From the Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011
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ABSTRACT |
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Asn64, Asp68,
Lys71, Lys72, and Asp74 of porcine
liver fructose-1,6-bisphosphatase (FBPase) are conserved residues and
part of a loop for which no electron density has been observed in
crystal structures. Yet mutations of the above dramatically affect
catalytic rates and/or AMP inhibition. The Asp74 Ala
and Asp74
Asn mutant enzymes exhibited 50,000- and
2,000-fold reductions, respectively, in kcat
relative to wild-type FBPase. The pH optimum for the catalytic activity
of the Asp74
Glu, Asp68
Glu,
Asn64
Gln, and Asn64
Ala mutant enzymes
shifted from pH 7.0 (wild-type enzyme) to pH 8.5, whereas the
Lys71
Ala mutant and Lys71,72
Met
double mutant had optimum activity at pH 7.5. Mg2+
cooperativity, Km for fructose 1,6-bisphosphate,
and Ki for fructose 2,6-bisphosphate were
comparable for the mutant and wild-type enzymes. Nevertheless, for the
Asp74
Glu, Asp68
Glu, Asn64
Gln, and Asn64
Ala mutants, the binding affinity
for Mg2+ decreased by 40-125-fold relative to the
wild-type enzyme. In addition, the Asp74
Glu and
Asn64
Ala mutants exhibited no AMP cooperativity, and
the kinetic mechanism of AMP inhibition with respect to
Mg2+ was changed from competitive to noncompetitive. The
double mutation Lys71,72
Met increased
Ki for AMP by 175-fold and increased Mg2+ affinity by 2-fold relative to wild-type FBPase. The
results reported here strongly suggest that loop 51-72 is important
for catalytic activity and the mechanism of allosteric inhibition of
FBPase by AMP.
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INTRODUCTION |
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Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase1), a key regulatory enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate and Pi in the presence of divalent metal ions such as Mg2+, Mn2+, and Zn2+ (1, 2). The activation of the enzyme by Mg2+ exhibits sigmoidal kinetics with a Hill coefficient of 2 at neutral pH, but is hyperbolic at pH 9.6 (3, 4). FBPase is inhibited synergistically by AMP (as an allosteric effector) and fructose 2,6-bisphosphate (Fru-2,6-P2; as a substrate analog) (5-7). In contrast, AMP and Fru-2,6-P2 act as strong activators for phosphofructokinase, a major regulatory enzyme in the opposing glycolytic pathway.
In mammals, FBPase is a homotetramer with a subunit Mr of 37,000 (8). Each subunit of the tetramer (designated C1, C2, C3, and C4) has an allosteric AMP domain (residues 1-200) and a catalytic Fru-1,6-P2 domain (residues 201-335), with the AMP-binding site being ~28 Å away from the substrate-binding site (9-11). Structures of AMP complexes of the enzyme define the T-state, whereas structures in the presence substrate analogs and without AMP represent the R-state. The structural transition (R- to T-state) involves a 17° rotation of the C1-C2 dimer with respect to the C3-C4 dimer and a 1.9° rotation of the AMP domain relative to the Fru-1,6-P2 domain within each subunit (10). The R- to T-state transition results in conformational changes at interfaces between subunits C1 and C2 and subunits C1 and C4 (as well as interfaces related to these by the symmetry of the tetramer). Metal-binding sites (up to three total) are at or near the active site at the interface between the two domains of the enzyme (12, 13). Mg2+ and AMP are mutually exclusive in their binding to FBPase (14, 15). In fact, AMP inhibition is nonlinear and noncompetitive with respect to Fru-1,6-P2 and nonlinear and competitive with respect to Mg2+. Yet crystallographic studies reveal similar metal coordination in the absence and presence AMP (either one Mg2+ ion or two Zn2+ or two Mn2+ ions), although small perturbations in the active site are attributed to the 1.9° rotation of the AMP relative to the Fru-1,6-P2 domain due to AMP binding (13).
