(Received for publication, October 3, 1996, and in revised form, December 4, 1996)
From the Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167
The role of the 190's loop of
fructose-1,6-bisphosphatase (Fru-1,6-P2ase) in the
allosteric regulation of Fru-1,6-P2ase has been
investigated through kinetic studies on three mutant enzymes, Glu-192
Ala, Glu-192
Gln, and Asp-187
Ala. AMP is an allosteric inhibitor, which binds to the regulatory sites and induces the R- to
T-state transition; for wild-type Fru-1,6-P2ase AMP
inhibition is cooperative with a Hill coefficient of 2.0. The
replacement of Asp-187, which forms an interaction across the C1:C2
monomer-monomer interface, with alanine did not change the catalytic
efficiency, and it had no effect on the cooperativity of AMP
inhibition; however, the apparent dissociation constant for AMP
increased more than 4-fold as compared to the value for the wild-type
enzyme. The replacement of Glu-192, which forms interactions across the
C1:C4 dimer-dimer interface, with Ala and Gln lowered
kcat from 21 s
1 for wild-type
enzyme to 15 s
1 and 13 s
1, respectively,
for the mutant enzymes, while their respective Km
values were not changed. However, these replacements did have dramatic
effects on AMP inhibition; first, cooperative AMP inhibition was lost;
second, the AMP inhibition was biphasic, which can be interpreted as
due to AMP binding to two classes of binding sites. The high affinity
class of sites corresponds to the regulatory sites, while the low
affinity class of sites may be the active sites. The results reported
here, combined with the structural and kinetic results from the Lys-42
Ala enzyme, strongly suggest that the C1:C4 dimer-dimer interface,
rather than the C1:C2 monomer-monomer interface, is critical for the propagation of the allosteric signal between the AMP sites on different
subunits; in addition, cooperative AMP inhibition is essential for the
enzyme to be fully inhibited by the binding of AMP to the allosteric
site.
Fructose-1,6-bisphosphatase (Fru-1,6-P2ase, EC
3.1.3.11),1 a key regulatory enzyme in
gluconeogenesis (1), catalyzes the hydrolysis of the anomer of
fructose 1,6-bisphosphate (Fru-1,6-P2) to
-D-fructose 6-phosphate and inorganic phosphate in the
presence of divalent metal ions. Pig kidney Fru-1,6-P2ase
is an allosteric enzyme composed of four identical subunits, each with
a molecular weight of 34,000 (2). Although the enzyme is normally in
the active R state, upon the binding of AMP it is transformed into the
completely inactive T state. X-ray structural data for both the R- and
T-states are available for pig kidney Fru-1,6-P2ase. The
AMP complexes (3) both in the absence and the presence of fructose
6-phosphate are identified as T-state structures, while those complexes
with bound fructose 6-phosphate (4) or fructose 2,6-bisphosphate
(Fru-2,6-P2) (5) are identified as R-state structures. The
tertiary structure of each monomer is composed of two folding domains
(Fig. 1), the AMP domain with the AMP binding site and
the FBP domain containing the active site.
The activity of pig kidney Fru-1,6-P2ase is negatively modulated by the allosteric inhibitor AMP and also by Fru-2,6-P2, which is a competitive inhibitor (6, 7). AMP binds to the allosteric site which is about 28 Å (3) from the active site and its inhibition is cooperative with a Hill coefficient of approximately 2.0. The enzyme requires divalent metal ions, such as Mn2+, Mg2+, and Zn2+ to achieve catalytic activity (6). Magnesium activation is also cooperative with a Hill coefficient of 2.0. There are two metal binding sites on each monomer. One metal binding site is defined as a structural site which is thought to be essential for subsequent substrate binding, and the other is defined as a catalytic site, thought to be essential for catalysis. The metal binding sites are located at the interface between the two domains of the enzyme (8) (Fig. 1).
During the transition from the R- to the T-state, the C1:C22 dimer rotates about 15° relative to the C3:C4 dimer, and within each monomer the AMP domain rotates about 1.9° and translates about 1.6 Å relative to the FBP domain. By comparing the T- and R-state structures, Lipscomb and co-workers (9) have proposed that, after the binding of AMP, the secondary structure of the AMP domain undergoes a series of rearrangements leading to a movement involving a relative rotation and translation of the AMP domain relative to the FBP domains. This movement disturbs the active site, which consists of a substrate binding region from the FBP domain and a metal binding region at the domain interface, in such a way that enzyme activity is inhibited. Inhibition is achieved by preventing coordinated binding of substrate and of one essential metal ion in the proper arrangement necessary for substrate hydrolysis. Despite progress made in explaining the allosteric mechanism, the mechanism of signal propagation between subunits during the allosteric transition remains unclear.
