From the Departments of Internal Medicine and
§ Biochemistry, University of Texas Southwestern Medical
Center, Dallas, Texas 75235, ¶ Research Service, Department of
Veterans Affairs Medical Center, Dallas, Texas 75216, and the
Department of Chemistry and Biochemistry, University of
Oklahoma, Norman, Oklahoma 73019
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ABSTRACT |
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Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase
(Fru-6-P,2-kinase/Fru-2,6-Pase) is a bifunctional enzyme,
catalyzing the interconversion of The bifunctional enzyme
fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase
(Fru-6-P,2-kinase1/Fru-2,6-Pase) participates
in glucose homeostasis by regulating the
intracellular concentration of fructose-2,6-bisphosphate
(Fru-2,6-P2). Fru-2,6-P2 is both a potent
physiological activator of 6-phosphofructokinase, and an in
vitro inhibitor of fructose-1,6-bisphosphatase (reviewed in Refs.
2-4). There is significant sequence homology among the Fru-2,6-Pase
domain, the phosphoglycerate mutases, and the acid phosphatases (5, 6).
We previously determined the crystal structure of the rat testis
Fru-6-P,2-kinase/Fru-2,6-Pase (RT2K-Wo), showing the enzyme to be a
head-to-head homodimer of 55-kDa subunits, with each monomer consisting
of independent kinase and phosphatase domains (7). The kinase domains
are in close contact, forming an extended hydrophobic core between
them, while the phosphatase domains are essentially independent of one
another. The structure of the isolated rat liver
Fru-6-P,2-kinase/Fru-2,6-Pase (RL2K) Fru-2,6-Pase domain has also been
determined (8), and as would be predicted by the 85% identity between
the Fru-2,6-Pase domains, the structures are nearly identical.
Comparison of the RT2K-Wo Fru-2,6-Pase domain structure with those of
yeast phosphoglycerate mutase (9) and rat acid phosphatase (10)
confirmed that these enzymes also have very similar tertiary structures
(7). These enzymes share a common catalytic mechanism involving a
covalent phosphohistidine intermediate (11-14) located in a conserved
sequence motif (ZXRHG(E/Q)XXXN, where
Z is a hydrophobic residue (15)). The sequence motif
253LCRHGESELN262 is found in the
RT2K phosphatase domain, where His256 is the catalytic
histidine. This conserved sequence motif, together with
Arg305, Glu325, and His390, forms a
larger conserved structural motif defining the catalytic center of all
three enzymes (7).
Mutagenesis and kinetic analyses on the rat liver isozyme (16-20) led
to a model of Fru-2,6-P2 binding and a potential
phosphatase reaction mechanism. Thus, the catalytic histidine
(His258 in RL2K, His256 in RT2K) was proposed
to make a nucleophilic attack on the 2-phosphate of
Fru2,6-P2, while positively charged side chains
(Arg257 and Arg307 in RL2K, Arg255
and Arg305 in RT2K) orient the 2-phosphate and stabilize
the transition state. His392 (His390 in RT2K)
was proposed to be coupled to Glu327 (Glu325 in
RT2K), making His392 doubly protonated, and capable of
donating a proton to the leaving group oxygen of Fru-6-P. A water,
activated by Glu327, would then attack the phosphoenzyme
intermediate, regenerating the enzyme and producing a free
PO4 ion (8, 14). This interpretation of the mutagenesis and
the proposed reaction mechanism are largely in agreement with the
RT2K-Wo and RL2K Fru-2,6-Pase domain structures, with the exception of
the role of His390, which is neither coupled to
Glu325, nor in a position to donate a proton to Fru-6-P. It
is important to note, however, that neither the RT2K-Wo nor the
original RL2K Fru-2,6-Pase domain structures contained bound ligands
(Fru-6-P or Fru-2,6-P2), which has hampered a thorough
characterization of the Fru-2,6-Pase mechanism. The report of a trapped
RL2K phosphoenzyme intermediate (21) and the work presented here
combine to remedy this deficiency.
To facilitate our own crystallographic studies of the RT2K kinase
reaction, we generated a mutant protein, H256A, that as reported for
the RL2K H258A mutant (16), would be devoid of phosphatase activity
(and allow us to soak Fru-2,6-P2 into crystals without
consumption by the Fru-2,6-Pase reaction). This enzyme unexpectedly
retains 17% of the RT2K-Wo Fru-2,6-Pase activity and led to a series
of experiments to assess the basis for this activity. The results of
mutagenesis and kinetic analyses on the Fru-2,6-Pase domain of the RT2K
isozyme are presented in a companion paper (1). We report here the
crystallographic structure of the Fru-2,6-Pase domain of the RT2K H256A
mutant enzyme. This structure includes bound Fru-6-P and
PO4 ligands in the active site. Based on a comparison of
the RT2K-Wo, H256A mutant, and the RL2K phosphoenzyme structures, we
propose catalytic mechanisms that explain the observed kinetic data for
both mutant and wild type enzymes.