Site-directed mutagenesis of the residues involved at subunit
interfaces of porcine liver FBPase revealed significant changes in the
kinetic mechanism of AMP inhibition and cooperativity (16-19). However, a detailed mechanism of catalysis and allosteric regulation based on changes in interacting residues has been elusive. Perhaps published structures of FBPase do not reveal all of the essential structural elements for catalysis and regulation. Mutation of Arg49, a residue that precedes a disordered loop (residues
54-71), causes dramatic changes in FBPase kinetics (17). Comparison of
the amino acid sequences of mammalian FBPases reveals
Gly58, Gly61, Asn64,
Asp68, Asp74, Lys71, and
Lys72 as conserved residues within this disordered
loop. Furthermore, loop 54-71 is proteolytically sensitive in all
known FBPases (20). To determine whether loop 54-71 plays a major role
in FBPase kinetics, mutant enzymes Asn64 Gln,
Asn64
Ala, Asp68
Glu, Lys71
Ala, Lys71,72
Met (double mutation),
Asp74
Ala, Asp74
Asn, and
Asp74
Glu were expressed in Escherichia
coli, purified to homogeneity, and evaluated by initial velocity
kinetics. The above mutations have profound effects on both catalysis
and regulation of FBPase.
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EXPERIMENTAL PROCEDURE |
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Materials--
Fru-1,6-P2,
Fru-2,6-P2, NADP, AMP, ampicillin, tetracycline, and
isopropyl--D-thiogalactopyranoside were purchased from
Sigma. DNA-modifying and restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were either from New England Biolabs Inc. or Promega.
Glucose-6-phosphate dehydrogenase and phosphoglucose isomerase were
supplied by Boehringer Mannheim. Tryptone, yeast extract, and agar were
from Difco. Other chemicals were of reagent grade or the
equivalent.
Mutagenesis of FBPase--
Mutations were accomplished by the
introduction of specific base changes into a double-stranded DNA
plasmid. Eight mutagenic primers
(5'-TGGCTCCACGCAGGTGACAGG-3',
5'-TGGCTCCACGGCCGTGACAGGT-3', 5'-GTGACAGGTGAGCAAGTGAAG-3',
5'-GTGATCAAGTGGCGAAGTTGGATG-3', 5'-GTGATCAAGTGATGATGTTGGATGT-3',
5'-GAAGTTGGCGGTCCTCTCCA3', 5'-GAAGAAGTTGGAAGTCCTCTC-3', and
5'-GAAGTTGGAGGTCCTCTCCAA-3') were used to mutate
Asn64 Gln, Asn64
Ala, Asp68
Glu, Lys71
Ala, Lys71,72
Met,
Asp74
Ala, Asp74
Asn, and
Asp74
Glu. A selective primer
(5'-CAGCCTCGCGTCGCGAACGCCAG-3') was used to change the unique
XhoI site to the original NruI site on the
pET-11a vector. The double-stranded FBPase expression plasmid (pET-FBP)
and mutagenic and selective primers were denatured, annealed, and
polymerized using the TransformerTM site-Directed
mutagenesis kit (21). Mutations were confirmed by
XhoI/NruI digestion and by fluorescent dideoxy
chain termination sequencing at the Nucleic Acid Facility at Iowa State
University. The plasmids with mutations were transformed into E. coli strain DF 657. The plasmid DNA was isolated from the
overnight culture, and sequence analysis was performed on the entire
FBPase gene.
Expression and Purification of Wild-type and Mutant FBPases-- The expression and purification of wild-type and mutant forms of FBPase were carried out as described previously (22) with slight modification. After centrifugation to remove cell debris, the supernatant was subjected to heat treatment, 30-70% ammonium sulfate precipitation, Sephadex G-200 column chromatography, and CM-Sepharose column chromatography. Wild-type and mutant FBPases eluted as a single peak from the CM-Sepharose column by a NaCl gradient from 20 to 400 mM in 10 mM malonate buffer, pH 6.0. Protein purity was evaluated by 12% SDS-polyacrylamide gel electrophoresis according to Laemmli (23). Protein concentrations were determined as described by Bradford (24) using bovine serum albumin as the standard.
Kinetic Studies--
Specific activity during purification was
determined by the phosphoglucoisomerase and glucose-6-phosphate
dehydrogenase coupled spectrometric assay at either pH 7.5 or 9.6 (1).
All other kinetic experiments were done at either pH 7.5 or 8.5 and
25 °C using a coupled spectrofluorometric assay (15). Initial rate data were analyzed using a computer program written in MINITAB language
with an value of 2.0 (25). Cooperativity was evaluated using either
the ENZFITTER program (26) or the MINITAB program.
