From the Structural Biology Core, Molecular Biology,
University of Missouri, Columbia, Missouri 65211, the
Department of Biochemistry, University of Texas Southwestern
Medical Center, Dallas, Texas 75390, ¶ Research Service,
Veterans Affairs Medical Center, Dallas, Texas 75216, and
** Pfizer Global Research and Development, Ann Arbor
Laboratories, Ann Arbor, Michigan 48105
Received for publication, September 5, 2002, and in revised form, October 11, 2002
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ABSTRACT |
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The crystal structures of the human liver
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in three different
liganding states were determined and compared with those of the rat
testis isozyme. A set of amino acid sequence heterogeneity from the two distinct genes encoding the two different tissue isozymes leads to both
global and local conformational differences that may cause the
differences in catalytic properties of the two isozymes. The sequence
differences in a Due to the two independent catalytic domains in a single
polypeptide chain, the bifunctional enzyme,
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-Fru(P)-2-kinase/Fru-2,6-P2ase),1
catalyzes both the synthesis and degradation of
fructose-2,6-bisphosphate (Fru-2,6-P2), a most potent
allosteric regulator with dual functions as follows: an activator of
phosphofructokinase and, simultaneously, an inhibitor of
fructose-1,6-bisphosphatase, the key rate-limiting enzymes of
glycolysis and gluconeogenesis, respectively (1-3). The functional
duality allows Fru-2,6-P2 to play a critical role in
control of the rates of both glycolysis and gluconeogenesis. An
elevated cellular concentration of Fru-2,6-P2 increases
glycolytic flux, whereas a lowered concentration of
Fru-2,6-P2 results in a net increase in gluconeogenic flux.
Because the liver is the major organ for vertebrate glucose
homeostasis, regulation of the activity in the liver isoform of
6-Fru(P)-2-kinase/Fru-2,6-P2ase and the resulting
Fru-2,6-P2 regulates glucose homeostasis in an organism
(1-5). Reflecting the diversity of tissues and their functions, at
least six different tissue-specific isoforms from five distinct genes
express varying ratios of kinase to bisphosphatase activity. A single
isoform generally predominates in each distinct tissue (1, 4).
The determination of crystal structures of the rat liver
Fru-2,6-P2ase domain (6) and the rat testis
6-Fru(P)-2-kinase/Fru-2,6-P2ase (7) has led a great
progress in our understanding of the catalytic mechanisms of this
enzyme system (8, 9). However, one of the most fundamental questions
about this enzyme system has remained unanswered. How are the
structures of the tissue-specific isozymes of
6-Fru(P)-2-kinase/Fru-2,6-P2ase related to their
differential functions in tissues that have specific physiological
roles and, accordingly, different optimums of glucose metabolism?
Reflecting such differences in roles, the two isozymes have been shown
to have different kinetic properties, summarized in Table
I (adapted from Ref. 10). Despite a
reasonably high amino acid sequence identity (74%), the liver isoform
kinase shows a 4-fold higher affinity for Fru-6-P but half the
Vmax compared with the testis form, whereas the
bisphosphatase has at least 20-fold increased affinity for
Fru-2,6-P2 and a 2-fold increase in
Vmax (10). These kinetic differences suggest
that glucose metabolism is uniquely related to the physiological roles
of the given tissues and not just a simple housekeeping function for
energy production (10, 12, 13). This is more evident by the fact that
only the liver isozyme of the two isozymes is phosphorylated by the
cAMP-dependent protein kinase upon the signal of low levels
of glucose and/or glucagon. The dephosphorylation is achieved by
xylulose 5-phosphate (Xyl-5-P)-dependent protein
phosphatase 2A upon the detection of insulin and/or high glucose levels
(14-18). Phosphorylation of the liver
6-Fru(P)-2-kinase/Fru-2,6-P2ase induces an as yet unknown
conformational change by which the predominant activity switches from
the kinase to the bisphosphatase that leads to a decrease in the
cellular concentration of Fru-2,6-P2 (15, 16) and hence
stimulating gluconeogenesis.
