The N-terminal Segment of Recombinant Porcine
Fructose-1,6-bisphosphatase Participates in the Allosteric Regulation
of Catalysis*
Scott W.
Nelson,
Feruz T.
Kurbanov
,
Richard B.
Honzatko, and
Herbert J.
Fromm§
From the Department of Biochemistry, Biophysics, and Molecular
Biology, Iowa State University, Ames, Iowa 50011
Received for publication, October 17, 2000
 |
ABSTRACT |
Residues 1-10 of porcine
fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in
different conformations, sensitive to the state of ligation of the
enzyme. Deletion of the first 10 residues of FBPase reduces
kcat by 30-fold and Mg2+ affinity
by 20-fold and eliminates cooperativity in Mg2+ activation.
Although a fluorescent analogue of AMP binds with high affinity to the
truncated enzyme, AMP itself potently inhibits only 50% of the enzyme
activity. Additional inhibition occurs only when the concentration of
AMP exceeds 10 mM. Deletion of the first seven residues
reduces kcat and Mg2+ affinity
significantly but has no effect on AMP inhibition. The mutation of
Asp9 to alanine reproduces the weakened affinity for
Mg2+ observed in the deletion mutants, and the mutation of
Ile10 to aspartate reproduces the AMP inhibition of the
10-residue deletion mutant. Changes in the relative stability of the
known conformational states for loop 52-72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms
of FBPase, including the thioredoxin-regulated FBPase from the chloroplast.
 |
INTRODUCTION |
Fructose-1,6-bisphosphatase (D-fructose
1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11;
FBPase1) catalyzes the
hydrolysis of fructose 1,6-bisphosphate (F16P2) to fructose
6-phosphate and inorganic phosphate (Pi) (1-3). The reaction facilitated by FBPase is subject to hormone and metabolite regulation, the net result of which is the tight coordination of FBPase
and fructose 6-phosphate 1-kinase activities (4). FBPase activity
requires divalent cations such as Mg2+, Mn2+,
or Zn2+, and plots of velocity versus metal ion
concentration are sigmoidal with a Hill coefficient of 2.0 (5-7). AMP
binds cooperatively (Hill coefficient of 2) 28 Å from the nearest
active site (8) and inhibits the enzyme, whereas F26P2
binds at the active site (9). Inhibition of FBPase by AMP and
F26P2 is synergistic. F26P2 can lower the
apparent inhibition constant for AMP by up to 10-fold (6).
FBPase is a tetramer of identical subunits (Mr = 37,000). To a first approximation, these subunits lie in the same plane
and occupy the corners of a square in one of the principal quaternary states of the mammalian enzyme (R-state). By past convention, subunit
C1 occupies the upper left-hand corner, with subunits C2-C4 following
in a clockwise direction. AMP causes a transition from the R-state to
the T-state, driving a 17° rotation of the C1-C2 subunit pair with
respect to the C3-C4 pair about a molecular 2-fold axis of symmetry
(10). Complexes of FBPase with AMP in the presence of
F16P2, F26P2, and fructose 6-phosphate are all in the T-state (9, 11, 12), whereas in the absence of AMP, the enzyme
has appeared in the R-state in crystal structures (13, 14).
Mutations in loop 52-72 and in the hinge preceding the loop (residues
50-51) greatly influence catalysis and AMP inhibition of FBPase and
together suggest the necessity of an engaged conformation for loop
52-72 for catalysis under physiological conditions (15, 16). The
engaged conformation, in which loop 52-72 interacts with the active
site, occurs in metal-product complexes of wild-type FBPase but only in
the absence of AMP (17). All residues of the engaged loop are in well
defined conformations associated with clear electron density in crystal
structures (13, 14). The disengaged conformation of loop 52-72 exists
in AMP complexes of wild-type FBPase. The disengaged loop is far from
the active site. Residues 52-60 lie near, and interact with, an
adjacent subunit (C1-C2 interaction), but residues 61-73 are
disordered, with no observable electron density in crystal structures
(14). In FBPase crystallized without metal cations (18) and in certain mutant forms of FBPase (to be discussed below in greater detail), loop
52-72 exists in yet another conformation in which residues 54-73 are
without electron density. This disordered state of the loop 52-72 is a
manifold of closely related conformations in which the loop itself
interacts weakly with the rest of the enzyme.