Structural data for the wild-type enzyme have suggested that the 190's
loop3 (Fig. 1) may play a crucial role for
allosteric regulation between subunits during the R- to T-state
transition. Previously, Lys-42, which interacts at the dimer-dimer
interface with several residues on the 190's loop, such as Ile-190,
Gly-191, and Glu-192 (9), was mutated to alanine (10). For the Lys-42
Ala enzyme, AMP inhibition was no longer cooperative, even though
the x-ray structure of this mutant enzyme showed no alterations at the
AMP binding site; this result has demonstrated that the dimer-dimer
interface is important for propagation of the AMP inhibition signal
(10). In order to obtain more detailed information concerning the role of the 190's loop in the regulation of the allosteric transition within each subunit and also between subunits, site-specific
mutagenesis has been performed on residue Glu-192, which forms hydrogen
bonds at the C1:C4 dimer-dimer interface, and on the residue Asp-187, which forms hydrogen bonds at the C1:C2 monomer-monomer interface. The
mutant enzymes Glu-192
Gln, Glu-192
Ala, and Asp-187
Ala
were expressed in Escherichia coli, and the consequences of these mutations on the kinetic properties of the enzyme were
investigated.
Materials
Agar, agarose, ampicillin, chloramphenicol, sodium dihydrogen
phosphate, and magnesium chloride were purchased from Sigma. Tris and
enzyme-grade ammonium sulfate were supplied by ICN Biomedicals. Tryptone and yeast extract were from Difco. The oligomers used to
construct the Glu-192 Ala and Glu-192
Gln mutations and some of
the DNA oligomers used as primers for DNA sequencing were synthesized
by Operon Technologies. All the reagents needed for DNA sequencing were
obtained from Amersham Corp. Restriction endonucleases, T4 DNA ligase,
T7 DNA polymerase, and T4 polynucleotide kinase were either from
Amersham or New England Biolabs, and used according to the supplier's
recommendations. DNA fragments were isolated from agarose gels with the
Geneclean II kit from Bio 101, Inc.
The E. coli K12 strain MV1190 ((lac-proAB),
supE, thi,
(sri-recA)
306::Tn10(tetr)/F
traD36, proAB,
lacIq, lacZ
M15) and the M13 phage M13K07
were obtained from J. Messing. The strain CJ236 (dut-1, ung-1,
thi-1, relA-1/pCJ105(Cmr)) was a gift of T. Kunkel,
while EK1601 (tonA22, ompF627(T2R),
relA1, pit-10, spoT1,
(fbp)287,
DE3) a
derivative of DF657 (11) was constructed in this laboratory (12).
E. coli strain XL1-Blue MRF
(
(mcrA)183,
(mcrCB-hsdSMR-mrr)172,
endA1, supE44, thi-1, recA, gyrA96, relA1, lac,
/F
proAB, lacIq,
lacZ
M15, Tn10(tetr)] was from Stragene (La
Jolla, CA).
Methods
Oligonucleotide SynthesisThe oligonucleotide used to
construct the Asp-187 Ala mutation and some of the primers required
for DNA sequencing were synthesized on an Applied Biosystems 381A DNA
synthesizer and purified by high pressure liquid chromatography
employing a DuPont Zorbax Oligo ion-exchange column.
Site-specific mutagenesis was performed on the
Fru-1,6-P2ase cDNA harbored on plasmid pEK284,
employing the method of Kunkel (13, 14). Uracil-containing
single-stranded DNA was obtained by infection of E. coli
strain CJ236 containing pEK284 with the helper phage M13KO7 (15).
Potential mutant candidates were initially identified by DNA sequence
analysis (16). The entire cDNA of Fru-1,6-P2ase of one
candidate was sequenced to ensure that the site-specific mutagenesis
had not introduced additional mutations other than the desired changes.
The resultant plasmids, pEK295, pEK372, and pEK373 contained only the
mutation at the codons corresponding to the Asp-187 Ala, the
Glu-192
Ala, and the Glu-192
Gln mutations, respectively.