hairpin loop in the kinase domain causes a
translational shift of several hydrophobic interactions in the dimeric
contact region, and its propagation to the domains interface results in
a 5° twist of the entire bisphosphatase domain relative to the kinase
domain. The bisphosphatase domain twist allows the dimeric interactions
between the bisphosphatase domains, which are negligible in the testis
enzyme, and as a result, the conformational stability of the domain is
increased. Sequence polymorphisms also confer small but significant
structural dissimilarities in the substrate-binding loops, allowing the
differentiated catalytic properties between the two different
tissue-type isozymes. Whereas the polymorphic sequence at the
bisphosphatase-active pocket suggests a more suitable substrate
binding, a similar extent of sequence differences at the kinase-active
pocket confers a different mechanism of substrates bindings to the
kinase-active pocket. It includes the ATP-sensitive unwinding of the
switch helix
5, which is a characteristic ATP-dependent
conformational change in the testis form. The
sequence-dependent structural difference disallows the liver kinase to follow the ATP-switch mechanism. Altogether these suggest that the liver isoform has structural features more appropriate for an elevated bisphosphatase activity, compared with that of the
testis form. The structural predisposition for bisphosphatase activity
in the liver isozyme is consistent with the liver-unique glucose
metabolic pathway, gluconeogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Comparison of the kinetic parameters of the isotype bifunctional
enzymes
Therefore, investigation of the structural differences in tissue
isoforms will provide a new insight into the structure/function relationship, which is differentiated according to the physiological roles of the tissues expressing those isozymes. To better understand this unique enzyme system and to provide a better target for drug design, the crystal structure of the human liver isoform, which has
kinetic properties nearly identical to the rat liver form of better
than 98% (as suggested by sequence identity) (11), was determined and
compared with the rat testis form, and the results are reported here.
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EXPERIMENTAL PROCEDURES |
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Preparation and Crystallization of the Human Liver Bifunctional
Enzyme--
The W0 mutant form of human liver
6-Fru(P)-2-kinase/Fru-2,6-P2ase (all Trp residues and Pro2
mutated to Phe and Arg, respectively) was employed for this study
because it has better expression in Escherichia coli with
the kinetic properties indifferent from those of the wild
type.2 The same mutant form
was used for the studies of the testis form (7). The protein was
expressed in E. coli BL21(DE3) and purified using DEAE and
Mono Q anion-exchange chromatography. Crystals were prepared by hanging
drop vapor diffusion at 12 mg ml1 protein (in 20 mM Tris-HCl, pH 7.25, 10 mM NaPi,
15% glycerol, 0.5 mM EDTA, 10 mM
dithiothreitol, 6 mM ATP
S·Mg or ADP, and 5-10 mM Fru-6-P) mixed 1:1 with a mother liquor of 50 mM Tris-HCl, pH 7.5, 20-25% ethylene glycol, and 8%
polyethylene glycol 4000. Crystals in a size of 0.2 × 0.2 × 0.05 mm were grown in 4-5 days.
Data Collection and Processing-- Crystals were submerged into liquid propane for flash freezing, and the low temperature (120 K) was maintained by an X-stream (MSC, The Woodlands, TX) cryogenic cooling device. Diffraction data were collected on an R-Axis IV image plate detector mounted on a Rigaku FR590 rotating anode generator, which was operated at 50 mA, 100 kV. The x-ray beam was focused using an Osmic confocal multilayer optic system. The data were integrated using DENZO and merged and scaled with SCALEPACK (20). The reduced data were formatted for the CCP4 suite (21), and 10% of the data were marked for free R-factor measurements in subsequent structure refinements. As shown in Table II, complete data sets with various resolution ranges depending on the liganding states were collected. All crystals belong to a C2 space group regardless of the bound ligands. The data statistics are summarized in Table II.
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Structure Determination and Refinement--
The structure of the
ATPS complex was determined first by the molecular replacement
method using X-PLOR (22). A single polyalanine form of the testis
structure (Protein Data Bank accession code 1bif) was used as a search
model. An X-PLOR rotation and subsequent translation search was
performed using a data subset between 15.0 and 4.0 Å. A self-rotation
search as well as the measurement of solvent fraction suggested a
non-crystallographic dimer of the liver enzyme in 1 asymmetric unit.
Assignment of the side chains and non-crystallographic dimerization was
made using O (23). The initial model went through iterated cycles of
manual model rebuilding and X-PLOR refinement, followed by two cycles
of "refmac" refinement (21). During this procedure, a single model
was the subject of all manual model corrections to prevent the
accumulation of structural errors developed during the refinement
cycles. When Rcrys/Rfree,
reached 0.254/0.289, one molecule of ATP
S per protein molecule was
built into the model, referring to an Fo
Fc omit map. As shown in Table II, the final model
has a Rcrys/Rfree factor
of 0.217/0.257 using a total of 7,545 scatterers, including solvent molecules, against 42,142 reflections. Of the 864 residues in the
dimer, 89.3% are within the "most favorable region" of main chain
dihedral angles distributions and none in the "disallowed region,"
when judged using "procheck" (21). The final structure was
deposited in the Protein Data Bank under the accession code of
1K6M.