Circular Dichroism Spectrometry-- CD studies on the wild-type and mutant FBPases were carried out in a Jasco J710 CD spectrometer in a 1-mm cell at room temperature using a 0.2 mg/ml concentration of the enzyme. Spectra were collected from 200 to 260 nm in increments of 1.3 nm, and each spectrum was blank-corrected and smoothed using the software package provided with the instrument.
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RESULTS |
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Purification of Wild-type and Mutant Forms of
FBPase--
Purification of mutant and wild-type enzymes followed
previous protocols (22). Mutant enzymes exhibited elution patterns similar to wild-type FBPase, except for Lys71 Ala and
Lys71,72
Met, which eluted from the CM-Sepharose column
at 100 mM NaCl. On the basis of SDS-polyacrylamide gel
electrophoresis, all enzymes exhibit identical mobilities
(Mr ~ 37, 500) with no evidence of proteolysis
(27) and purity greater than 95% (data not shown). Activity ratios (pH
7.5:9.6) for the wild-type, Lys71
Ala, and double
mutant Lys71,72
Met enzymes confirmed the absence of
proteolysis.
Secondary Structure Analysis-- The CD spectra of wild-type and mutant FBPases are essentially superimposable from 200 to 260 nm (data not shown), indicating the absence of global structural alteration changes caused by the mutations.
Catalytic Rates of FBPase Mutants--
Initial rate studies were
done at saturating concentrations of Fru-1,6-P2 or
Mg2+ that do not cause substrate inhibition. Kinetic
parameters are in Table I. The
kcat values for the Asp74 Ala
and Asp74
Asn mutants decreased 50,000 and 2,000 times,
respectively, whereas a shift in the pH of optimum activity from pH 7.5 to 8.5 was exhibited by the Asp74
Glu mutant with only
a 20-fold reduction in kcat. A
similar alteration in the pH of optimum activity occurred with mutants Asn64
Gln, Asn64
Ala, and
Asp68
Glu. On the other hand, replacements
Lys71
Ala and Lys71,72
Met did not
alter the pH of optimum activity. In Table I, the kinetic parameters
for the Asp74
Glu, Asn64
Gln,
Asn64
Ala, and wild-type FBPases were measured at pH
8.5, and for mutants Asp68
Glu, Lys71
Ala, and Lys71,72
Met at pH 7.5. A slight change in
Fru-1,6-P2 affinity was observed in all mutants relative to
wild-type FBPase.
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Mg2+ Activation--
The activity of wild-type FBPase
as a function of Mg2+ concentration is sigmoidal at neutral
pH with a Hill coefficient of ~2, but is hyperbolic at pH 9.6 (3, 4).
The Hill coefficient for Mg2+ and the Ka
for Mg2+ of the wild-type and mutant forms of FBPase were
determined here at a saturating Fru-1,6-P2 concentration
(30 µM) at pH 7.5 or 8.5. Hill coefficients at pH 8.5 dropped from 2.2 for the wild-type enzyme to ~1.4 for the
Asp74 Glu, Asn64
Gln, and
Asn64
Ala mutants, and at pH 7.5 from 2.0 to 1.4 for
the Asp68
Glu mutant. Increases of 126-, 51-, 47-, and
39-fold in Ka values for Mg2+ were found
for the Asp74
Glu, Asp68
Glu,
Asn64
Gln, and Asn64
Ala mutants,
respectively, relative to the wild-type enzyme. The Lys71
Ala and Lys71,72
Met mutant enzymes as well as
wild-type FBPase showed sigmoidal kinetics for Mg2+ at pH
7.5. On the other hand, the affinity for Mg2+ at pH 7.5 increased 2-fold for the Lys71
Ala and
Lys71,72
Met mutant enzymes relative to wild-type
FBPase.
Fru-2,6-P2 Inhibition--
Fru-2,6-P2 is a
competitive inhibitor of Fru-1,6-P2 and competes with the
substrate for the active site of FBPase (7, 29). Ki
values for Fru-2,6-P2 decreased by <4-fold for all mutant
enzymes compared with the wild-type enzyme. On the other hand, the
Lys71,72 Met mutant exhibited a 7-fold increase in
Ki for Fru-2,6-P2.
