(Received for publication, June 10, 1994; and in revised form, October 21, 1994)
From the
Site-directed mutagenesis of an amino acid residue in the
substrate binding site of porcine fructose-1,6-bisphosphatase was
carried out based on the crystal structure of the enzyme (Zhang, Y.,
Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W. N.(1993) Biochemistry 32, 1844-1857). A mutant enzyme form of
fructose-1,6-bisphosphatase, G122A, was purified and characterized by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular
dichroism spectrometry (CD), and initial rate kinetics. There were no
discernible differences between the secondary structures of the
wild-type and the mutant enzyme on the basis of CD data. Altering
Gly-122 to alanine caused a significant decrease in the enzyme's
activity and affinity for Mg. The k
for this mutant enzyme was only about 5% of that of wild-type
fructose-1,6-bisphosphatase, and the K
for Mg
was about 20-fold higher than that
of the wild-type enzyme. The K
for AMP
was increased 77-fold in the case of the mutant enzyme; however, the
Hill coefficient was unaltered. Most importantly, it was observed that
replacement of Gly-122 with alanine caused the total loss of
cooperativity for Mg
. It is concluded that Gly-122 is
essential for Mg
cooperativity and important for
binding of Mg
and AMP as well as for enzyme activity.
It is well known that fructose-1,6-bisphosphatase (FBPase) ()is a crucial step in the regulation of
gluconeogenesis(1, 2, 3) . The enzyme
catalyzes the hydrolysis of fructose 1,6-bisphosphate
(Fru-1,6-P
) to fructose 6-phosphate (Fru-6-P) and inorganic
phosphate and exhibits a rapid equilibrium random Bi Bi kinetic
mechanism(4) . The reaction is competitively inhibited by
fructose 2,6-bisphosphate (Fru-2,6-P
) and noncompetitively
inhibited by AMP(4) . These two compounds are also involved in
the activation of phosphofructokinase(5) . Therefore, it is
thought that Fru-2,6-P
plays crucial roles in the
regulation of glycolysis and gluconeogenesis. AMP is also an important
element in the regulation of FBPase because it acts synergistically
with Fru-2,6-P
to inhibit FBPase(5) . The role of
AMP is thought to prevent divalent metal binding to FBPase, and
divalent cations such as Mg
and Mn
are absolutely required for FBPase
activity(4, 6) . One of the functions of
Fru-2,6-P
is to keep AMP on the enzyme (make it stickier) (7) , thus enhancing the action of AMP.
FBPase has long been
recognized to be a metal-requiring enzyme(8) . Univalent
cations can activate the enzyme(9) , but divalent cations are
absolutely required for enzyme activity(8) . The
crystallographic structure, reported by the Lipscomb
group(10) , suggests that two divalent cations are bound at the
metal-binding site. Benkovic et al.(11, 12) suggested that catalysis requires the
sequential addition of metal and substrate in the order, structural
metal, substrate, and catalytic metal to form a catalytically competent
quaternary complex of
enzyme-M-M
-Fru-1,6-P
. Nimmo and
Tipton (13, 14) showed that pH could affect the
divalent metal kinetics of bovine liver FBPase, i.e. the plots
of velocity versus Mg
are sigmoidal at
neutral pH, but they are hyperbolic at pH 9.6. Furthermore,
cooperativity with respect to metal ion binding and its activation of
the mammalian FBPase reaction has long been
recognized(11, 13, 14, 15) .
Recently, Chen et al.(16) and El-Maghrabi et al.(17) carried out site-directed mutagenesis experiments in
the metal binding sites of mammalian FBPase. Their investigations
support the suggestion that there are two metal binding sites
associated with FBPase.
FBPase from mammalian liver and kidney, two highly gluconeogenic tissues, is a homotetramer(18) . Alteration of the amino acid residues in the metal binding site can affect not only the cooperativity and ligand affinity of metal ions but also the enzyme's affinity for AMP (16) . These observations suggest that metal binding sites and AMP binding sites may somehow communicate with each other.