Protein Preparation and Crystallization--
The preparation and
purification of the Wo form of RT2K was described previously (22, 23).
A single point mutation of the Wo enzyme, converting His256
to Ala was generated and purified by the same procedure (1). Crystallization was performed by the hanging drop vapor diffusion method. The conditions for crystallization were a modified version of
those reported previously (24). Briefly, 10 mg/ml of protein (in 50 mM Tris·PO4, pH 7.5, 5% glycerol, 40 µM EDTA, 40 µM EGTA, 800 µM
dithiothreitol, 0.4% PEG300, 1.7 mM Fru-6-P, 1.7 mM AMP-PNP, 1% Data Collection and Processing--
Crystals were serially
transferred into solutions of surrogate mother liquor (19% PEG 4000, 10 mM MgCl2, 90 mM succinate, pH
6.0, 50 mM Tris·HCl, pH 7.5, 1% Phasing by Molecular Replacement, Model Building, and
Refinement--
The rotation search and PC refinement were carried out
in XPLOR, using a polyalanine model derived from the RT2K-Wo crystal structure (7). Because of the extensive interaction between monomers in
the original P3121 crystal form, we anticipated that the
global dimer structure would be unchanged in this new P1 crystal packing and thus used the entire dimer as a search model, rather than
separate searches for the two monomers. The molecular replacement was
unambiguous, with a rotation function peak 7.4 Protein Crystallization and Structure Solution--
Crystals of
the RT2K H256A mutant protein were grown by the hanging drop vapor
diffusion method as described under "Experimental Procedures."
Crystals grew with a rod-shaped morphology and were found to belong to
space group P1. The unit cell dimensions are a = 61.7 Å, b = 73.5 Å, c = 76.7 Å; Fru-6-P Binding in the Active Site--
In our previous structure
of RT2K-Wo, there was not a Fru-6-P molecule bound to the Fru-2,6-Pase
domain, despite its presence in the crystallization mixture. Instead,
there were phosphate ions bound in the presumed binding sites for the
2- and 6-phosphates of Fru-6-P. In the structure of the RT2K H256A
mutant reported here, there is clear electron density for Fru-6-P in
both monomers (Fig. 1). The refined
coordinates of the Fru-6-P molecule in the binding site (Fig.
2) reveal that the Fru-6-P traverses a
crevice composed of Ile267, Glu325,
Tyr336, Arg350, Lys354,
Tyr365, Gln391, and Arg395. The
polar residues form hydrogen bonds with the Fru-6-P hydroxyls and
6-phosphate (Table II and Fig. 2), while
Ile267 forms a Van der Waals interaction by stacking with
the fructose ring (Fig. 3).
Tyr336, Arg350, Lys354, and
Tyr365 interact with the 6-phosphate in exactly the same
way that the free phosphate was bound in the RT2K-Wo structure. Eight
of the nine oxygens of Fru-6-P are involved in direct hydrogen bonds to
the protein or to well ordered waters, which are in turn bound by
protein. Fru-6-P-O-1 interacts with two water molecules, which are in
turn bound to main chain carbonyls and side chains. Fru-6-P-O-2, which
is of course the site of bond cleavage from the 2-phosphate during the
Fru-2,6-Pase reaction, forms a hydrogen bond with Glu325,
making Glu325 a prime candidate for a catalytic residue, as
will be discussed below. The main chain nitrogen of Gly268
interacts with the Fru-6-P-O-3 hydroxyl, while the nitrogen of Gln391 forms two hydrogen bonds to fructose oxygens (O-5
and O-6). Only Fru-6-P-O-4 is not involved in a hydrogen bond
interaction, and that is because the stacking of Ile267
with the fructose ring includes an interaction with the C-4-O-4 bond,
preventing a close approach of any other protein groups that might
interact with Fru-6-P-O-4. This mode of Fru-6-P binding (a combination
of many hydrogen bonds and hydrophobic stacking) is typical for
carbohydrate binding proteins that bind their substrate in an internal
pocket (type I proteins, as defined by Quiocho (31) and Vyas (32)) and
promotes both tight binding and substrate specificity.