Kinetics of AMP Inhibition--
AMP is an allosteric regulator of
FBPase (30). The action of AMP inhibition is nonlinear and
noncompetitive with respect to Fru-1,6-P2 (31), but
nonlinear and competitive relative to Mg2+ for wild-type
FBPase at either neutral or alkaline pH (15). AMP binding to FBPase is
cooperative with a Hill coefficient of ~2 (4). The wild-type and
Lys71 Ala, Lys71,72
Met,
Asp68
Glu, and Asn64
Gln FBPases
exhibited competitive inhibition patterns for AMP relative to
Mg2+, whereas the Asp74
Glu and
Asn64
Ala mutants showed noncompetitive inhibition.
Fig. 1 shows double-reciprocal plots of
1/velocity versus 1/[Mg2+]2 at
various fixed concentrations of AMP for the Lys71,72
Met mutant of FBPase. The data of Fig. 1 are consistent with a
steady-state random mechanism represented by Equation 1,
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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DISCUSSION |
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Amino acid sequences of all known mammalian FBPases are well
conserved, particularly in segment 52-72, where 10 amino acids are
highly conserved, suggesting an important functional or structural role. In all published crystal structures of FBPase (9-13), however, little or no electron density is present for residues 52-72, which is
indicative of conformational disorder or proteolytic damage. The latter
possibility is unlikely as microsequencing detected no proteolysis
(10). Reduced kcat values for the
Asp74 Ala, Asp74
Asn, and
Asp74
Glu FBPases by 50,000-, 2,000-, and 20-fold,
respectively, along with the shift in the pH of optimum activity from
neutral to alkaline for the latter mutant, demonstrate the importance of position 74 in catalysis. Given the sensitive nature of position 74, conformational changes in the adjacent segment 52-72 could leverage
significant alterations in the catalytic properties of FBPase.
Mutations Asn64
Ala, Asn64
Gln, and
Asp68
Glu, for instance, change the pH of optimum
activity from neutral to alkaline and decrease
kcat values from 3- to 4-fold relative to that
of the wild-type enzyme, and the double mutation Lys71,72
Met elevates the Ki for AMP nearly 175-fold. The
activity profiles of the Asp74
Glu, Asn64
Ala, Asn64
Gln, and Asp68
Glu
mutants are very similar to those obtained with the proteolyzed enzyme
in which residues 1-64 and 1-25 are missing (32-34).
The analysis above is strengthened by the crystal
structure2 (2.3-Å
resolution, R-factor = 0.165, R-free = 0.24) of recombinant wild-type porcine liver FBPase with the products
Fru-6-P, Pi, and Zn2+, which reveals strong
electron density for loop 52-72 and a significant network of hydrogen
bonds involving Asp68 , Asn64, and other
residues of the active site. In the proposed catalytic mechanism of
hydrolysis of Fru-1,6-P2 (12, 13), a water molecule, bound
to a metal cation, is activated for a nucleophilic attack of the
1-phosphate. The side chains of Asp74 and
Glu98, relative to the attacking water and to the metal ion
at the "catalytic" site (called metal site 2 in crystal structures
of FBPase) (Fig. 4), are in position to
act as general base catalysts. The distance between OD2 of
Asp74 and OE2 of Glu98 and the cation at site 2 is ~3.3 Å, but neither side chain is in the inner coordination
sphere of that cation. Replacement of Glu98 by glutamine
resulted in a 1,600-fold reduction in kcat
relative to wild-type FBPase (35). The Asp74 Asn
mutation caused a decrease in catalytic rate of the same order of
magnitude, whereas the Asp74
Ala mutation resulted in
almost the total loss of catalytic activity. Thus, Asp74
and Glu98 may act in concert to remove a proton from the
attacking water molecule.
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Asn64 and Asp68 are also in the active site
(Fig. 5), with the former hydrogen
bonding with Glu97 and Glu98 and the latter
with Arg276. Evidently, Asn64 maintains
Glu97 and Glu98 in proper orientations for
metal binding and the activation of the attacking water molecule.
The above is consistent with the reduction in the Hill coefficient for
Mg2+ and the 50-fold decrease in affinity for
Mg2+, exhibited by the Asn64 Ala and
Asn64
Gln mutants relative to wild-type FBPase.