The subsequent structures of the liver isozyme complexed with either
ADP or Pi were determined by using the structure of the ATPS complex as the starting model and by following the structural refinements procedures similar to that used for the ATP
S complex. But, to guarantee a freedom to structural refinements, the indices of
reflections in the free data were kept as those of the ATP
S complex.
The B-factor refinement of the Pi complex was stopped after
one cycle of group B-factor refinement, and the solvent molecules were
not added to the structure due to a limited resolution. The statistics
of reflection data and structure refinements are summarized in Table
II.
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RESULTS AND DISCUSSION |
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Similarity of Overall Folding in the Liver and Testis
Isoforms--
The crystal structures of the liver isozyme reveal a
head-to-head homodimer of 432 amino acid residues per monomer (residues 39-470) (Fig. 1a and 2).
Depending on the liganding conditions, each subunit contains an
ATPS, ADP, or Pi molecule bound to the kinase domain,
and two inorganic phosphates, regardless of the liganding conditions,
bound to the bisphosphatase domain (Table II). No electron density is
interpretable for the N-terminal regulatory fragment of residues 1-38,
in which Ser-32, the cAMP-dependent phosphorylation site
(24-27), and a number of amino acid sequence differences are located
(Fig. 2). The structure of this fragment thus remained undetermined in both the liver and testis isozymes.
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As predicted from a reasonably high sequence identity (74%) of tissue isozymes (Fig. 2), the liver bifunctional enzyme has a structure very similar to that of the testis isoform, consistent with similarities in their catalytic mechanisms (7, 9-11, 28). As shown in Fig. 1b, where the ribbon diagrams of the two isozyme kinase domains are superimposed, the overall folding of the liver form is very similar to that of the testis form. The similarity in folding patterns is maintained throughout both the kinase and phosphatase domains. The main structural characteristics are conserved in the liver form, i.e. the major dimeric interactions through the kinase domains, the constellation of catalytic residues in the active sites of each domain, and the manner of intramonomer domain-domain interactions.
All this together allowed us to assign the secondary structural motifs of the liver form according to those of the testis form (7) with no ambiguity, and for convenience of comparison, the amino acid sequences are compared with the assigned secondary structures in Fig. 2. Unless specifically mentioned, for the entire text of this report, all the amino acid sequence and numbers are according to the liver isozyme. For example, T224Q is Thr-224 in human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and is Gln in the same position of the testis isozyme.
Bisphosphatase Twist and Differentiated Dimeric
Interface--
Although the number is small, the amino acid sequence
differences between the two tissue isozymes seem to cause a variety of
conformational differences. That includes a twist of the bisphosphatase domain relative to the kinase domain, resulting in an altered dimeric
interface. Despite the fact that the amino acid residues involved in
this interface are relatively well conserved, the bisphosphatase domain
of the liver form is twisted by 5° around the dimeric axis from its
position in the testis form (Fig. 1b). We considered three
possibilities of this twist as follows: an indirect effect of the
mobile N terminus, a difference in crystal contacts between the two
isoforms, and an intrinsic difference coming from the amino acid
sequence. A possibility of an effect of phosphorylation of Ser-32 at
the mobile N terminus is ignorable since the enzyme was expressed in
bacterium and purified in the absence of any protein phosphatase
inhibitor such as fluoride. To test its relevance to liganding states
and crystal contacts, the three structures of the testis isoform all in
different crystal contacts and liganding states (ATPS, AMP-PNP, and
Pi) (7, 9, 28) and the three structures of the liver form
in three different liganding states (ATP
S, ADP, and Pi)
were cross-compared by superimposing onto each other. The positional
differences of C
s were calculated and presented as Table
III. The liver form ADP complex and the
testis form AMPPNP complex were excluded from this table, because they
were so similar to the liver form ATP
S complex and the testis form
ATP
S complex, respectively, with a difference range of <0.5 Å. It
is evident that the overall difference (~2 Å) between the two tissue
isozymes is mainly caused by the bisphosphatase domain (~3 Å). This
difference is not significantly affected by the liganding states, which
cause less than 1 Å in magnitude. Legitimacy of the positional
difference was tested by rotating the testis bisphosphatase domain by
5°, and the result, which is 2.8 Å, was found to be close to the
value in the table. Because no different space group with reasonable
resolution is available for the liver form, it is impossible to
directly infer the effect of crystal contacts on the liver form.