Phosphate Binding at the 2-Phosphate Pocket--
A phosphate ion
is bound in the 2-phosphate pocket of both monomers in the crystal. The
phosphate is in hydrogen bonding distance to the side chains of
Arg255, Glu325, Arg305,
His390, Asn262, and the main chain nitrogen of
Gln391, as well as being in contact with Fru-6-P-O-2. A
superposition of the H256A and RT2K-Wo structures shows that the
phosphate occupies a different position in the two enzymes. In the
H256A structure, the ion shifts (by an average distance of 1.46 Å for
the two monomers) to occupy the space created by the loss of
His256. While the phosphate still contacts the same set of
protein atoms, most of the hydrogen bonds are longer in the H256A
mutant (Table III). The phosphate in the
H256A structure has, of course, lost its interaction with
His256 but has gained an interaction with Fru-6-P.
Modeling Fru-2,6-P2 Binding in the Active
Site--
Superposition of the H256A crystal structure with the
RT2K-Wo structure allows us to generate a model of
Fru-2,6-P2 in the phosphatase active site. This is possible
because the positions of the 6-phosphate and protein atoms in the
active site have not changed (root mean square coordinate change for
143 atoms = 0.20 Å) beyond the intrinsic coordinate error of the
H256A model (0.35 Å). Thus, by combining the protein and 2-phosphate
analogue from the RT2K-Wo structure with the Fru-6-P from the H256A
structure, we achieve an excellent approximation of the
Fru-2,6-P2 complex (Fig. 3). The arrangement of
protein/Fru-6-P hydrogen bonds and Van der Waals interactions described
above holds the Fru-2,6-P2 molecule in the ideal
orientation for an in-line associative transfer of the 2-phosphate to
His256. Thus, the crystallographically determined location
of Fru-6-P in the active site (and the model of Fru-2,6-P2)
allow us to re-evaluate the proposed catalytic mechanism in more detail.
Fru-6-P and Fru-2,6-P2 Binding--
The structure
presented here represents the first structure of a Fru-6-P-bound form
of an intact Fru-2,6-Pase domain in a dimeric
Fru-6-P,2-kinase/Fru-2,6-Pase. Fru-6-P is a potent inhibitor of
Fru-2,6-Pase activity at physiologic concentrations of Fru-6-P (Ki = 51 nM, hepatocyte concentration of
20-50 µM). In the case of the RT2K isozyme, this should
result in an inhibited Fru-2,6-Pase activity and a net production of
Fru-2,6-P2 and consequent driving force for glycolysis in
tissues that express this isozyme. Thus, the elucidation of the mode of
binding of this potent inhibitor is itself significant. Recently, a
structure of the truncated form of an isolated RL2K Fru-2,6-Pase domain
was determined with a covalent phosphohistidine intermediate in the
presence of Fru-6-P (21). The position of the Fru-6-P molecule is
notably different in the two structures. While it is impossible to say
that one conformation is correct and the other incorrect, there are
several points that argue that the conformation of Fru-6-P reported
here is more likely to be the native conformation. First, the RL2K structures are of a truncated protein that is missing 30 C-terminal amino acids. The analogous stretch of polypeptide in our RT2K structure
passes within 3.5 Å of the Fru-6-P molecule, at Thr443.
The carbonyl oxygen of Thr443 is in fact hydrogen-bonded to
a water, which in turn coordinates the 1-hydroxyl of Fru-6-P (see Fig.
2). In addition, the region of RL2K structure that should have been
occupied by the truncated protein is instead involved in a crystal
contact, such that amino acids from a neighboring molecule are within
5.8 Å of the Fru-6-P. One of these amino acids (Gln342)
actually interacts with Gln393 (the analogue of
Gln391 in RT2K). Remember that Gln391 makes two
hydrogen bonds with Fru-6-P that are important in maintaining its
position in the active site (see Fig. 2). Last, the Fru-6-P in the RL2K
structure is in an unusual conformation compared with two Fru-6-P and
two Fru-1,6-P2 structures from the Cambridge small molecule
data base. In contrast, the Fru-6-P conformation reported here closely
resembles the extended, low energy conformations determined by small
molecule crystallography. Finally, recall that Ile267 forms
a typical stacking interaction with the fructose ring in the structure
reported here. This stacking interaction is absent in the RL2K structure.
The determination of a Fru-6-P-bound form of the RT2K H256A
Fru-2,6-Pase domain has allowed us to build a reliable model of the
Fru-2,6-P2-bound enzyme. As seen in Fig. 3, the active site of the Fru-2,6-Pase domain is perfectly tailored to accommodate Fru-2,6-P2. The active site serves as a molecular ruler,
measuring the length of the bound substrate along an axis defined by
the line between the Fru-6-P 2-hydroxyl and His256 (5.5 Å in the RT2K-Wo structure; see Fig. 4). In
this space, there is room for one phosphate, one covalent bond to
phosphate (1.6-1.8 Å), and one noncovalent interaction (~3.2 Å).