X-ray diffraction results of FBPase have pinpointed a number of amino acid residues that are associated with substrate binding(10) . These findings (10) suggest that the main chain NH group of the Gly-122 residue forms a bifurcated hydrogen bond to the ester oxygen and a 1-phosphoryl oxygen of the substrate. Thus, this residue is postulated to have the function of fixing the 1-phosphoryl group in the required position for metal binding and catalysis.
To gain some insight into
the residues involved in substrate binding and catalysis, we have
prepared a mutant of FBPase at position 122 by site-directed
mutagenesis and have studied its properties. In this study, we report
that replacement of Gly-122 with alanine causes the total loss of
cooperativity of Mg and very significant decreases in
enzyme activity, AMP binding, and affinity for metal ions.
Figure 1: SDS-PAGE analysis of purified wild-type and mutant G122A porcine FBPase. All samples were run on a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250. Lanes 1 and 4, protein standard; lane 2, G122A; lane 3, wild-type. Molecular mass of protein standards: A, 66 kDa; B, 45 kDa; C, 36 kDa; D, 29 kDa; E, 24 kDa; F, 20 kDa; G, 14 kDa.
The effects
of Fru-2,6-P inhibition on Mg
with the
G122A mutant form of FBPase were also studied. The K
increased with the increase of Mg
concentration
as described previously(24) . At 5 mM Mg
, the K
for
Fru-2,6-P
is about 22 µM.
The binding of AMP to wild-type FBPase is known to exhibit cooperativity(27) . The kinetic data for AMP inhibition with the two mutant forms of FBPase gave excellent fits to a cooperative model (28) in which the Hill coefficient is 2.0 (data not shown). Thus, the Gly-122 residue is not directly involved in the cooperativity of AMP binding to FBPase.
Figure 2:
Plot of
reciprocal of initial velocity in arbitrary fluorescent units against
reciprocal of Mg concentration for G122A FBPase. The
concentrations of Fru-1,6-P
are 25 µM (
), 15 µM (
), 10 µM (
), 6 µM (+), and 3 µM (
). The lines are theoretical based on when n = 1, and the points are experimentally
determined.
where V, V, A, B, K
, K
, and K
represent the initial velocity, maximum
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; n represents the Hill coefficient for Mg
with FBPase. When n = 1, there is no
cooperativity; when n = 2, the binding of
Mg
to FBPase is cooperative, with a Hill coefficient
of 2. is the fundamental initial rate equation for the
sequential kinetic mechanism shown in . (
)
On the other hand, the kinetic data in the absence of inhibitors
with wild-type FBPase (shown in Fig. 3) gave excellent fits to when n = 2 and did not fit well to when n = 1. Note that the only difference
in the rate equations between the G122A mutant enzyme and the wild-type
enzyme is the term n. The data shown in Fig. 3are
consistent with previous reports that show that binding of divalent
metal ions to FBPase exhibits
cooperativity(11, 13, 14, 15) ,
whereas the findings illustrated in Fig. 2exclude the
possibility of cooperativity for Mg with the G122A
mutant enzyme. Had Mg
ions bound cooperatively to
FBPase, the data would have fit to much better when n = 2 than when n = 1. On the basis of these
results and the Hill coefficient obtained for G122A, it is reasonable
to conclude that the Gly-122 residue is required for the cooperativity
observed for Mg
with FBPase.
Figure 3:
Plot of reciprocal of initial velocity in
arbitrary fluorescent units versus reciprocal of
[Mg]
for wild-type FBPase. The
concentrations of Fru-1,6-P
are 9.0 µM (
), 5.0 µM (
), 3.0 µM (
), 2.0 µM (+), and 1.2 µM (
), The lines are theoretical based on when n = 2, and the points are experimentally
determined.
The central finding of this report involves the complete loss
of cooperativity for Mg when FBPase Gly-122 is
mutated to alanine. It is known that mammalian FBPase is a divalent
metal-requiring enzyme (8) and that the Hill coefficient is 2.0
at neutral pH (13, 14) but only 1.0 at alkaline
pH(4, 13, 14) . Crystallographic data (10) suggest that two metal ions such as Mn
and Zn
are associated with the enzyme; however,
with Mg
, only one metal ion was found to bind FBPase.