AMP binds between helices H1 and H2 of FBPase and causes modest
movements in both helices relative to the rest of the subunit (14). The
0.9-Å movement of helix H2 along its axis directly influences loop
52-72 (13), whereas the 1.5-Å movement of helix H1 could influence
loop 52-72 of an adjacent subunit (14). AMP may stabilize the
disengaged conformation of loop 52-72 through helix H1 by facilitating
contacts between residues 52-59 of the loop and residues 1-10 of the
N terminus. Residues belonging to this N-terminal segment are either
disordered (residues 1-6) or exist in different conformations in the
R- and T-state of FBPase (residues 7-10). In addition, several of
these N-terminal residues are conserved throughout FBPases from
eukaryotes, suggesting a functional significance, as yet unconfirmed.
Here we present the changes in functional properties of FBPase due to
deletion and point mutations in the N-terminal segment (residues
1-10). Mutations, which directly or indirectly influence positions 9 and 10, cause large perturbations in catalysis and/or AMP inhibition. A
simple model in which the quaternary states of FBPase differentially
stabilize specific conformations of loop 52-72 accounts for the
properties of wild-type and mutant FBPases presented here and in other studies.
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EXPERIMENTAL PROCEDURES |
Materials--
F16P2, F26P2,
NADP+, and AMP were purchased from Sigma. DNA-modifying and
restriction enzymes, T4 polynucleotide kinase, and ligase were from
Promega. Glucose-6-phophate dehydrogenase and phosphoglucose isomerase
came from Roche Molecular Biochemicals. Other chemicals were of reagent
grade or equivalent. Escherichia coli strains BMH 71-18
mutS and XL1-Blue came from CLONTECH and Stratagene, respectively. The FBPase-deficient E. coli
strain DF 657 came from the Genetic Stock Center at Yale University.
Mutagenesis of Wild-type FBPase--
Mutations were accomplished
by deletion of or specific base changes in double-stranded plasmid
using the TransformerTM site-directed mutagenesis
kit (CLONTECH). The mutagenic primers are as
follows: 10DEL,
5'-GAAGGAGATATACATATGGTCACCCTAACCCGCTTCGTCATGGAG-3'; 7DEL,
5'-GTTTAACTTTAAGAAGGAGATATACATAATGACCAATATCGTCACCCTAACCCGCCTCG-3'; Ile10
Asp,
5'-ACACCAATGACGTCACCC-3'; Ile10
Met 5'-ACACCAATATGGTCACCC-3'; Asn9
Ala, 5'-CCTTCGACACCGCTATCGTCACCC-3';
Thr8
Ala,
5'GGCCTTCGACGCCAATATCGTC-3'; Asp7
Ala, 5'CGGCCTTCGCCACCAATATCGTC-3';
Phe6
Trp,
5'-GACCAGGCGGCCTGGGACACCATTATC-3'; and
Asp2
Ala,
5'-ACATATGACGGCCCAGGCGGC3' (codons spanning
deletions and codons for point mutations are underlined in bold
typeface). The selection primer for mutagensis, which changed an
original NruI site on the plasmid into a XhoI
site was 5'-CAGCCTCGCCTCGAGAACGCCA-3' (digestion site underlined in bold typeface). The mutations and integrity of the constructs were confirmed by sequencing the promoter region and the entire open reading frame. The Iowa State University sequencing facility provided DNA sequences using the fluorescent dye-dideoxy terminator method.
Expression and Purification of Wild-type and FBPase
Mutants--
Protein expression and purification were performed as
described previously (16). To avoid contamination of recombinant FBPase by endogenous enzyme, a FBPase-deficient strain of E. coli
was used in the expression of the enzymes. Protein purity and
concentration was confirmed by SDS-polyacrylamide gel electrophoresis
(19) and by the Bradford assay (20), respectively. The initial five amino acid residues of the 10DEL and 7DEL mutants were determined by
cycles of automated Edman degradation performed by the Iowa State
University protein facility.
Circular Dichroism (CD) Spectroscopy--
CD spectra of
wild-type and mutant FBPases were recorded at room temperature on a
Jasco J710 CD spectrometer in a 1-cm cell using a protein concentration
of 0.35 mg/ml. Three scans of each spectrum were collected from 200 to
260 nm in increments of 1.3 nm and averaged. Each averaged spectrum was
blank-corrected using the software package provided with the instrument.