Thus, Fru-2,6-P2 is accommodated, with a covalent
Fru-6-P-2-phosphate bond and the 2-phosphate in Van der Waals contact
with His256. After the catalytic transfer of the
2-phosphate to His256, the ruler is still satisfied, with a
covalent phosphohistidine and a noncovalent interaction between the
transferred 2-phosphate and Fru-6-P. However, there is not enough room
for either the combination of phosphohistidine, hydrolytic water, and
Fru-6-P or for a noncovalently bound phosphate ion with a Fru-6-P
molecule. In those cases, the ruler would be measuring two noncovalent
interactions, a distance that cannot be accommodated. The arrangement
of molecules in the RL2K trapped phosphohistidine structure described
above (E-P·H2O·Fru-6-P) is an arrangement
that violates this molecular ruler concept. This violation is only
allowed because of the unnatural position of Fru-6-P in the binding
pocket, which is a consequence of the C-terminal truncation of the RL2K
Fru-2,6-Pase domain.
Correlation of Structures with Kinetics--
The molecular ruler
concept predicts an ordered reaction mechanism (Scheme I), where
Fru-6-P release must precede phosphohistidine hydrolysis. After Fru-6-P
has dissociated, a water molecule would be accommodated in the active
site for hydrolysis of the E-P intermediate. This model is
consistent with previous kinetic experiments where it has been reported
that the rate of phosphoenzyme formation is greater than the overall
reaction rate (33), that Fru-6-P inhibits the Fru-2,6-Pase reaction
(34), that Fru-6-P release is the rate-limiting step in the overall
reaction (35), and that Pi accelerates E-P
hydrolysis by competing for Fru-6-P binding (presumably at the
6-phosphate binding site) (33). In our own studies, we showed that
Fru-6-P release and phosphohistidine breakdown occur in parallel in the
wild type enzyme (1). Together, the structural and kinetic data
indicate that E-P hydrolysis does not occur until after
Fru-6-P has dissociated from the enzyme, and that E-P
hydrolysis is fast (Scheme 1).
There have been several models of Fru-2,6-Pase catalysis proposed,
based first on mutagenesis directed by the homology of the Fru-2,6-Pase
domain to the phosphoglycerate mutase family (16, 17) and later based
on crystal structures (1, 7, 8, 21). These studies have all concluded
that all of the amino acids that define the 2-phosphate binding pocket
(His256, His390, Arg255,
Arg305, Asn262, and Glu325) are
influential in catalysis. Specifically, His256 has
conclusively been demonstrated to act as a nucleophile that attacks the
2-phosphate, resulting in a His256 phosphohistidine
intermediate (1, 13, 14, 21). His256 is perfectly placed
for an in-line attack that would break the O-2-P-2 bond of
Fru-2,6-P2 with an inversion of phosphate geometry (Fig.
3). Our own data on the retention of significant catalytic activity in
the H256A mutant (1) are contradictory to previous findings in the RL2K
enzyme (16) and indicate that this phosphoenzyme intermediate is not an
obligatory intermediate for the enzyme. Arg255 and
Arg305 have been proposed to neutralize the charge of the
2-phosphate and/or to stabilize the transition state (1, 18),
consistent with their location as equatorial ligands to the
2-phosphate. Glu325 has been proposed to act both to
promote the protonation of His390 and to polarize a water
molecule as a nucleophile for attack on the phosphohistidine
intermediate (8, 17, 21). The capacity of Glu325 to alter
the pKa of His390 is doubtful, since the
ionizable groups of these amino acids are not near each other (see
Figs. 2 and 3). The role for Glu325 as a catalytic base in
phosphohistidine hydrolysis is consistent with both its location in the
structure and the available kinetic data on Glu325 mutants
(1, 17). Finally, His390 has been proposed to act as a
catalytic acid, donating a proton to the leaving group O-2 of Fru-6-P
(8, 16). This, however, is inconsistent with the structure, since
His390 is not in the vicinity of the leaving group (Fig.
3), nor does it exist in an environment that would promote its
protonation. Our kinetic data from the H390A mutant protein are also
inconsistent with its role as a proton donor to the leaving group, in
that this mutant protein demonstrates a lag in phosphate release after Fru-6-P release (1), indicating that His390 is involved in
phosphohistidine hydrolysis. Thus, the role of H390, the mechanism of
catalysis in the H256A mutant, and the identity of the proton donor for
the leaving group O-2 of Fru-6-P remain unresolved from previous
findings. We believe that the Fru-2,6-Pase kinetic data (as cited
above) and the structural findings reported here lead to a complete
catalytic mechanism for the wild type enzyme and a distinct mechanism
for the H256A mutant enzyme.
