(Received for publication, October 11, 1995; and in revised form, December 1, 1995)
From the
Mutation of Arg-15, Glu-19, Arg-22, and Thr-27 of porcine liver
fructose-1,6-bisphosphatase was carried out by site-directed
mutagenesis. These residues are conserved in all known primary
sequences of mammalian fructose-1,6-bisphosphatase. On the basis of the
crystal structure of the enzyme, Arg-15, Glu-19, and Arg-22 are located
at the interface of the two dimers (C1-C2 and C3-C4), and Thr-27 is in
the AMP binding site. The wild-type and mutant forms of the enzyme were
purified to homogeneity and characterized by initial rate kinetics and
circular dichroism (CD) spectrometry. No discernible differences were
observed between the secondary structures of the wild-type and mutant
forms of fructose-1,6-bisphosphatase on the basis of CD data. Kinetic
analyses revealed similar k values for mutants
R15A, E19Q, R22K, and T27A of fructose-1,6-bisphosphatase; however, a
2-fold increase of k
was observed with R22M
compared with that of the wild-type enzyme. Small changes in K
values for fructose-1,6-bisphosphate
were found in the five mutants. 4-6-fold decreases in K
values for fructose 2,6-bisphosphate
and 5-9-fold decreases in the binding affinity of Mg
relative to the wild-type enzyme were exhibited by R15A and E19Q.
No alteration of Mg
cooperativity was found in the
five mutants. Significant changes in K
values for AMP were obtained in the case of R22K (30-fold)
and T27A (1300-fold) with a Hill coefficient of 2.0. Replacement of
Arg-22 with methionine, however, caused the total loss of AMP
cooperativity without changing AMP affinity. Modeling of the mutant
structures was undertaken in an attempt to define the functional role
of Arg-22. These studies link specific interactions between subunits in
fructose-1,6-bisphosphatase to observed properties of cooperativity.
Fructose 1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11),
FBPase()) is an allosteric enzyme located at a crucial
control point in carbohydrate metabolism. In the presence of divalent
metal ions, the enzyme catalyzes the hydrolysis of
fructose-1,6-bisphosphate (Fru-1,6-P
) to fructose
6-phosphate (Fru-6-P) and inorganic phosphate (P
). The
reaction is noncompetitively inhibited by AMP and competitively
inhibited by Fru-2,6-P
, and the action of these two
inhibitors is synergistic(1, 2, 3) . These
two compounds are involved in the activation of phosphofructokinase (4) and are responsible for the coordinated regulation of
glycolysis and glyconeogenesis(5, 6) .
FBPase is
composed of four identical subunits, each of which contains a substrate
and AMP binding domain separated by 28 Å, and metal binding sites
in close proximity to the substrate binding
site(7, 8, 9) . AMP binding is cooperative
with a Hill coefficient of approximately 2 (10, 11, 12, 13) . In addition, AMP
inhibition exhibits ``half of the sites''
reactivity(14) . Cooperativity with respect to metal ion
binding to FBPase has also been
recognized(10, 15, 16) . The activity of
FBPase, as a function of Mg concentration, is
sigmoidal at neutral pH but hyperbolic at pH 9.6. Furthermore, AMP and
Mg
are mutually exclusive in their binding to the
enzyme (10, 17) . X-ray crystallographic studies have
shown that an R- to T-state transition of FBPase is triggered by the
binding of AMP, causing a shift of binding sites for metals to
unfavorable positions (18, 19) . The mutation of
residues at the metal binding sites of FBPase affects the
Mg
and AMP affinity(20) ; however,
significant changes in Mg
cooperativity and affinity
and/or AMP affinity were also a consequence of the mutation of active
site residues(21, 22, 23) . In addition, AMP
cooperativity is completely lost when Glu-29, a residue in the AMP
domain, is replaced by Gln (24) .
X-ray diffraction studies of FBPase from porcine kidney have pinpointed a number of amino acid residues located at the interface of dimers that might contribute to cooperativity during the R- to T-state transition. In an attempt to associate functional properties of FBPase to specific interactions of the dimer interface, Arg-15, Glu-19, Arg-22, and Thr-27, which is located in the AMP binding site but interacts with Arg-22 in the T-state of FBPase, were the subjects of experiments in directed mutation. The kinetic properties of R15A, E19Q, R22K, R22M, and T27A were investigated. The most important finding of this investigation is that the replacement of Arg-22 with methionine causes a total loss of AMP cooperativity and increases the turnover number of the enzyme as measured by initial rate kinetics.