However, comparisons between the structures of the testis form, which
have three different space groups (P1 for the Pi
complex, C2 for the AMPPNP complex, and
P3121 for the ATP
S complex), allowed an
indirect observation. When these three structures were compared, the
positional differences caused by the different crystal contacts are
neither domain-specific nor significant (<0.8 Å). This observation
becomes more convincing by the fact that the major source of crystal
contacts in both isoforms is provided by the conserved dimeric kinase
interactions. All this together suggests that the rotated
bisphosphatase is a featuring conformation of the liver form.
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Moreover, it was found that the amino acid sequence differences in the
kinase domain can cause a twist of the liver form bisphosphatase domain. As in the testis form, the kinase/bisphosphatase interface of
the liver enzyme includes a structure in which one hairpin between
1-5 and
1-6 (residues 215-230 in the liver form and 213-228
in the testis form) in the kinase domain forms a set of hydrophobic
interactions with the following helix
7 in the kinase domain, which
in turn makes tight interactions with the bisphosphatase helix
13
(7). A major portion of this junction also serves as the dimeric
interface through intermonomeric kinase domain interactions (Fig.
3a). Sequence heterogeneity
introduced at the three residues, T224Q, R225S, and Q232A, of this
hairpin may have caused the bisphosphatase domain twist and, also,
affected the dimeric interactions. As shown in Fig. 3a,
the hairpin loop in the liver type forms a new intermolecular salt
bridge with Glu-208 of the dimeric partner molecule, because of a newly
introduced positive charge in Arg-225, and this tends to pull the loop
upward. This upward movement is strengthened by T224Q, which forms a
new hydrogen bond with the carbonyl O of Asp-221 in the same subunit, losing the intersubunit hydrogen bonding with Arg-230. Q232A also helps
this structural change by introducing a new hydrogen bond to Ser-215.
As a result, the hairpin structure is rotated by ~3° and shifted by
0.4-0.6 Å with respect to that of the testis form. To adjust for this
structural change and to maintain tight hydrophobic interactions with
the hairpin, the helix
7 has made a translational shift (Fig.
3b), which is helped by a new hydrogen bond between Q232A
and Ser-215 (Fig. 3a). The shift of
7 induces a similar type of movement in
13. In this way, a small shift of the
-hairpin is propagated to the rest of the structural components of
the junction between the kinase and bisphosphatase domains, eventually causing a 5° swing of the bisphosphatase domain (Fig.
1b).
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Intramolecular rotation of the bisphosphatase domain appears to enhance the overall stability of the bisphosphatase domains in the liver form by introducing new dimeric interactions between the Fru-2,6-P2ase domains. As summarized in Table IV, the conformation distinguishes the liver isozyme from the testis form, causes a 4-fold increase in the buried surface area between the two bisphosphatase domains, and decreases the distances in the dimeric kinase/bisphosphatase interaction as well. Interestingly, neither the increased contact areas nor the decreased distances between the two bisphosphatase domains are caused by amino acid sequence differences within the bisphosphatase domain. The polymorphic sequences in the kinase domain that induce the twist between the kinase and bisphosphatase domains are the sole cause of increases in the dimeric interactions between the bisphosphatase domains.
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When deviations from mean B-factors of the testis ATPS complex and
the liver ATP
S complex are plotted, significant differences in the
stability of the 6-Fru(P)-2-kinase (residues 39-249) and Fru-2,6-P2ase (residues 250-470) domains of the two
isozymes are revealed (Fig. 4). A very
similar pattern was also shown in other structures (data not shown for
clear comparisons). This comparison dramatically shows that the
Fru-2,6-P2ase domain of the liver isozyme is the more
stable half of the monomer liver isozyme. Conversely, the
6-Fru(P)-2-kinase domain of the testis isozyme is the more stable
domain. This distinct domain stability is not an artifact of different
crystal contacts between the two isoforms. As shown on the bottom of
the plot, most of the crystal contacts are shared, and the several
unique contacts do not explain the unique stability of the
tissue-specific isoforms. This difference was apparent throughout the
structure refinement process, where the liver bisphosphatase domain
always had an overall more interpretable density map than the kinase
domain, opposite the situation seen in the testis structures. The liver
bisphosphatase domain is more stable than its own kinase domain and the
testis bisphosphatase domain. On the contrary, the testis kinase domain
is more stable than its bisphosphatase domain and the liver kinase
domain. However, it is unclear whether the increase in the buried
surface area by the twisted bisphosphatase causes the liver
bisphosphatase domain to be more stable.