Wild Type Mechanism--
Fig. 5
shows a Fru-2,6-Pase catalytic cycle where Glu325 is the
sole amino acid involved in proton exchanges. At the beginning of the
cycle (E·Fru-2,6-P2 from Scheme I),
His256 attacks the phosphate, leading to a pentacoordinate
transition state, with the excess negative charge of the transition
state being stabilized by Arg255, Asn262,
Arg305, and His390. A protonated
Glu325 then donates a proton to the leaving Fru-6-P. As
discussed above, Fru-6-P dissociation must precede the next step, where
the ionized Glu325 polarizes a water molecule for attack on
the phosphohistidine intermediate, regenerating the ground state of the
enzyme. The attractive features of this model are its simplicity and
its consistency with both the kinetic and structural data. The
proximity of Glu325 to the 2-oxygen of Fru-6-P and/or the
model of Fru-2,6-P2 and the kinetic data from
Glu325 mutants that show a drastic loss of Fru-2,6-Pase
activity (1, 17) make Glu325 a prime candidate for a major
role in catalysis. Glu325 is well positioned for proton
transfer to the 2-oxygen of Fru-6-P (Fig. 3, and Table II) and is well
positioned to polarize water for attack on the phosphohistidine
intermediate (21). The role of the amino acids that are equatorial
ligands in transition state stabilization is consistent with the
decreases in kcat/Km that are
observed when they are mutated to amino acids that cannot fill this
role. Where it has been studied, mutation of these equatorial ligands
clearly affects both transition states (i.e.
phosphohistidine formation and phosphohistidine hydrolysis). Note that
His390, which had previously been designated as a proton
donor, is now simply another equatorial ligand. This is consistent with
the kinetic data, where His390 mutations have effects
similar to the mutation of other equatorial ligands. Most important
though, is the observation that the H390A mutation causes a significant
lag in Pi release after Fru-6-P release (1). This lag in
Pi release is consistent with the role of
His390 in transition state stabilization but not consistent
with His390 as a proton donor to Fru-6-P.
There is one significant problem with this mechanism. The crystal
structure clearly shows that Glu325 is capping the N
terminus of an
One approach to address this apparent Glu325 paradox would
be pH titration kinetics of phosphohistidine formation in the wild-type and active site mutant enzymes to determine the pKa
values for Glu325 and other active site residues. Pilkis
and co-workers reported the pH titration of
kcat/Km for wild type, H392A,
and E327Q mutant forms of the RL2K enzyme (36). Their results with the
wild type eznyme indicated two ionizable groups with
pKa values of 6.1 and 8.4, which we would interpret
as the pKa of a Fru-2,6-P2 phosphate and
an unidentified residue of the protein, respectively. The titration
profiles of the H392A and E327Q mutants clearly showed a partial change
in the low end pKa with an increase in the
pKa. The curves were complex however, suggesting the
possibility of compensating charge interactions. It is evident that
further experiments in this area will be necessary to clarify the role
of ionizable groups in the Fru-2,6-P2ase active site.
H256A Catalysis: Hydrolysis Versus Phosphohistidine 390--
The
discovery that the H256A mutant of RT2K has 17% of the wild type
bisphosphatase activity has led to two alternative explanations of the
mechanism (1). Either the enzyme is able to utilize His390
as an alternate to His256 as a nucleophile to form a
phosphohistidine 390 intermediate, or another as yet undetermined
mechanism exists for the H256A mutant. Examination of the active site
structure (Fig. 3) leads to the conclusion that the transfer of a
phosphate to His390 is quite unlikely. As noted above,
His256 is ideally positioned for the in-line transfer of
phosphate from Fru-2,6-P2. His390, on the other
hand, is nearly 90° off this transfer axis, ideal positioning for an
equatorial ligand to the transition state intermediate, but horribly
suited for an in-line phosphotransfer. In order to accomplish a
phosphotransfer to His390, either by direct in-line attack
or by an attack from the side followed by pseudorotation (37, 38), the
substrate and/or His390 would have to be repositioned in
the active site. The position of His390 is of course
constrained by the fold of the protein and thus could not move from its
equatorial location to an axial position. Manual remodeling of Fru-6-P
in the active site to a position that would lead to an axial
His390 led only to steric clashes of Fru-6-P with protein.
From this evidence, combined with the inability to detect a
phosphohistidine intermediate in the H256A mutant by several methods
(1), we conclude that a phosphohistidine 390 intermediate pathway is
not at all likely.