Mutations were confirmed by NruI/XhoI digestion and by fluorescent dideoxy chain-termination sequencing at the Nucleic Acid Facility at Iowa State University. The mutagenesis plasmid was finally transformed into Escherichia coli DE 657, a strain deficient in the FBPase gene.
Protein concentration was assayed as described by Bradford (27) with bovine serum albumin (from Sigma) as the standard. The protein purity was determined by using 12% SDS-polyacrylamide gel electrophoresis.
where v, V, A, B, K
, K
, and K
represent initial velocity, maximal velocity,
the concentration of free Mg
, the concentration of
free Fru-1,6-P
, the Michaelis constant for
Mg
, the Michaelis constant for Fru-1,6-P
,
and the dissociation constant for Mg
, respectively,
and where n represents the cooperativity for Mg
with FBPase. Table 2shows that wild-type and the five
mutants possess similar Hill coefficients for Mg
.
Mg
activation of the enzymes was sigmoidal in all
cases (data not shown). The kinetic data for these enzymes also gave
excellent fits to with n = 2. On the basis
of these results, it is probable that Arg-15, Glu-19, Arg-22, and
Thr-27 may not be directly involved in the Mg
cooperativity. A 2-, 5-, and 9-fold increase in K
values for Mg
were found in T27A, E19Q, and
R15A, respectively, relative to wild-type FBPase.
Figure 1:
Plot of reciprocal of initial velocity
in arbitrary fluorescent units versus reciprocal of
[Mg]
for R22K FBPase. The
concentrations of AMP are 0 (
), 2.0 µM (+), 3.0
µM (
), and 4.5 µM (
). The
coupled spectrofluorescence assay was performed at 25 °C in 50
mM Hepes buffer (pH 7.5) containing 0.1 M KCl, 10
µM Fru-1,6-P
. The lines are
theoretical based upon when n = 2, and the points were experimentally determined. The inset shows a plot of the slope of the family of curves in Fig. 1versus [AMP]
.
where v, V, A, B, I, K
, K
, K
, K
, K
, K
, and K
represent initial velocity, maximal velocity,
the concentration of Mg
, Fru-1,6-P
, AMP,
the Michaelis constants for Mg
and
Fru-1,6-P
, dissociation constants for Mg
,
and the dissociation constants for AMP from the enzyme-AMP, the
enzyme-AMP-AMP, the enzyme-Fru-1,6-P
-AMP, and the
enzyme-Fru-1,6-P
-AMP-AMP complexes, respectively. n represents the Hill coefficient for AMP with FBPase. When n = 2, the binding of AMP to FBPase exhibits cooperativity;
on the other hand, there is no cooperativity when n =
1. In the case of wild-type FBPase and R15A, E19Q, and T27A mutant
FBPases, the kinetic data from experiments similar to those shown in Fig. 1all fit best to when n = 2
(data not shown). Thus, AMP cooperativity was not altered with these
mutants. Replacement of Arg-22 with Met, however, yielded the kinetic
data shown in Fig. 2, which gave an excellent fit to when n = 1 but did not fit as well to when n = 2. The ``Goodness of
Fit'' was 5% when n = 1 and 12% when n = 2. is consistent with the random mechanism
along with the following interactions:
Figure 2:
Plot of reciprocal of initial velocity in
arbitrary fluorescent units versus reciprocal of
[Mg]
for R22M FBPase. The
concentrations of AMP are 0 (
), 0.5 µM (+), 1.0
µM (
), 2.0 µM (
), and 3.0
µM (
). The coupled spectrofluorescence assay was
performed at 25 °C in 50 mM Hepes buffer (pH 7.5)
containing 0.1 M KCl, 10 µM Fru-1,6-P
. The lines are theoretical based
upon when n = 1, and the points were experimentally determined. The inset shows a plot of
the slope of the family of curves in Fig. 2versus [AMP].
It is clear from that I, a competitive
inhibitor for substrate A will be a noncompetitive inhibitor of the
other substrate (B) in the reaction. As expected, when a
double-reciprocal plot of 1/velocity against 1/(Fru-1,6-P)
was made at different concentrations of AMP for wild type, R15A, E19Q,
R22K, or T27A, a family of intersecting lines was obtained to the left
of the 1/v axis (data not shown). These data were best fit to when n = 2. In the case of R22M, the best
fits were obtained when n = 1 in . These
results suggest that the positive charge on the side chain of Arg-22 is
essential for maintaining AMP cooperativity. Mutation of Thr-27 to Ala,
which interacts with O2A of the phosphate group of AMP, caused a
significant decrease (1300-fold) in AMP affinity for FBPase without
changing the AMP cooperativity. A dramatic change of AMP binding and
cooperativity, however, has been reported for the Glu-29
Gln
mutant of FBPase(24) . Glu-29 is also directly ligated to the
phosphoryl portion of AMP.