In order to express the wild-type and mutant pig kidney Fru-1,6-P2ases, the corresponding phagemids were transformed into E. coli strain EK1601. E. coli strain EK1601 has a deletion in the chromosomal fbp gene and can be induced to produce T7 RNA polymerase (12). In this manner the pig kidney Fru-1,6-P2ase expressed from the plasmid could not be contaminated with the E. coli Fru-1,6-P2ase expressed from the chromosome.
Enzyme PurificationBacteria were cultured with vigorous
agitation at 37 °C in M9 medium supplemented with 0.5% casamino
acids and ampicillin at 100 µg/ml. Induction of T7 RNA polymerase was
initiated by addition of 0.4 mM
isopropyl--D-thiogalactopyranoside (Sigma). After
further cultivation for 16-22 h, cells were harvested by centrifugation and broken open by a freeze-thaw procedure (17). Purification of mutant enzyme was accomplished by the method previously described (12).
A spectrophotometric, coupled-enzyme assay was employed to measure Fru-1,6-P2ase activity (18). Standard conditions (2 mM magnesium chloride) were used to determine specific activity. Digital absorbance values were collected and fit to a straight line by computer, using data beyond the coupling lag period. In all assays, the enzyme was added last, after incubation and thermal equilibration of the coupling enzymes, magnesium (and inhibitor) components. One unit of enzyme activity causes the reduction of 1 µmol of NADP/min at 30 °C.
Methods used to determine the kinetic model parameters were described
previously (19). The AMP inhibition curves of the Glu-192 Ala and
Glu-192
Gln Fru-1,6-P2ase were biphasic and therefore
had to be analyzed differently from the data for the wild-type enzyme.
The data for both phases of the curve did not exhibit cooperativity and
therefore could be fit by the sum of two hyperbolic functions (10).
Multiple determinations of initial rates of catalysis at a specific set
of component concentrations provided measures of assay precision.
Weighted mean data were used.
Concentrations of the wild-type and all the mutant enzymes were determined using the Lowry-Peterson method (20, 21) with bovine serum albumin as the standard.
Other MethodsSDS-polyacrylamide gel electrophoresis was used to judge enzyme homogeneity (22). Concentrations of Fru-1,6-P2 and NADP were checked by performance in the coupled assay, of AMP using 15.4 as the millimolar extinction coefficient at pH 7.0 and 259 nm, and of Fru-2,6-P2 by partial acid hydrolysis and analysis of fructose 6-phosphate (23).
The kinetic data for
wild-type and the three mutant enzymes are summarized in Table
I. For each enzyme, the substrate saturation curve is
hyperbolic with inhibition exhibited at high substrate concentrations.
Analysis of the kinetic data was performed by using a nonlinear least
squares method incorporating a term for substrate inhibition. The
Asp-187 Ala enzyme (Fig. 2) had the same
kcat as the wild-type enzyme; however, the
Km for this enzyme (0.7 µM) was
slightly lower than the corresponding value for the wild-type enzyme
(1.4 µM). Both the Glu-192
Ala and the Glu-192
Gln enzymes (Fig. 2) have lower kcat values, 15 s
1 and 13 s
1, respectively, compared to 21 s
1 for the wild-type enzyme. However, the
Km values of the mutant enzymes were close to that
of the wild-type enzyme (Table I).
|
Influence of Mg2+ on the Wild-type and the Mutant Fru-1,6-P2ases
Wild-type Fru-1,6-P2ase
requires divalent metal ions such as Mg2+ to achieve
catalytic activity, and the activation effect is cooperative with a
Hill coefficient of approximately 2 (6). For wild-type Fru-1,6-P2ase, the concentration of Mg2+ needed
to activate the enzyme to half its maximum activity is 0.34 mM; however, the corresponding values for mutant enzymes were 0.95 mM, 1.16 mM and 0.49 mM
for the Glu-192 Ala, Glu-192
Gln, and Asp-187
Ala enzymes,
respectively. There were also slight differences in the values of the
Hill coefficients between the wild-type and the mutant enzymes. The
Hill coefficient of wild-type Fru-1,6-P2ase was 1.8 ± 0.5; however, the corresponding values for the mutant enzymes were
1.4 ± 0.1, 1.4 ± 0.1, and 1.7 ± 0.1 for the Asp-187
Ala, Glu-192
Ala, and Glu-192
Gln enzymes, respectively
(Table I). The lower Hill coefficients indicate that the magnesium
activation for the mutant enzymes were not as cooperative as it was for
the wild-type enzyme.
