimicking structural
features of the ketone (DHAP) and aldehyde (DGAP) substrate,
respectively. The comparison of the mode of binding of these two
compounds suggests that the precise details of the active-site geometry
concerning substrate binding change as the reaction proceeds from
substrate to product (3). This is correlated with the structural
differences between an -hydroxy ketone and an
-hydroxy aldehyde,
because the planar carbonyl function is transferred from the C2
position to the C1 position. In particular, it has been shown that the
side chains of Lys13 and His95 create a
stabilizing oxyanion hole for the ketone carbonyl group, whereas
Asn11 and His95 form such an oxyanion hole for
the carbonyl group of the aldehyde substrate (3). In the apo structure,
a water molecule is bound in the latter pocket. PGA is a transition
state analogue in which the methanol group of the ketone substrate
(DHAP) is replaced by a hydroxyl group. The O12 atom mimics the ketone
moiety, whereas the O11 atom mimics the C1 atom of DHAP from which
Glu167 abstracts a proton. The O11 atom of PGA points to
both the O
1 and O
2 of Glu167, implying that it
interacts with the syn orbitals of this carboxylate moiety
(Fig. 5), which is the optimal geometry for proton abstraction (18). In
the major conformation, O12 of PGA binds to N
of Lys13
and N
2 of His95, whereas in the minor conformation the
O12 of PGA is bound at the expense of induced strain in the molecule
(the C1-C2 dihedral is 0°), with low occupancy in the second
oxyanion hole. The disorder of PGA in this structure correlates with
the differences in the mode of binding of PGH and IPP, resembling the
situation as the reaction proceeds from DHAP to DGAP.
A key feature of the active site is a very short hydrogen bond between
the carboxylate groups of PGA and Glu167. The O-O distance
for the usual hydrogen bond between two oxygen atoms, with the hydrogen
being firmly attached to one of them, is around 2.8 Å (43), in good
agreement with the O-O distances highlighted in Fig. 6. However, the
distance between O11 of PGA and O2 of Glu167 is 2.61 and
2.55 Å, when considering the major and minor conformations, respectively. This is significantly shorter than a normal hydrogen bond
and similar to the O-O distance expected for a low barrier hydrogen
bond (LBHB) (43). An LBHB concerns a short and very strong hydrogen
bonding interaction, in which the hydrogen is more diffusely
distributed between the two atoms. The possible role of LBHBs in the
mechanism of TIM as well as other enzymes has been discussed since 1993 (43-45). NMR studies have shown that, in the complex of TIM with
PGH, there exists an LBHB between the NOH hydrogen and the catalytic
glutamate (46). Clearly, the glutamate side chain and ligand geometry
can flip between different conformations allowing for strong
interactions between the glutamate and different atoms of the
substrate. This apparently allows the side chain to shuttle protons
directly between the two carbons of the substrate (as in the classic
mechanism) as well as via the oxygen atoms (as in the criss-cross
mechanism). The former, which explains the tritium incorporation from
DHAP to DGAP (15), requires that the proton transfer between the
oxygens is mediated by the His95 side chain (32, 47). In
this TIM·PGA complex, the N
2 of His95 is
hydrogen-bonded to O12 of PGA (2.74 Å) and interacts with O11 at a
distance of 3.21 Å (Fig. 6). The electron density suggests that N
2
is protonated and N
1 is unprotonated. As seen in Fig. 5, the
anisotropic B-factors of His95 indicate that the
histidine side-chain motion is a concerted motion in the plane of the
imidazole ring. When examining the mode of binding of IPP and PGH (3),
it can be seen that such dynamic properties would facilitate proton
transfer between the two substrate oxygen atoms via N
2 of
His95, as has been observed to occur (32).
The atomic resolution structure discussed here provides precise
structural information on the TIM·PGA complex. The observed dynamic
properties of the side chains of Glu167 and
His95 agree with the known proton-shuttling roles of these
residues. PGA is a true transition state analogue, because it has the
same planarity and charge as the intermediate formed from the substrate after proton abstraction by the catalytic glutamate. This structure appears to mimic the active-site geometry of the enzyme·substrate complex, trapped after the very first proton abstraction step by
Glu167 has been initiated. The Glu167 side
chain shares a proton with the ligand donor atom, and no further proton
shuttling has occurred. Additional proton transfer via the
His95 side chain seems also not to have happened. The true
substrate differs from PGA such that the O11(H) group is the C1(H)-OH
moiety in the ketone substrate (DHAP). The LBHB-like hydrogen bond
between O11 of PGA and the catalytic glutamate indicates that the
active-site geometry favors transition state stabilization required for
proton transfer from the C1(H)-OH moiety of DHAP to Glu167
and thereby facilitates efficient catalysis of the proton-shuttling steps. The observed structural heterogeneity in the active site is an
important feature of the mode of binding of PGA, because it correlates
with conformational changes taking place during the reaction. Atomic
resolution structures of TIM complexed with other transition state
analogues will be required to obtain a complete understanding of the
structural rearrangements occurring in the active site during the
proton transfer steps of the catalytic cycle.
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ACKNOWLEDGEMENTS |
---|
The excellent support by Dr. Xiao-Dong Su and Dr. Yngve Cerenius on I711 (MAX-Lab, Lund) and Dr. Paul Tucker on BW7B (EMBL/DESY, Hamburg) is gratefully acknowledged. We thank Dr. Petri Kursula for helpful discussions on the manuscript and helping with the figures. The technical assistance of Ville Ratas with protein purification and crystallization is highly appreciated.
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FOOTNOTES |
---|
* This work was funded by the Academy of Finland (grant number 44198).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 1N55) 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 addressed. Tel.: 358-8-553-1199;
Fax: 358-8-553-1141; E-mail: rik.wierenga@oulu.fi.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M211389200
2 P. Kursula, unpublished.
3 A. Philippsen (2001) www.bioz.unibas.ch/~xray/dino.
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ABBREVIATIONS |
---|
The abbreviations used are: TIM, triosephosphate isomerase; DGAP, D-glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; E65Q, the L. mexicana TIM variant, in which Glu65 has been replaced by Gln; IPP, 2-(N-formyl-N-hydroxy)aminoethyl phosphonate; LBHB, low barrier hydrogen bond; PGA, 2-phosphoglycolate; PGH, phosphoglycolohydroxamate; YGGS, the region Tyr210-Gly211-Gly212-Ser213 of loop 7 in TIM; r.m.s.d., root mean square deviation.
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