Mutation of Arg276 to methionine reduces activity by
2,000-fold, abolishes Mg2+ cooperativity, and alters the
kinetic mechanism of FBPase (36). Evidently, the
Asp68-Arg276 hydrogen bond is a determinant
for many of the kinetic properties of FBPase; its loss could
destabilize loop 52-72 and impair the catalytic function of
Asp74.
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AMP induces a modest structural change in the helix (H2) immediately preceding loop 52-72, and in all AMP-bound complexes of FBPase, loop 52-72 is disordered (13, 28). The new crystal structure has an ordered loop in the absence of AMP. Hence, AMP could inhibit FBPase by the disruption of interactions between loop 52-72 and the active site. As Asn64 and Asp68 probably stabilize metal binding to the active site, the observed competition between AMP and Mg2+ in kinetics and NMR investigations may stem from the displacement of loop 52-72 from the active site due to the AMP-induced perturbation of helix H2.
The 175- and 10-fold increases in Ki of AMP
inhibition with respect to Fru-1,6-P2 for the
Lys71,72 Met and Lys71
Ala enzymes,
respectively, could originate from the destabilization of loop 52-72
in its disordered, AMP-induced conformation. AMP may be less effective
in the displacement of loop 52-72 of the double mutant (in which
positions 71 and 72 are methionine instead of lysine) because of the
unfavorable thermodynamics of exposing two methionyl side chains to the
solvent. Indeed, the double mutant apparently exhibits preference
toward the active R-state, relative to the less active or inactive
T-state. Its kcat/Km
increases 3-fold, and its Ka for Mg2+
decreases 2-fold, whereas its Ki for AMP and
Fru-2,6-P2 increases 175- and 7-fold, respectively.
In fact, the double mutant may represent the kinetic properties of
FBPase locked into the R-state.
Mutations that bring about a change in the kinetic mechanism of FBPase from competitive to noncompetitive (AMP inhibition relative to Mg2+) are possible in the context of a steady-state random mechanism (16, 17) and now can be understood in terms of a conformational mechanism. To a first approximation, loop 52-72 is the instrument by which AMP exerts its allosteric effect on the active site. In the wild-type system with a fully functional loop, AMP and Mg2+ (presumably at site 2) are probably antagonists with respect to the conformation that they stabilize for loop 52-72. Hence, in wild-type FBPase, loop 52-72 strongly couples the AMP- and Mg2+-binding sites. In some mutants of loop 52-72, however, the strong coupling of the AMP and the cation site is diminished; AMP binds and perturbs the metal at site 2, thereby causing inhibition, but is no longer completely effective in displacing the cation from a catalytically productive association with the active site.
A rational basis for an uncompetitive mechanism is more challenging,
however. Four separate mutations (Asp74 Glu,
Asn64
Ala, Asn64
Gln, and
Arg49
Cys) change AMP inhibition with respect to
Fru-1,6-P2 from noncompetitive to uncompetitive. The
uncompetitive mechanism implies that AMP binds as an inhibitor only
when Fru-1,6-P2 is productively bound at the active site.
Hence, these mutations may stabilize a conformation of loop 52-72 that
does not favor the binding of AMP (as an inhibitor), but permits the
association of Fru-1,6-P2 with the active site. Productively bound Fru-1,6-P2 could then induce a
conformational change in loop 52-72 that re-establishes the coupling
between the AMP and the substrate-binding site. A more detailed
explanation of the above phenomenon, however, must await
crystallographic and ligand binding studies of mutant FBPases.
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FOOTNOTES |
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* This work was supported by Research Grant NS 10546 from the National Institutes of Health and Grant MCB-9603595 from the National Science Foundation. This is Journal Paper J-17874 of the Iowa Agriculture and Home Economics Experiment Station (Ames, IA), Project 3191.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Biophysics, 1210 Molecular Biology Bldg., Iowa State University,
Ames, IA 50011. Tel.: 515-294-6116; Fax: 515-294-0453; E-mail:
hjfromm{at}iastate.edu.
1 The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; Fru-1,6-P2, fructose 1,6-bisphosphate; Fru-2,6-P2, fructose 2,6-bisphosphate.
2 J.-Y. Choe, H. J. Fromm, and R. B. Honzatko, manuscript in preparation.
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REFERENCES |
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