For the wild-type enzyme, Fru-2,6-P2 is a
competitive inhibitor of the substrate (24), and all the mutant enzymes
investigated here showed the same characteristic. The
Ki value for wild-type Fru-1,6-P2ase was
0.065 µM, which is very close to the value determined for
the Glu-192 Ala enzyme (Ki = 0.064 µM) and the Glu-192
Gln enzyme (Ki = 0.059 µM). However, the Ki for the
Asp-187
Ala enzyme (0.11 µM), was slightly higher
than that of the wild-type enzyme.
AMP
is an allosteric inhibitor for pig kidney Fru-1,6-P2ase,
and it binds to an allosteric site, about 28 Å distant from the substrate binding site (3). For wild-type Fru-1,6-P2ase,
AMP inhibition is cooperative with a Hill coefficient of approximately 2 and a dissociation constant of 2.8 µM. For the Asp 187 Ala enzyme (see Fig. 4), AMP inhibition was still cooperative with the same Hill coefficient as that of the wild-type enzyme. However, the
dissociation constant was 13.4 µM, which is about 4-fold
higher than the corresponding value for the wild-type enzyme.
In contrast to the Asp-187 Ala enzyme, AMP inhibition was altered
drastically for the Glu 192
Gln and the Glu-192
Ala enzymes
(see Fig. 4). First, AMP inhibition was not cooperative for either of
these mutant enzymes. Second, the inhibition was biphasic and the
enzymes could only be fully inhibited at high concentrations of AMP.
Thus, both the high and low affinity sites must be saturated in order
for complete inhibition to be obtained. The high affinity binding sites
could be saturated at approximately 200 µM of AMP,
resulting in 90% inhibition of the Glu-192
Ala enzyme. The AMP
dissociation constant for the high affinity sites was 1.6 µM; however, the corresponding value for the low affinity sites could not be determined accurately due partially to the fact that
the remaining activity of this phase was very low.
The Glu-192 Gln enzyme also exhibited biphasic AMP inhibition;
however, saturation of the high affinity binding sites resulted only in
40% inhibition, as compared with 90% for the Glu-192
Ala enzyme.
Moreover, the dissociation constant of the high affinity sites was 19 µM, which was about 10-fold higher than the corresponding value for the Glu-192
Ala enzyme, while the dissociation constant for the low affinity binding sites was 14 mM.
Based on the x-ray structures of wild-type
Fru-1,6-P2ase (3, 4) and previous kinetic and x-ray crystal
structure studies of the Lys-42 Ala enzyme (10), we speculated that
the 190's loop (C1), which is located at the central part of the
tetramer and forms multiple interactions with Lys-42 (C4), may be
important for signal transduction across the interfaces during the R-
to T-state transition (9). Both residues 187 and 192 from the 190's
loop form interface interactions with residues from the neighboring
subunits (Fig. 3). Therefore, in order to elucidate the
role of the 190's loop in allosteric regulation, three mutant enzymes,
the Glu-192
Ala, the Glu-192
Gln and the Asp-187
Ala
enzymes were generated and their kinetic characteristics were
determined.
Mutations at Residues 187 and 192 Have Only a Minor Effect on Catalytic Efficiency of the Mutant Enzymes
In the structure of
the wild-type enzyme, the side chain carboxyl group of Asp-187 (C1)
forms hydrogen bonds across the C1:C2 monomer-monomer interface (9);
the side chain of Glu-192 (C1) forms hydrogen bonds with Thr-39 (C4)
and Lys-42 (C4) across the C1:C4 dimer-dimer interface in both the R-
and T-states (see Fig. 3). Since the 190's loop is about 30 Å distant
from the substrate binding site, only minor effects on the activity
were anticipated by these amino acid substitutions. For all three
mutant enzymes presented here, kcat and
Km values were close to the corresponding values for
the wild-type enzyme. In addition, none of the mutations significantly
altered the Ki value for the competitive inhibitor
Fru-2,6-P2. In fact, the Ki values for
the Glu-192 Ala and Glu-192
Gln enzymes are almost identical to
those of the wild-type enzyme. These results are consistent with the
proposal that the active site is not altered by mutations at the 190's
loop.