Kinetic Experiments--
Assays for the determination of
specific activity, kcat, and activity ratios at
pH 7.5/9.5 employed the coupling enzymes phosphoglucose isomerase and
glucose-6-phosphate dehydrogenase (1). The reduction of
NADP+ to NADPH was monitored spectroscopically at 340 nm.
All other assays used the same coupling enzymes but monitored the
formation of NADPH by its fluorescence emission at 470 nm using an
excitation wavelength of 340 nm. All kinetic assays were performed at
room temperature. Initial rates were analyzed using programs written either in the MINITAB language using a
value of 2.0 (21) or by
ENZFITTER (22). The kinetic data were fit to several models, and the
best fits are reported below.
Steady-state Fluorescence Measurements--
Fluorescence data
were collected using a SLM 8100C fluorimeter from Spectronic
Instruments. The single tryptophan of the Trp-6 mutant was excited
selectively using a wavelength of 295 nm. Fluorescence emission spectra
were recorded in steps of 1 nm from 310 to 400 nm with a slit width of
2 mm for both excitation and emission and represent the average of five
such scans. Fluorescence from AMP-PNP employed excitation and
emission wavelengths of 400 and 535 nm, respectively. Conditions under
which specific spectra were recorded are provided in the text and
figure legends. Each data point for the titration experiments is an
average of 15 1-s acquisitions. Enzyme concentrations ranged from 0.3 to 0.5 µM. All spectra were corrected for dilution and
inner filter effects (absorbance of light by AMP and AMP-PNP) using
the formula (23),
|
(Eq. 1)
|
where Fc is the corrected fluorescence,
F is the fluorescence intensity experimentally measured,
B is the background, Vi is the volume of
the sample for a specific titration point, Vo is the
initial volume of the sample, Aex is the
absorbance at the wavelength of excitation (295 nm for AMP and 410 for
AMP-PNP), and Aem is the absorbance at the
wavelength of emission (535 nm for AMP-PNP). As a control, ligands
caused no change in fluorescence emission after correction from a
solution of tryptophan (100 µM) in Hepes buffer (20 mM, pH 7.5).
AMP-PNP titration data were analyzed by nonlinear least squares fits
using the following equation.
|
(Eq. 2)
|
where
Fmax is the change in
fluorescence caused upon the addition of ligand L,
Fo is the fluorescence in the absence of ligand,
Kd is the dissociation constant, and n is the Hill coefficient.
 |
RESULTS |
Expression, Purification, and Secondary Structure Analysis of
Wild-type and Mutant FBPases--
Wild-type and mutant FBPases
have identical mobilities in chromatographic and electrophoretic
separations and are at least 95% pure by SDS-polyacrylamide gel
electrophoresis. The pH 7.5/9.5 activity ratios of mutant enzymes
(excluding the 7DEL and 10DEL mutants) indicated the absence of
proteolyzed enzyme. Results from amino acid sequencing of the 7DEL and
10DEL mutants are consistent with their cDNA, except for the
absence of the N-formyl methionine. Furthermore, the
sequencing data revealed a single N terminus with no background signal
indicative of proteolysis. The CD spectra of wild-type and mutant
FPBases were essentially superimposable from 200 to 260 nm (data not shown).
Catalytic Rates and Michaelis Constants for Mg2+ and
F16P2 for Wild-type and Mutant FBPases--
Initial rate
kinetic studies employed substrate concentrations saturating in
F16P2 (20 µM) and Mg2+ (10 times
Ka-Mg2+) but not so high as to cause
inhibition. The deletion mutants have a decreased turnover relative to
wild-type FBPase, reducing kcat by 4- and
28-fold for the 7DEL and 10DEL mutants, respectively (Table
I). These deletions also caused a 20-fold
increase in the Ka for Mg2+
and eliminated (10DEL) or reduced (7DEL) Mg2+
cooperativity. The 7DEL and 10DEL mutants did not influence the Km for F16P2. Single point mutations had
little affect on kcat relative to the wild-type
enzyme; the mutant Asn9
Ala decreased Mg2+
affinity and reduced metal cooperativity. Other point mutations cause
only minor changes in kinetic parameters, except for (in some cases)
the Ki for AMP.
AMP Inhibition of Wild-type and FBPase Mutants--
AMP
allosterically inhibits FBPase (5). Complete AMP inhibition of
Ile10
Asp and 10DEL FBPases requires ~1500-fold more
AMP than for the wild-type enzyme. In addition, AMP inhibition is
biphasic (Fig. 1). The data presented in
Fig. 1, except that for the Ile10
Asp and 10DEL
FBPases, were fit to Equation 3,

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Fig. 1.