Replacement of Gly-122 with alanine caused a 19-fold decrease in
enzyme activity and a significant decrease of the enzyme's
affinity for Mg. This is expected because the main
chain NH group of Gly-122 is thought to act as a donor to form a
bifurcated hydrogen bond with the O1 oxygen of the fructofuranoside
ring and an oxygen of the 1-phosphoryl group of the
substrate(10) . Therefore, this bifurcated hydrogen bond is
believed to anchor the 1-phosphoryl group in the required position for
both metal binding and catalysis(10) . In addition, the
bifurcated hydrogen bond may contribute to catalysis by making the
phosphoester bond weaker such that nucleophilic attack by
OH
on the 1-phosphorus atom would be facilitated.
A significant finding associated with this report is that the Hill
coefficient of the G122A mutant FBPase for Mg is
about 1.0. This conclusion is alluded to from kinetic data obtained in
the absence of inhibitors. These results suggest that altering Gly-122
of FBPase to alanine essentially causes the complete loss of
Mg
cooperativity. Furthermore, the affinity of this
mutant form of FBPase for metal ions decreased markedly. From these
results, we conclude that the Gly-122 residue of mammalian FBPase is
essential for both cooperativity and binding of Mg
to
the enzyme. An understanding of this important finding requires further
investigations (e.g. solution of the crystal structure of
G122A FBPase); however, modeling studies were undertaken to provide
some insight into the effect of substituting alanine for glycine at
position 122. Gly-122 is located in a 3-residue loop between strand B3
(residues 113-118) and helix H4 (residue 123-127). If one
compares the various crystallographic complexes of FBPase, it is
obvious that residues 121-127 are subject to conformational
changes during catalysis(27) : root mean square deviation for
backbone atoms of these residues ranges between 0.29 and 0.70 Å
(between native FBPase and FBPase complexed with Fru-1,6-P
,
with
-methyl-Fru-1,6-P
, or with Fru-6-P,
respectively). Most interestingly, Gly-122 is at a hinge point between
strand B3 and helix H4.
The active site region of FBPase, based on
the crystal structure of the enzyme(10) , is shown in Fig. 4. If Gly-122 (Fig. 4A) is replaced by
Ala-122 (Fig. 4B), the -methyl group is located in
the Fru-1,6-P
binding site, close to the C1 carbon of the
substrate or its analog. Ala-122 can reduce the flexibility of the
122-127 region or lock the enzyme in a conformation which
suppresses cooperativity for Mg
. In addition, binding
of Mg
causes repositioning of the 1-phosphoryl group
of Fru-1,6-P
or its analog,
-methyl
Fru-1,6-P
(27) . Based on these structural studies,
the following events can be proposed: when the substrate binds, the
1-phosphoryl group adopts an initial conformation. The binding of
Mg
in the metal site moves the 1-phosphoryl group
into a position that allows cleavage of the P-O bond, yielding the
formation of Fru-6-P. The
-methyl group of Ala-122 can lock the
1-phosphoryl group in a defined conformation when substrate binds and
this affects the hinge movement of helix H4, disrupting some
interactions that are required for signal transmission between
monomers.
Figure 4:
A, stereo model of the active site of
FBPase as defined in the crystallographic study of porcine FBPase
complexed with the substrate analogue -methyl-Fru-1,6-P
and Mg
(10) . For clarity the binding
residues of the 6-phosphate group have been omitted. The backbone NH of
Gly-122 is hydrogen bonded to oxygens O1 and O2 of the 1-phosphate
group (2.8 and 2.5 Å, respectively). The backbone CO of Gly-122
interacts with the backbone NH of Ser-124 and Asn-125 and also with the
side chain NH of Asn-125. The Mg
ion is coordinated
to the O2 oxygen of the 1-phosphate group and to residues Glu-97,
Asp-118, and Glu-280. B, stereo model of the catalytic site
showing the effect of the alanine substitution at position 122. If the
position of backbone atoms are kept as in the crystallographic complex
shown in A, the
-methyl group of Ala-122 is only 3.4
Å from the C1 carbon of
-methyl-Fru-1,6-P
and
only 2.4 Å from the side chain of Asn-125. Slightly different
positions of residues 122-125 are required in order to
accommodate the presence of a
-methyl group in residue 122. The
energy-minimized model (B) shows that interactions of the
backbone NH of Ala-122 with the oxygen O1 and O2 of the 1-phosphate
group of the substrate analog are maintained. The
-methyl group of
Ala-122 is 4 Å from the C1 carbon of the substrate analog and 3.8
Å from the side chain of Asn-125.