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Differentiated Substrate-binding Loops in the Kinase
Domain--
Outside of the N-terminal regulatory motif (residues
1-38), polymorphic amino acid sequences between the liver and testis isozymes are most frequent in the substrate binding loops of the kinase
domain: the Fru-6-P loop (residues 80-120) and the ATP loop (residues
160-200) (Fig. 2). In the testis isozyme, it was shown that the last
turn of helix 5 (residues 162-175) undergoes unwinding upon binding
of ATP to recruit Lys-172 to the bridge oxygen between the
- and
-phosphates of the bound ATP (9, 29). This ATP-driven conformational
change has been suggested to be transmitted to the Fru-6-P loop via the
loop coupling composed of the salt bridges between Asp-177, Arg-78, and
Arg-193 and the hydrogen bonds between Arg-181 and the carbonyl oxygens
of Tyr-85 and Lys-86 (9, 29). Thus, the ATP binding was suggested to be
a prerequisite for the subsequent binding of Fru-6-P to the kinase
domain in the testis kinase reaction, following the ordered binding of substrates.
Like the testis form and as shown in Fig.
5a, the structure of the liver
form also has a "switch-on" conformation induced by the bound ATP.
Neither of the two additional structures of the liver enzyme complexed
with ADP or Pi shows the "switch-off" conformation,
which is shown by the testis enzyme upon bindings of ADP or
Pi (Fig. 5b). Furthermore, substitution of
cysteine for Arg-183 in the liver isozyme (C183R) breaks the outer lobe coupling of the ATP and Fru-6-P loops (Fig. 5c). It is
believed that the sequence differences in this region stabilize the
"switch-on" conformation of the liver enzyme. Mutation in G164E
appears to greatly enhance the hydrophobic interactions between I165V
and Val-222 by introducing more flexibility into the first turn of the
switch helix (5), and as a result, the distance between Ile-165 and
Val-222 is shortened to 3.8 Å from 4.9 Å. In addition, a different sequence at E168A introduces a new hydrogen bond to the main chain N of
Gln-172. Furthermore, the residue differences of V188A, L189T, and
L193M add new sources of interactions to a local hydrophobic pocket
made of Tyr-180, Leu-175, Phe-192, and Ile-170 (Fig. 5a), which has been shown to be important in the binding of ATP and Fru-6-P
(9).
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These sequence-related structural differences altogether appear to
stabilize the switch-on conformation of the liver isoform by
locking down the switch-on conformation and to isolate the Fru-6-P loop
from the conformational changes in the ATP loop. To have the
"off" (ATP-free) conformation suggested in the testis enzyme, the new hydrophobic interactions introduced by V188A, L189T,
and L193M have to be broken. Thus, it appears that the helix (5) of
the liver form would experience a much higher energy barrier than that
of the testis isoform when it returns to the ATP-free conformation.
Because of the additional hydrogen bond through the side chain of E168A
and the strengthened local hydrophobic interactions, it is likely that
the ATP-induced switch-on conformation of the liver form is more stable
than that of the testis form. The two substrate loops are mutually
independent from the conformations of the other, resulting in an
enhanced affinity for Fru-6-P. This observation allows the conclusion
that the substrate binding to the liver kinase follows a random
fashion, as originally suggested by the previous kinetic studies of the
liver form (30), and that stabilized ATP-induced switch-on conformation
together with the decoupled substrate loops may contribute to a 4-fold
higher affinity of the liver kinase for Fru-6-P (1, 10).
Differentiation in the Bisphosphatase Domain--
As mentioned
earlier, a prominent feature of the liver isozyme is the increased
stability of the bisphosphatase domain induced by the bisphosphatase
twist and the concomitant increase in dimeric interactions between the
bisphosphatase domains (Table IV). In addition, polymorphic amino acid
residues apparently contribute to an enhanced stability of loops
involved in substrate binding to the bisphosphatase domain. The
sequence change at R266K introduces a new salt bridge between Arg-266
and Glu-262. In addition, a change at S273P stabilizes the loop
(residues 260-280), which surrounds the 2-P position of the
bisphosphatase-active site, by forming a new hydrogen bond with the
main chain N of Leu-263 (Fig. 6).