The major finding of this report is the complete loss of AMP
cooperativity and increased enzymatic activity when FBPase Arg-22 is
mutated to Met. It has long been recognized that the binding of AMP to
wild-type FBPase exhibits
cooperativity(10, 12, 13, 24) . The
kinetic data of AMP inhibition with wild-type, R15A, E19Q, R22K, and
T27A FBPases gave excellent fits to a cooperativity model. In these
cases, the Hill coefficient for the nucleotide is 2.0. On the other
hand, R22M shows a loss of AMP cooperativity with a Hill coefficient of
1.0 for AMP. AMP is a competitive inhibitor of Mg and
noncompetitive inhibitor of Fru-1,6-P
at neutral pH with
the five mutant forms of FBPase as well as with the wild-type enzyme.
Data contained in this report suggest that mutation of porcine
FBPase Arg-22 to Met causes the complete loss of cooperativity without
changing AMP affinity; however, the affinity for AMP decreased markedly
in the case of R22K without altering AMP cooperativity. We conclude
that the Arg-22 residue of mammalian FBPase is essential for
simultaneous cooperativity and binding of AMP. An understanding of this
finding requires further investigations (e.g. solution of the
crystal structure of R22M FBPase); however, modeling studies were
undertaken to provide some insights into the effect of mutation of
Arg-22 to Met and Lys. Fig. 3is a stereoview of the subunit
C1-C4 interface of FBPase in the T-state from the porcine kidney FBPase
crystal structure reported by Ke et al.(38) . AMP
molecules and most of helix H1 and H2 are shown. In the T-state of
FBPase, NE of Arg-22 of subunit C1 is hydrogen bonded (3.1 Å) to
O of Thr-27, a residue located at the AMP binding site of subunit C4.
Atoms N and OG1 of Thr-27 are hydrogen bonded to O2A of the phosphoryl
group of AMP. To determine the possible conformational changes in the
area of the mutation, we used computer modeling to compare energy
minimized R22M and R22K mutants with the wild-type enzyme. The
10-Å area around residue 22 of both subunits C1 and C4 was free
to change, while the rest of the molecule was fixed. The conformation
of residues in the energy minimized area changed slightly, but the
changes were principally identical in all three models. This is why in
our further analysis we kept all residues fixed to their positions in
the x-ray structure except residues 22 and 27 and the nearest water
molecules. In the R22M mutant, the side chain of Met-22 retained the
conformation of the side chain of Arg (Fig. 4A). Thus,
the only obvious change in subunit interactions is the loss of hydrogen
bonding from NE of Arg-22 to O of Thr-27. The kinetic data suggest that
the hydrogen bond is necessary for cooperativity but not AMP binding
affinity. The importance of the interactions between Glu-19, Glu-29,
and Arg-22 is not obvious from the x-ray structure. For example, Glu-19
is hydrogen bonded to Arg-22, but the symmetry related pair
Glu-19- Arg-22 is not. Moreover, the Glu-19-Arg-22
interaction is within the same subunit and has no direct relation to
intersubunit contacts. The interaction of Arg-22 with Glu-29 (and vice
versa) may be more important, but these groups do not make a direct
contact (Arg-22 NH is 4.74 Å from Glu-29 OE1). In the
case of the R22K mutant, NZ of Lys is shifted 1.4 Å further from
its C
atom than the NE of Arg. This difference puts NZ of Lys-22
close enough to OG1 of Thr-27 of the symmetry-related subunit so that
they form a hydrogen bond (Fig. 4B). Our modeling
suggests that the new hydrogen bond perturbs the interaction between
OG1 of Thr-27 and the phosphoryl group of AMP (the distance changes
from 2.8 Å in the wild-type to 3.2 Å in the mutant enzyme).
This finding correlates with kinetic data for the R22K and T27A
mutants. In T27A, a complete loss of one hydrogen bond (from OG1 of
Thr-27 to O2A of AMP) reduced its affinity for AMP by 1300-fold. The
putative weakening of the same hydrogen bond in the R22K mutant also
reduces AMP affinity but to a lesser extent. Despite the different
positions for NZ of Lys-22 and NE of Arg-22, each side chain maintains
an interaction with carbonyl 27. This interaction should be present as
well in the T27A mutant. Thus, apparently, subtle changes in the
position of OG1 of Thr-27 influences AMP affinity, whereas the loss of
hydrogen bond to O of Thr-27 (as in R22M) results in the complete loss
of cooperativity.