AMP inhibition of wild-type and mutant
FBPases. AMP titrations are of wild-type ( ), Ile10
Asp ( ), and 10DEL ( ) FBPases in saturating F16P2
(20 µM) and a Mg2+ concentration equal to the
Ka for Mg2+ of each enzyme. See text for
details regarding the fitted curves.
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|
|
(Eq. 3)
|
where v is the observed velocity at a specific
concentration of AMP, Vo is the fitted velocity in
the absence of AMP, I is the concentration of AMP,
IC50 is the concentration of AMP that causes
50% inhibition, and n is the Hill coefficient. The biphasic
curves of the Ile10
Asp and 10DEL mutants were fit to
Equation 4,
|
(Eq. 4)
|
where v, Vo, and I are as
above in Equation 3, and IC50-high and
IC50-low represent concentrations of AMP that
cause 50% relative inhibition due to the ligation of high and low
affinity sites, respectively. Ligation of the high affinity sites, as
shown below, is cooperative with a Hill coefficient of 2, whereas the cooperativity with respect to the ligation of the low affinity sites is
an adjustable parameter (n) in Equation 4. The fitted value
for n is 0.68 ± 0.09 and 0.39 ± 0.05 for the
Ile10
Asp and 10DEL enzymes, respectively. The positive
cooperativity for the high affinity interaction of AMP (the exponent
for the I/IC50-high term is 2) is justified by
the analysis of kinetics data depicted in Fig.
2 using low concentrations of AMP (0-10 µM). These (as well as data for FBPases that do not
exhibit biphasic inhibition by AMP) fit very well to Equation 5
(goodness of fit between 2.6 and 5.3%),
|
(Eq. 5)
|
where v, Vm, A, I,
Ka, Ki, and n
represent, respectively, initial velocity, maximal velocity, Mg2+ concentration, AMP concentration, the Michaelis
constant for Mg2+, the dissociation constant for AMP from
the enzyme-AMP complex, and the Hill coefficient for AMP. All mutants
(except 10DEL) exhibited both Mg2+ and AMP cooperativity
(n = 2). The 10DEL mutant retained AMP cooperativity
(n = 2), but Mg2+ cooperativity was
eliminated (n = 1).

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Fig. 2.
Kinetic mechanism of AMP inhibition of
Ile10 Asp and 10DEL FBPases.
AMP competes with Mg2+ in both the 10DEL (A) and
Ile10 Asp (B) mutants. Concentrations of AMP
are 0 ( ), 1 ( ), and 1.5 µM ( ) (A) and
0 ( ), 2 ( ), and 4 µM ( ) (B).
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|
F26P2 Inhibition of Wild-type and Mutant
FBPases--
F26P2 is a substrate analog that binds at the
active site of the enzyme. The Ki values for
F26P2 listed in Table I were determined by fitting the data
(not shown) to Equation 2, a model for linear competitive inhibition
(goodness of fit between 2.1 and 3.6%),
|
(Eq. 6)
|
where v and VM are as above,
I is the concentration of F26P2, B is
the concentration of F16P2, Kb is the
Michaelis constant for F16P2, and Ka is
the dissociation constant for F26P2 from the
F26P2-enzyme complex. The mechanism of F26P2 inhibition is the same for all mutant and the wild-type FBPases.
Fluorescence Emission from Phe6
Trp and
AMP-PNP--
As wild-type FBPase has no tryptophan residues, the
substitution of tryptophan for phenylalanine at position 6 introduces a
unique spectroscopic probe. The kinetic properties of Phe6
Trp FBPase are nearly identical to those of the wild-type
enzyme (Table I). Furthermore, the fluorescence emission spectrum of the Phe6
Trp mutant changes by only 7% in its emission
maximum in the presence and absence of saturating AMP, with no
observable change in the wavelength of maximum emission.
The fluorescent AMP analogue, AMP-PNP, and AMP inhibit wild-type FBPase
by identical kinetic mechanisms, with nearly identical kinetic
parameters (16). AMP-PNP has the advantage compared with AMP of
exhibiting a significant increase in fluorescence emission upon its
binding to the allosteric pocket of FBPase. Hence, for mutants of
FBPase that exhibit a significant loss in AMP inhibition due to
mutation, one can use AMP-PNP to distinguish whether the change
is due to impaired binding or to an impaired mechanism of allosteric
inhibition. In the cases of the 10DEL (Fig.