An alternative hypothesis is that the H256A mechanism involves the
direct hydrolysis of Fru-2,6-P2 by water. Due to the
deletion of His256, there is a hole in the enzyme where the
imidazole ring used to be. In the crystal structure, this hole is
occupied by a free phosphate as described above (Fig. 2 and Table III).
When we model Fru-2,6-P2 bound to the enzyme by
superimposing the H256A and RT2K-Wo structures, this hole would be
empty in the H256A substrate complex. Since vacuums do not exist in
proteins, this space will be filled, and most likely by a water
molecule. Like the imidazole of His256, this water molecule
would be in the perfect position for an in-line transfer of the
phosphate. The equatorial ligands to the phosphate remain in the same
position in both the RT2K-Wo and H256A structures, so they would be
competent to stabilize this hydrolytic transition state in the mutant
enzyme. The best experiment to verify this hypothesis would be to
utilize a synthetic Fru-2,6-P2 that has been triply labeled
at the 2-phosphate with 16O, 17O, and
18O, generating a chiral phosphate. In the event of a
direct hydrolysis, there will be an inversion of the phosphate
stereochemistry, while for a two-step process involving a
phosphohistidine intermediate, there will be retention of
stereochemistry. Such an experiment was used to verify the
phosphohistidine intermediate in phosphoglycerate mutase (39).
-D-fructose-
6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate
(Fru-2,6-P2) at distinct active sites. A mutant rat
testis isozyme with an alanine replacement for the catalytic histidine
(H256A) in the Fru-2,6-Pase domain retains 17% of the wild type
activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal structure of H256A to a resolution of 2.4 Å by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the
important interactions in substrate/product binding. A superposition of
the H256A structure with the RT2K-Wo structure reveals no
significant reorganization of the active site resulting from the
binding of Fru-6-P or the H256A mutation. Using this superposition, we
have built a view of the Fru-2,6-P2-bound enzyme and
identify the residues responsible for catalysis. This analysis yields
distinct catalytic mechanisms for the wild type and mutant proteins.
The wild type mechanism would lead to an inefficient transfer of
a proton to the leaving group Fru-6-P, which is consistent with a view
of this event being rate-limiting, explaining the extremely slow turnover (0.032 s
1) of the Fru-2,6-Pase in all
Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-octyl glucoside) was mixed 1:1 with a
well solution of 17% polyethylene glycol 4000, 60-90 mM
succinate, pH 6.0, 10% glycerol, and 10 mM
MgCl2. Crystals were grown and stored at 4 °C.
-octyl glucoside, 10 mM Fru-6-P, and 1 mM AMP-PNP) with increasing
concentrations of glycerol (10-20% in 5% steps). These cryoprotected
crystals were flash frozen in liquid propane and subsequently
maintained at 120 K in a dry nitrogen stream using an X-Stream crystal
cooler (Molecular Structure Corp). Data were collected on a DIP-2020
image plate detector (MacScience) mounted on a Rigaku rotating anode
generator operated at 50 mA, 100 kV, with double mirror focusing
(MacScience). Diffraction intensities were integrated using the program
Denzo (25). All data were merged and scaled in Scalepack (25) and
formatted for subsequent use in XPLOR (26). Automatic indexing in Denzo indicated that the crystals belong to space group P1, and
postrefinement in Scalepack led to unit cell dimensions
a = 61.74 Å, b = 73.51 Å,
c = 76.70 Å;
= 116.9°,
= 99.31°, and
= 105.2°. Assuming two molecules per asymmetric unit, the calculated
Matthews coefficient (27) is 2.54 Å3/Da.
above the mean.
Rigid body minimization of this rotation solution using data from 20- to 2.8-Å resolution led to an Rfree of 0.51 for the polyalanine model and an Rfree of 0.42 with
all side chains included. To minimize model bias, initial electron
density maps were calculated with the polyalanine molecular replacement
phases and data from 30 to 2.4 Å, using SIGMAA (28) weighting as
implemented in XPLOR. Model rebuilding was accomplished using the
program O (version 6.1 (29)). The major changes in the structure are related to changes in the ligand-binding state of both catalytic domains and changes due to crystal packing. Refinement in XPLOR included the use of noncrystallographic restraints. Four groups were
defined (kinase-tight, kinase-loose, phosphatase-tight, and phosphatase-loose), using a weight of 100 for the tighter and 50 for
the looser restraints. Rebuilding, positional, and B-factor refinement
yielded the final model reported here with indicators of model quality
as reported in Table I. Coordinates have been deposited with the
Protein Data Bank (30), with accession code 2bif.