Figure 3: Stereo drawing of the adjacent AMP binding sites of two subunits of wild-type FBPase. The coordinates for the drawing were taken from the x-ray structure of porcine kidney FBPase complexed with AMP ((38) , Protein Data Bank entry 4FBP). The symmetry axes between subunits is perpendicular to the picture. Subunit C1 is drawn with thin lines, and subunit C4 is drawn with thick lines. One set of 2-fold related hydrogen bonds is shown with dotted lines. Other distances are shown with dashed lines.
Figure 4: Superpositon of the wild-type structure on (A) the energy-minimized model for the R22M mutant and on (B) the energy-minimized model for the R22K mutant. Thin lines represent the wild-type structure and the fixed part of the mutant model, thick lines represent the energy minimized residues of the mutant, and bold lines represent the AMP ligand. The residues numbers from subunit C1 are prefaced with #. Distances corresponding to hydrogen bonds of the wild-type enzyme are shown with dotted lines. Other distances are shown with dashed lines.
According to the x-ray diffraction data(19) , AMP induces localized conformational changes, which lead to the global rearrangement of the C1-C4 (C2-C3) subunit interface. This rearrangement involves significant movement of side chains along the C1-C4 (C2-C3) interface and the formation of new hydrogen bonds among residues at that interface. The rearrangement is putatively responsible for the cooperativity of AMP binding and the stabilization of the T-state conformation of FBPase. The kinetic properties of the three mutants can be explained simply by the disruption of interactions in the T-state. Studies of the R22M mutant demonstrates that despite the reorganization of significant numbers of hydrogen bonds during the transition to the T-state, the elimination of interactions produced by the mutation of a single residue abolishes AMP cooperativity. The loss of AMP cooperativity in R22M, without a concomitant change in AMP affinity and inhibition, demonstrates that these properties can be separated. Based on the mechanism of allosteric regulation of FBPase by AMP(19) , we can suggest two mechanisms for the loss of cooperativity: 1) the loss of cooperativity is associated with the destabilization of the T-state. As a result, a subunit with bound AMP is transformed from the R-state to the T-state but without altering the conformation of other subunits; 2) the binding of AMP to the mutant still forces all the subunits into the T-state, but the lack of an Arg-22-Thr-27 interaction leaves the symmetry-related AMP binding sites unaltered.
In the absence of AMP, k of the R22M is higher than that of the
wild-type enzyme. The increase in activity of R22M may be associated
with changes in the conformation of the R-state due to the loss of two
hydrogen bonds from the guanidinium of Arg-22 in the mutant FBPase. In
the R-state, NH1 of Arg-22 is hydrogen bonded to the carbonyls of
Arg-110 and Glu-108, each belonging to a neighboring subunit (C1-C4 or
C2-C3 interfaces). Alternatively, FBPase may exist as an equilibrium of
R- and T-states, with a significant amount of T-state even in the
absence of AMP. The loss of hydrogen bonds in the T-state mutants would
shift the equilibrium to the R-state and, thus, increase the activity.
Both alternatives can provide an explanation of the observed
Mg
cooperativity. On one hand, Mg
cooperativity may be due to the transformation of FBPase to some
``activated'' R-state after it binds one Mg
ion, whereas, on the other hand, Mg
cooperativity may be explained by the shift of the equilibrium
between R- and T-states toward the R- state.
The hydrogen bonding
between the side chains of Arg-15 and Ser-87 (C1-C4 and C4-C1) is
abolished in the Ala-15 mutant, thus causing the increase in
Fru-2,6-P binding affinity (6-fold) and in the K
for Mg
(9-fold) relative to
that of wild-type FBPase. A similar effect was found with the E19Q
mutant, which exhibits about 5-fold changes in Fru-2,6-P
and Mg
binding affinities. X-ray diffraction
studies(18, 19) have demonstrated that the relative
movements between the Fru-1,6-P
and AMP domains during the
R- to T-state transition also shifts the metal binding sites to
unfavorable positions, the result of which is the inhibition of enzyme
activity.
This study demonstrates that residues located at the
interfaces of subunits C1 and C4 and of subunits C2 and C3 have
different effects on AMP binding, substrate binding, and Mg binding, i.e. Arg-22 is essential for AMP cooperativity
and affinity, and Arg-15 and Glu-19 effect Mg
and
Fru-2,6-P
binding. This report also shows that the helix H1
(residues 12-24) are somehow involved in the communication
between the active site and the AMP site during the R- to T-state
transition, a finding consistent with the results of x-ray diffraction
studies (19) .