3) and Ile10
Asp (data
not shown) FBPases, dissociation constants for AMP-PNP are comparable
with that determined for the wild-type enzyme (16).

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Fig. 3.
AMP-PNP titration of the 10DEL mutant.
Fluorescence of AMP-PNP observed in the presence of 0.5 µM 10DEL. A wavelength of 400 nm was used for excitation,
and fluorescence emission was monitored at 535 nm. A.U.,
arbitrary units.
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 |
DISCUSSION |
Crystal structures of FBPase from mammalian sources and the
chloroplast have little or no electron density for the N-terminal region of the enzyme. Yet certain amino acids in this region, particularly residues 9-10, are conserved over a wide range of organisms. As porcine FBPase has no tryptophan, incorporation of a
tryptophan for Phe6 introduces a unique spectroscopic
probe. The functional properties of Phe6
Trp FBPase are
comparable with those of the wild-type enzyme, and fluorescence
emission in the presence and absence of AMP is about the same.
Evidently, the first six residues of porcine FBPase have no functional
role in catalysis or metabolite regulation of catalysis.
Position 7 may be the first residue from the N terminus to directly or
indirectly influence the functional properties of FBPase. Asp7 is the first residue defined by electron density in
crystal structures of wild-type FBPase (14). The 7DEL construct
exhibits significant changes in functional properties (Table I), in
particular Ka-Mg2+ is elevated 15-fold
relative to that of wild-type FBPase. Asp7
Ala FBPase,
however, has functional properties similar to those of the wild-type
enzyme. Hence, the functional changes due to the 7DEL construct may be
the consequence of an indirect perturbation on the conformation of
residues 9-10 and/or the conformational properties of loop 52-72 in
the R-state (AMP absent). Interestingly, the mutation of the
Lys50 to proline (a hinge element for loop 52-72)
eliminates AMP inhibition by the disruption of the allosteric mechanism
and also causes a 15-fold elevation in
Ka-Mg2+ (16). Other mutations in the
segment 50-55 cause little effect on AMP inhibition but increase
Ka-Mg2+ by 10-fold or
more.2
The impact of the 10DEL construct on the functional properties of
FBPase is even more extreme than is that of the 7DEL mutant. The low pH
activity ratio is consistent with the effects of the mutation of
Lys50 to Pro (16) and of the truncation of 25 residues from
the N terminus by proteolysis of FBPase (24). The low pH-activity ratio
could result from the proteolysis of loop 52-72 (25); however,
N-terminal sequencing of the 10DEL mutant excludes this possibility.
Instead, the 15-fold increase in Ka-Mg2+
and the 30-fold decrease in kcat is consistent
with the failure of loop 52-72 to achieve an engaged conformation in
the R-state of the 10DEL construct. The effects of the 10DEL mutant can
be explained in part by the side chain at position 9. Asn9
Ala FBPase exhibits a 10-fold increase in its
Ka-Mg2+ but no change in
kcat or the pH-activity ratio relative to the wild-type enzyme. In R-state crystal structures, Asn9
hydrogen bonds with Arg15 (Fig.
4). The
Asn9-Arg15 interaction probably stabilizes
residues 7-11 in their R-state conformation. These residues pack
between helix 3 and loop 187-194. Loop 187-194 interacts through
hydrogen bonds and nonbonded contacts with hinge residues preceding
loop 52-72 (Fig. 4). The loss of the
Asn9-Arg15 hydrogen bond may lead to a long
range conformational change, which destabilizes the engaged
conformation of loop 52-72. Indeed, the disordered conformation of
loop 52-72 appears hand in hand with disorder in residues 1-11 in
R-state crystal structures (9, 18).

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Fig. 4.
Structural elements important to allosteric
regulation in the R-state of FBPase. The tetramer as viewed down
one of its three, mutually perpendicular symmetry axes in the R-state
(top). Loop 52-72 is in the engaged conformation. Residues
near the hinge of this loop in subunit C4 pack against loop 182-194 of
subunit C3, which in turn pack against residues 7-11 of the N-terminal
segment preceding helix H1 of subunit C3 (bottom). Residues
7-11 lie in a groove bordered by helix H3 of subunit C2 and loop
182-194 of subunit C3. Orientations of top and bottom illustrations
are the same. This illustration was drawn with MOLSCRIPT (33).