Based on a comparison of the wild-type R- and T-state structural data, Zhang et al. (9) found that AMP domains have extensive interactions with each other across the interfaces. They also found that during the R- to T-state transition almost all the secondary structural changes occur within the AMP domain. These authors proposed that small initial changes at any one of the AMP domains could be quickly and efficiently transmitted to neighboring AMP domains (9). However, the pathway for intersubunit signal transduction, whether it be through the C1:C2 monomer-monomer interface and or the C1:C4 dimer-dimer interface cannot be determined by merely evaluating the structural data for the wild-type enzyme.
By combining the structural data from the wild-type enzyme with
functional data obtained from mutant enzymes, it is possible to shed
light on the mechanism of signal transduction in pig kidney Fru-1,6-P2ase. For the Asp-187 Ala enzyme, AMP
inhibition is cooperative (Fig. 4), although the enzyme
is about 4-fold less sensitive to AMP than the wild-type enzyme. For
the Glu-192
Ala and the Glu-192
Gln enzymes (Fig. 4), AMP
inhibition was altered more significantly. First, for these two enzymes
AMP inhibition was no longer cooperative, and second, AMP inhibition
was biphasic as has been seen previously for the Lys-42
Ala enzyme
(10).
The two residues mutated in this work participate in different
intersubunit interactions. Asp-187 forms interface interactions exclusively at the C1:C2 monomer-monomer interface, whereas Glu-192 forms interactions only across the C1:C4 dimer-dimer interface. The
Asp-187 Ala mutation had no effect on the cooperativity associated
with AMP inhibition, while the two mutations at Glu-192 abolished the
cooperativity associated with AMP inhibition. Previously, Lys-42 (10),
which forms hydrogen bonds across the C1:C4 dimer-dimer interface with
residues on the 190's loop including 192, was mutated to Ala, and the
resulting mutant enzyme also lost cooperativity associated with AMP
inhibition. Grazi et al. (25) studied the kinetic properties
of matrix-bound Fru-1,6-P2ases. They found that the
matrix-bound tetramer retained most of the catalytic activity but
became half-desensitized to AMP. The matrix-bound dimer possessed half
the specific activity of the tetramer; however, it was almost
completely desensitized to AMP. All these results strongly suggest that
signal transduction between the AMP binding sites of
Fru-1,6-P2ase occurs mostly via a pathway involving Glu-192 and Lys-42 across the C1:C4 dimer-dimer interface, rather than a
pathway across the C1:C2 interface.
For the Glu-192 Ala and Glu-192
Gln enzymes, in addition to the
loss of cooperativity associated with AMP inhibition, the AMP
inhibition was found to be biphasic, as was previously observed for the
Lys-42
Ala enzyme (10). The dissociation constant for the high
affinity binding sites of the Lys-42
Ala enzyme was 18 µM, and the corresponding value for the low affinity binding sites was 11 mM. At a concentration of less than
100 µM AMP, which would be adequate to saturate only the
high affinity sites, a T-state crystal of the Lys-42
Ala enzyme was
obtained and its structure determined (10). Analysis of the x-ray
structural data indicated that all the AMP sites were fully occupied.
Furthermore, the structure of the Lys-42
Ala enzyme showed clearly
that the allosteric site was not altered by the mutation (10).
Additionally, Lipscomb and co-workers (5) were able to crystallize the
wild-type enzyme in the R state, with the competitive inhibitor
Fru-2,6-P2 and one of the products fructose 6-phosphate (4)
bound to the allosteric site, which suggests that comparable binding of
AMP to the active site at high concentrations might be possible. Lu et al. (10) concluded that the high affinity binding sites
were associated with the binding of AMP to the regulatory sites, while the low affinity sites were associated with the binding of AMP to the
active sites. However, for wild-type enzyme the binding of AMP at the
active site cannot be detected by standard activity measurements
because the enzyme is completely inactivated at concentrations of AMP
that would not be sufficient to bind significantly to the active sites.
For the Glu-192
Ala and Glu-192
Gln enzymes, AMP binding at the
allosteric sites cannot completely inactivate the enzyme. Only by
binding of AMP at both the high and low affinity sites can these mutant
enzymes be completely inhibited.