RESULTS
= 116.9°,
= 99.31°, and
= 105.2°, and there are two
monomers (the functional dimer) in the asymmetric unit. These crystals
diffract to 2.4 Å, and data were collected from specimens frozen at
cryogenic (120 K) temperatures. The structure was solved by molecular
replacement using the reported RT2K-Wo structure as a search model. A
summary of the overall data and model qualities is presented in Table I. The overall structure of the RT2K
dimer is similar to that reported earlier (7), i.e. a close
interaction between kinase domains, with essentially independent
phosphatase domains tethered to the kinase dimer. Because there are two
monomers in the asymmetric unit, we have determined two independent
structures of the monomer. For the kinase domains, the two copies are
different, with subtle conformational changes, and different ligands
bound.2 The two phosphatase
domains are quite similar in conformation and bound ligands, although
one copy has generally lower B-factors (monomer A average B-factor = 50.1; monomer B average B-factor = 32.3), due to closer crystal
packing for the phosphatase domain of monomer B. For this reason,
subsequent results and discussion will generally be restricted to the
phosphatase domain of monomer B.
Structure determination statistics
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Fig. 1.
Electron density of Fru-6-P bound in the
Fru-2,6-Pase active site. A stereo view of the final molecular
model of Fru-6-P bound in the Fru-2,6-Pase active site is shown, with
several neighboring amino acids labeled. The electron density mesh is
drawn at the 4.5 contour of an Fo
Fc difference map, calculated after Fru-6-P was
omitted, and the model was subjected to 2,500 K simulated annealing in
XPLOR (26). This figure was generated using the program O
(29).
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Fig. 2.
Fru-6-P interactions in the Fru-2,6-Pase
active site. A stereo view of the Fru-2,6-Pase active site is
shown, with the protein bonds colored tan, and
the Fru-6-P and phosphate bonds magenta. The various
interactions between protein and Fru-6-P that account for the affinity
and specificity of the active site are shown. The Fru-6-P 6-phosphate
is shown at the top, bound by interactions with
Arg350, Lys354, Tyr365, and
Arg395. The fructose portion of Fru-6-P is bound by both
hydrogen bonds and a hydrophobic stacking interaction, where
Ile267 can be seen to stack with the fructose ring and
Fru-6-P-O-2. The amide nitrogen of Gln391 has a bidentate
interaction with the fructose at O-5 and O-6. The main chain nitrogen
of Ile267 is hydrogen-bonded to Fru-6-P-O-3. The two water
molecules that form hydrogen bonds between Fru-6-P-O-1 and the protein
are shown as isolated red spheres. One of these
waters interacts with Fru-6-P, Tyr336, and the main chain
carbonyl of Thr443. The other water interacts with
Asn262 and the main chain carbonyl of position 266. The
free phosphate (analogous to the 2-phosphate of Fru-2,6-P2)
is shown at the bottom, bound by Arg255,
Asn262, Arg305, and His390. The
identities of the side chains are indicated with the one-letter amino
acid designation and position in the RT2K protein sequence. The side
chain of Thr443 has not been included so as not to obscure
the view of F-6-P. Not labeled and at the back of the image are
Arg255, Glu325, and His390,
interacting with the Fru-6-P molecule and/or the free phosphate.
Hydrogen bonds between the Fru-6-P, phosphate, and protein are drawn as
black lines. This figure was produced using
MOLSCRIPT (40) and rendered in Raster3D (41).
Fru-6-P interaction with the RT2K H256A mutant
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Fig. 3.
Model of Fru-2,6-P2 bound to the
RT2K-Wo active site. We have constructed a model of
Fru-2,6-P2 binding to the Fru-2,6-Pase active site based on
the coordinates of the RT2K-Wo structure (protein and 2-phosphate drawn
with tan bonds) and the coordinates of Fru-6-P
from the H256A structure (Fru-6-P drawn with cyan
bonds). We have not repositioned either the 2-phosphate or
the Fru-6-P, so there is a small (0.9-Å) gap between the Fru-6-P-O-2
and the phosphate oxygen. This gap would obviously not exist in
Fru-2,6-P2, since these represent the same oxygen. The
catalytic His256 is positioned in-line with the O-2-P
bond, while Asn262, His390, and
Arg255 are arranged perpindicular to that line, in a
position to interact with the equatorial oxygens in the pentacoordinate
transition state. Arg305 is not shown, to reduce the
clutter in the figure but would be positioned in the foreground,
interacting with the phosphate oxygen that is shown interacting with
His390. Glu325 is shown hydrogen-bonded with
Fru-6-P-O-2 and also interacting with the N terminus of helix 14
(labeled as 392 and 393). Ile267 is
included to clearly demonstrate the stacking interaction between this
side chain and the Fru-6-P. The identity of the side chains are
indicated with the one-letter amino acid designation and position in
the RT2K protein sequence. This figure was produced using
MOLSCRIPT (40) and rendered in Raster3D (41).