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Unlike the 7DEL construct, which has little effect on allosteric
inhibition by AMP, the 10DEL mutation significantly alters the maximum
level of AMP inhibition. The 25-residue truncated form of FBPase is not
inhibited by AMP (24). As some of the 25 residues are part of the AMP
binding pocket (14), the effect of limited proteolysis is of little
surprise. In contrast, the first 10 residues are remote from the site
of AMP binding, and indeed, AMP-PNP binds with nearly identical
affinity constants to 10DEL and wild-type FBPases. Changes in the
functional properties due to the 10DEL mutant, then, must result from
perturbations in the allosteric mechanism of AMP inhibition. Although
no single point mutation reproduced the functional properties of the
10DEL construct in the absence of AMP, the mutation of
Ile10 to aspartate reproduced the phenomenon of 50%
maximal inhibition by AMP. As noted below, the hydrophobic side chain
at position 10 is critical to the stability of the disengaged
conformation of loop 52-72 in the T-state.
The following model is a basis for understanding AMP inhibition in
wild-type FBPase and in mutant FBPases. Hereafter, T-state and R-state
refer to quaternary arrangements of subunits in FBPase, distinguished
by the 17° rotation about a molecular symmetry axis. In addition, the
subunits in the T- and R-states can adopt different tertiary
conformations, the most significant of which involve conformational
changes in loop 52-72. In the T-state of wild-type FBPase, loop 52-72
is in the disengaged conformation. The T-state subunit arrangement
stabilizes the disengaged conformation by interactions, which involve
residues 50-60 of subunit C1 with residues 187-194 and 9-11 of
subunit C2 (Fig. 5). Point mutations in
these structural elements profoundly influence AMP inhibition (16, 26,
27), AMP cooperativity (26, 27), F26P2 inhibition (16, 27),
and/or metal affinity (16). The R-state subunit arrangement does not
stabilize the disengaged loop conformation. As a consequence loop
52-72 occupies an engaged conformation or a disordered conformation.
The engaged conformation was first observed in the context of a
product-Zn2+ complex of the wild-type enzyme (13). The
disordered conformation of loop 52-72 appears in FBPase structures
crystallized in the absence of metal activators (18).

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Fig. 5.
Structural elements important to allosteric
regulation in the T-state of FBPase. Shown here are segments of
two polypeptide chains (residues 9-89) viewed down an axis of
molecular 2-fold symmetry toward the interface buried between subunits
C1-C2 and subunits C3-C4 (top). The side chain of
Ile10 from subunit C2 makes nonbonded contacts with
Thr12 and Ile194, both from subunit C2, and
Tyr57 and Ile59, both from loop 52-72 of
subunit C1 (bottom). Orientations of top and bottom
illustrations are the same. This illustration was drawn with MOLSCRIPT
(33).
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Catalysis occurs at neutral pH if loop 52-72 can cycle between its
engaged and disordered conformations. A loop that is always engaged is
a dead-end complex. A loop that cannot achieve the engaged conformation
results in low metal affinity and little or no activity at neutral pH.
Assuming loop 52-72 exchanges between its engaged and disordered
conformations, the free energy differences between these conformational
states must be small, that is, the R-state maintains small free-energy
differences between disordered and engaged conformations of loop
52-72. In contrast, the T-state subunit arrangement selectively
stabilizes a new conformation for loop 52-72 (disengaged
conformation), which de-populates the disordered/engaged loop
conformations. The decline in the catalytic rate of T-state FBPase is
directly related to the differences in free energy between the
engaged/disordered loop conformations and the disengaged loop
conformation. For the wild-type enzyme, the free energy difference is
large; hence, little or no catalysis occurs. For specific mutants of
FBPase that selectively destabilize the disengaged loop conformation,
the free energy difference is less, the engaged/disordered loop
conformations are more populated, and hence, a measurable level of
catalysis occurs. Biphasic AMP inhibition should appear whenever a
mutation selectively destabilizes the disengaged loop conformation of
the T-state. FBPase mutants with biphasic AMP inhibition
(Lys42
Ala, Glu192
Ala,
Glu192
Gln, Lys50
Asn,
Lys50
Pro/Tyr57
Trp, and
Ile10
Asp) exhibit plateau activities, which vary from
10 to 70% full activity (16, 26, 27). As suggested by Kantrowitz and co-workers (28), the activity most likely comes from FBPase in the
T-state subunit arrangement. Here we add that some fraction of the
mutant FBPases have engaged/disordered loop conformations in the
presence of AMP, which are responsible for the observed turnover. The
free energy relationships of the model are summarized in Fig.