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The amino acid difference at residue E347L pushes Glu-347 away from the
hydrophobic pocket consisting of E347L, Leu-351, and Ile-455 and
includes Tyr-359 instead (Fig. 6). The rearranged hydrophobic pocket
shifts the loop 358-361 such that a salt bridge between Arg-360 and
Asp-369 becomes more stable. The distance of this salt bridge is
shortened from 3.5 to 2.7 Å, and as a result, the B-factors of Arg-360
and Asp-369 decrease by ~27 and 7 Å2, respectively, from
those of the testis form. In addition, a new salt bridge between
Glu-335 and Lys-363 is formed by the enhanced dimeric bisphosphatase
interactions, and as a result, the B-factors are decreased by 15 and 19 Å2 from those of the testis form. This conformational
change makes the loop (residues 335-363) more stable, which serves as
a lid of the active site of the bisphosphatase domain (6, 7) and harbors a number of residues interacting with the 6-phosphate moiety of
substrate and product (31, 32). This loop has served as a popular
target of the earlier structure/function studies and has been shown to
be critical in the proper binding of both Fru-6-P and
Fru-2,6-P2. The site-directed mutants, Arg-352 Ala, Lys-356
Ala, and Arg-360
Ala, showed more than 100-fold
decreases in binding of both Fru-6-P and Fru-2,6-P2. Thus,
in addition to the domain stabilization by the twist, it is likely that
stabilization of both the 2-P and the 6-P loop structures by the amino
acid sequence of the liver isozyme plays an important role in the
20-fold increase in its affinity for Fru-2,6-P2 compared
with that of the testis form, reflecting the differences in their
kinetic and allosteric properties, consistent with their different
physiological roles (1-3, 11-13).
Physiological Significance--
Sequence differences between the
human liver and rat testis isozymes result in both local and global
differences in the structures of these isozymes. The local
conformational differences in the liver form kinase domain result in a
loose coupling (or decoupling) of the Fru-6-P- and ATP-binding loops
and a stabilized switch-on conformation even without ATP. These
structural differences may allow the Fru-6-P binding to be independent
of ATP binding unlike the testis form, in which ATP-binding is
prerequisite for Fru-6-P binding. This structure-related functional
difference may be related to the physiological roles of the tissues
that harbor these isozymes. To regulate the blood glucose levels, the
liver appears to harbor an isozyme that is more sensitive to the
changes in Fru-6-P concentration, which is a more accurate indicator of
glucose concentration than that of ATP. Meanwhile, the testis isozyme,
which functions to maintain a steady level of energy production,
appears to adopt a structure more sensitive to the changes in ATP
concentration, a more accurate indicator of the cellular energy level
than Fru-6-P. Stabilization of the whole bisphosphatase domain by the
"domain twist" and its Fru-2,6-P2-binding loops by the
local sequence differences in the liver isozyme appears to enhance its
affinity for Fru-2,6-P2. Considering that the
Fru-2,6-P2 concentration is a critical factor in
determining the flux through the glucose pathways in liver, it is
reasonable that the liver bisphosphatase domain has a keen sensitivity
to the concentration of Fru-2,6-P2 to actively engage in
such a mission. However, by having lower affinity for
Fru-2,6-P2, the testis isozyme is likely to have the
bisphosphatase domain functioning not to determine direction of the
pathways but to regulate only the rate of glycolysis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK52089 (to C. A. H.) and DK16194 (to K. U.).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 1K6M) 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: Structural Biology Core, Molecular Biology, 125 Chemistry Bldg., University of Missouri, Columbia, MO 65211. Tel.: 573-884-5767; Fax: 573-882-2754; E-mail: leeyon@missouri.edu.
Published, JBC Papers in Press, October 11, 2002, DOI 10.1074/jbc.M209105200
2 K. Uyeda, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
6-Fru(P)-2-kinase/Fru-2, 6-P2ase,
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase;
Fru-2, 6-P2, fructose 2,6-bisphosphate;
Fru-2, 6-P2ase, fructose-2,6-bisphosphatase;
Fru-6-P, fructose 6-phosphate;
PFK, phosphofructokinase;
FBPase, fructose-1,6-bisphosphatase;
PP2A, protein phosphatase 2A;
Xyl-5-P, xylulose 5-phosphate;
ATPS, adenosine
5'-O-(thiotriphosphate);
AMP-PNP, adenosine
5'-(
,
-imino)triphosphate.
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REFERENCES |
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