Comparison of PO4 interaction in RT2K-Wo and RT2K-H256A mutant
(monomer B)
DISCUSSION
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Fig. 4.
The "molecular ruler" of the Fru-2,6-Pase
active site. Based on the position of Fru-6-P in the Fru-2,6-Pase
active site, there is a 5.5-Å distance between the reactive nitrogen
of the catalytic histidine and the 2-OH of Fru-6-P (shown as the
dark bar at the top). This distance
can accommodate either an E·Fru-2,6-P2 or
E-P·Fru-6-P complex but would preclude either an
E-P·H2O·Fru-6-P or
E·P·Fru-6-P complex. The dark bars
below each complex represent ideal bond lengths or
approximate Van der Waals contact distances.
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Scheme 1.
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Fig. 5.
Fru-2,6-Pase catalytic cycle. The
proposed mechanism begins with formation of the
E·Fru-2,6-P2 complex shown in the
upper left panel. His256
attacks the 2-phosphate, resulting in a tetrahedral intermediate that
is stabilized by its interactions with Arg305,
Arg255, His390, and Asn262 as shown
in the upper right panel. A proton
derived from Glu325 then joins the leaving group Fru-6-P as
the O-2-P bond is broken. The resulting E-P·Fru-6-P
complex is stable, and Fru-6-P release from the active site is
rate-limiting. After Fru-6-P diffuses out of the active site, a water
molecule will diffuse in. Glu325 then activates the water
as a nucleophile to attack the E-P intermediate as shown in
the lower right panel. The
E-P hydrolysis requires the formation of another tetrahedral
intermediate, again stabilized by Arg305,
Arg255, His390, and Asn262 (not
shown). Finally, the free phosphate remains bound in the 2-phosphate
binding site (lower left panel) until
it is displaced by another molecule of Fru-2,6-P2,
restarting the cycle.
-helix (
14, amino acids 391-400, Fig. 3). Such a
protein environment would shift the pKa of
Glu325 such that the existence of a protonated
Glu325 is unlikely, making it a poor choice for the proton
donor in Fru-6-P release. On the other hand, this is in fact an enzyme with a low turnover (i.e. the wild type RT2K Fru-2,6-Pase
reaction has a kcat of 0.032 s
1).
Because the biological role of the enzyme is regulatory and is not a
step in a biosynthetic pathway, it makes some teleological sense that
the enzyme would be slow. As such, a rare protonation of
Glu325 and subsequent proton donation to Fru-6-P could be
tolerated. This idea of a slow Glu325 protonation is not in
agreement with the kinetic evidence, however, since phosphohistidine
formation has been reported to be 2 orders of magnitude faster than the
overall rate of catalysis, with the Fru-6-P release rate-limiting (34,
35). It is possible that Fru-2,6-P2 binding to the active
site shifts the Glu325 pKa via the
interaction of the 2-phosphate with the N terminus of helix
14 (Fig.
3).
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ACKNOWLEDGEMENTS |
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We thank Cu Nguyen and Yang Li for excellent technical assistance in the preparation of the enzyme, and the Howard Hughes Medical Institute x-ray facility for access to diffraction equipment.
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FOOTNOTES |
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* This work was supported by grants from the Welch Foundation, the American Heart Association, Texas Affiliate (to C. A. H.), and the Department of Veterans Affairs and National Institutes of Health Grants DK16194 (to K. U.) and GM36799 (to P. F. C.).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 structure factors (code 2bif) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
** To whom correspondence should be addressed: Dept. of Internal Medicine, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8884.
The abbreviations used are:
Fru-6-P, 2-kinase,
fructose-6-phosphate,2-kinase; Fru-2, 6-Pase,
fructose-2,6-bisphosphatase; Fru-2, 6-P2,
fructose-2,6-bisphosphate; Fru-6-P, -D-fructose-6-phosphate; AMP-PNP, 5'-adenylyl
imidodiphosphate; RL2K, rat liver isozyme of
fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase; RT2K, rat
testis isozyme of
fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase; RT2K-Wo, RT2K with all four tryptophans mutated to phenylalanine; H256A, a mutant form of RT2K-Wo with an additional histidine 256 to
alanine mutation.
2 M. H. Yuen and C. A. Hasemann, manuscript in preparation.
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REFERENCES |
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