6.

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Fig. 6.
Changes in relative free energy levels of
three loop conformations in the R- and T-states of wild-type and
Asp10 FBPases. Bold lines indicate the energies
of the engaged and disengaged conformations of loop 52-72, whereas the
bundle of fine lines represents the disordered loop, which
is in a manifold of conformational states with nearly equivalent free
energies. In the wild-type enzyme, the R to T transition selectively
stabilizes the disengaged conformation of loop 52-72 over the engaged
and disordered conformational states. In the Ile10 Asp
mutant, the T-state does not stabilize a disengaged conformation for
loop 52-72. The separation in free energy between the engaged
conformation and the disordered conformation in the T-state
( G in figure) may influence the observed level of
catalytic activity in the presence of saturating AMP.
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From the model above, mutant FBPases with biphasic AMP inhibition must
have a less stable, disengaged conformation of loop 52-72 in the
T-state. In fact, structural evidence supports this claim. The
disengaged loop in the T-state of wild-type FBPase has interpretable
electron density up to residue 60, with the residues 50-60
participating in interactions across the C1-C2 subunit interface
(14). The mutation of Lys42 to alanine results
in biphasic AMP inhibition with 70% relative activity at the plateau
(26). The crystal structure of the AMP complex of the Lys42
Ala mutant is in the T-state, but loop 52-72 is poorly ordered from residues 54 to 72 (28). In the crystal structure of the AMP complex of Ile10
Asp2 the enzyme is again
in the T-state, and loop 52-72 is without electron density from
residues 54 to 72. Hence, loop 52-72 in the Ala42 and
Asp10 mutants is not in the disengaged conformation of
wild-type FBPase but, rather, in a disordered conformation similar to
that of the R-state apoenzyme. If the disengaged structure is
de-populated in favor of disordered conformations, then perhaps under
the appropriate conditions an engaged conformation for loop 52-72 may
occur in the T-state of such mutants.
The thioredoxin-mediated formation of a disulfide bond between
Cys153 and Cys173 in chloroplast FBPase (29)
putatively stabilizes the position of a loop that excludes the binding
of metal activators and the engaged conformation of loop 61-81 (which
corresponds to loop 52-72 of mammalian FBPase) (30). FBPase from the
chloroplast evidently does not bind AMP (30, 31). Yet existing crystal structures of reduced and oxidized chloroplast FBPase have nearly the
same quaternary arrangement of subunits, resembling most closely the
T-state of mammalian FBPase. Hence, as illustrated by FBPase from the
chloroplast, a T-state subunit arrangement by itself does not exclude
catalysis. Interestingly, position 18 of the chloroplast enzyme, which
corresponds to position 10 of mammalian FBPase, is isoleucine, this
residue being widely conserved among FBPases. Furthermore, in the
inactive, oxidized form of chloroplast FBPase, loop 61-81
(corresponding to loop 52-72 of the mammalian enzyme) is in the
disengaged conformation (30), whereas the equivalent loop in the
active, reduced form of chloroplast FBPase is in the disordered
conformation (32). Evidently, oxidation and reduction of the disulfide
bond in chloroplast FBPase influences the relative stability of the
disengaged loop-conformation in a manner similar to that of AMP in
mammalian FBPases. The observation of corresponding conformational
states in mammalian and chloroplast FBPases infers a common, early
mechanism of FBPase regulation that has diverged through evolution.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Research Grant NS 10546 and National Science Foundation Grants MCB-9603595 and MCB-9316244. This is Journal Paper 19128 of the Iowa
Agriculture and Home Economic Experiment Station, Ames, 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.
Present address: Dept. of Molecular and Cell Biology, University of
California, Berkeley, California 94720.
§
To whom correspondence should be addressed. Tel.: 515-294-4971;
Fax: 515-294-0453; E-mail: hjfromm@iastate.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009485200
2
C. Iancu, H. J. Fromm, and R. B. Honzatko, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
FBPase, fructose-1,6-bisphosphatase;
F16P2, fructose
1,6-bisphosphate;
F26P2, fructose 2,6-bisphospate;
Pi, orthophosphate;
CD, circular dichroism;
AMP-PNP, 2'-(or
3')-O-(trinitrophenyl) adenosine 5'-monophosphate.
 |
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