As shown in Fig. 4, even
though the apparent AMP dissociation constant of the Glu-192 Ala
enzyme for the allosteric sites is about half the value for the
wild-type enzyme, activity could not be fully inhibited by saturating
the high affinity allosteric sites. The corresponding value for the
Asp-187
Ala enzyme is more than four-fold higher than the wild-type
enzyme; however, it could be fully inhibited by binding of AMP to the
allosteric site and it retained the cooperativity associated with AMP
inhibition. Previously, kinetic studies on the Arg-22
Ala enzyme
(19) have shown that although this mutant enzyme's apparent
dissociation constant is about 10-fold higher than the wild-type
enzyme, AMP inhibition is still cooperative with a Hill coefficient of
2. Furthermore, the mutant enzyme could be completely inhibited by saturating the allosteric sites with AMP. All these results strongly support the notion that cooperativity is required for complete inactivation of Fru-1,6-P2ase by the binding of AMP to the
allosteric sites.
AMP inhibition of the Glu-192 Gln and the Lys 42
Ala enzymes is
so similar that their inhibition curves are almost superimposable (Fig.
5), which suggests that these two mutations had almost
the same effects on the allosteric regulation between the allosteric and the metal binding sites. However, the replacement of Glu-192 by Ala
or Gln results in two mutant enzymes that are almost identical kinetically, except for their inhibition by AMP. Besides the
approximate 10-fold difference between their apparent AMP dissociation
constants for the allosteric site, the extend of inhibition is about
90% for Glu-192
Ala enzyme whereas it is about 40% for the
Glu-192
Gln enzyme. These mutations would alter the hydrogen bond
interaction between Glu-192 (C1) and Lys-42 (C4), which is important
not only for allosteric regulation between the AMP binding sites across the dimer-dimer interface, but also for signal transduction between the
AMP binding site and the metal binding sites within each subunit. In
addition, because of the differences in their side chains, these
mutations would have different affects on the charge neutralization of
the two closely spaced Lys-42 residues. In this manner, the signal
transduction pathway connecting the AMP binding site and the metal
binding sites is interrupted differently in the Glu-192
Ala and
Glu-192
Gln enzymes.
It is important to maintain the distinction between cooperative and
allosteric phenomena, while recognizing that they are often found
together (26). By definition, allostery allows one kind of small
molecule to regulate the action of a protein on another kind of
molecule. For the Glu-192 Ala and the Glu-192
Gln enzymes, even
though AMP no longer inhibits cooperatively, within each subunit the
AMP binding site can still communicate with the metal binding sites,
and for this reason AMP can still inhibit the enzyme. However, the
mutations at Glu-192 do affect the communication pathways in such a way
that the binding of AMP at the allosteric site cannot completely
inhibit these mutant enzymes. Thus, the enzyme requires cooperative AMP
inhibition in order to achieve the full regulation effect between the
allosteric site and the metal cation binding sites.
x-ray crystallographic structural data for wild-type Fru-1,6-P2ase showed that there are two metal binding sites in each monomer (8), which are located between the FBP and the AMP domains (Fig. 1). Direct binding (27, 28) and steady-state kinetic studies (6) have established the sequence of binding of metal cations and the substrate in the order: structural metal cation, substrate, then catalytic metal cation. All three mutant enzymes presented here exhibit dramatic altered AMP inhibition, but are also less sensitive to magnesium activation, for which their Hill coefficients are decreased from 2 for wild-type enzyme to around 1.5 for these mutant enzymes. The alteration of residue 187 and 192 might cause a small local structural reorientation of the 190's loop, which would result in the observed changes in the magnesium activation of the mutant enzymes.
The mechanism of allosteric regulation between the magnesium sites has been studied before. Direct binding studies (28) showed that after the binding of four structural metal ions per tetramer, 2 moles of ligand result in the binding of 4 catalytic metal ions, suggesting a positive cooperative interaction between sites on different subunits. Nevertheless, since within each subunit the structural metal binding site must be occupied before the second metal cation can bind to the catalytic site (6), intrasubunit cooperativity cannot be ruled out.
SummaryThe results presented here, combined with the
previous kinetic and structural results from Lys-42 Ala enzyme
(10), strongly suggest that the signal transduction pathway between the
AMP binding sites takes place across the C1:C4 dimer-dimer interface,
mediated by residues of the 190's loop. Furthermore, cooperative AMP
inhibition is required in order to achieve full communication between
the allosteric and the metal binding sites in
fructose-1,6-bisphosphatase.