It is well documented that the mechanism of regulation of
FBPase involves AMP and Fru-2,6-P, which are potent
synergistic inhibitors of the
enzyme(28, 29, 30, 31) . Fromm and
co-workers (4, 7, 25) have shown that AMP and
metal ions are mutually exclusive in their binding to the enzyme. X-ray
diffraction studies suggested that the metal binding sites are far from
the allosteric sites for AMP(32) . The data shown in Table 1indicate that the K
for AMP of the
G122A mutant is much higher than that of the wild-type enzyme. It is
obvious that converting Gly-122 to alanine not only affects the
enzyme's affinity for divalent metal ions, it also affects the
enzyme's affinity for AMP. These results are consistent with
previous suggestions that the metal binding sites and the allosteric
sites for AMP can somehow communicate with each
other(4, 7, 28) . When AMP binds to the
enzyme, the coordination sphere of Mg
is affected as
follows; side chains of residues Glu-97, Asp-118, and Asp-121 belonging
to strand B3 move from their R state conformation 1.79 Å
(OE1), 1.71 Å (OD2), and 4.10 Å (OD1). Residues Lys-274 and
Glu-280 are also affected upon binding(27) . From our results,
Gly-122, located in the hinge region between strand B3 and helix H4,
seems to be directly involved in the signal transmission pathway for
communication between metal binding sites and allosteric sites.
The
binding of AMP to wild-type FBPase exhibits cooperativity(28) .
Site-directed mutagenesis at the AMP binding site led to a total loss
of cooperativity for AMP with FBPase(28) . The kinetic data of
AMP inhibition with G122A FBPase gave excellent fits to a cooperative
model. This finding suggests that the Gly-122 residue may not be
involved in the cooperativity of AMP, although it is absolutely
required for Mg cooperativity.
It is believed that
Fru-2,6-P has two functions in regulating FBPase. One is
that it is a potent competitive inhibitor of the substrate and competes
with Fru-1,6-P
at the enzyme's active
site(24, 25, 31, 33) . The second
function is to enhance the effect of AMP by making AMP
``stickier'' to the enzyme(7) . AMP is known to be a
potent competitive inhibitor of Mg
(28) and
to prevent divalent metal ion binding to the enzyme, i.e. the
two ligands are mutually exclusive in their binding to FBPase.
The
findings of this report demonstrate that Gly-122 is essential for the
well established cooperativity and divalent metal binding affinity of
porcine liver FBPase. The x-ray diffraction investigations of FBPase by
the Lipscomb group (34) has provided a basis for AMP
cooperativity. The enzyme functions as a dimer of dimers in which R and T states exist. In the case of AMP binding, the T state is induced, whereas the substrate adds to the R state of the enzyme. This model provides a rational explanation
for a Hill coefficient of 2 for AMP. On the other hand, an explanation
of divalent metal ion binding seems to be less well understood. Two
binding sites for either Mn or Zn
have been demonstrated; however, the x-ray diffraction studies
can pinpoint only a single Mg
ion per monomer. In
addition, kinetic studies at pH 9.6 (4) accord with the latter
finding, i.e. only 1 Mg
per monomer and no
cooperativity. It is well established that at neutral pH,
Mg
binding is cooperative and the Hill coefficient is
2(11, 13, 14, 15) . The data shown
in Fig. 3confirm this finding. The origin of Mg
binding cooperativity remains unclear even though the metal
binding sites of FBPase were markedly altered by site-directed
mutagenesis(16) . Although the k
of the
mutant FBPase was decreased by at least 3 orders of magnitude relative
to the wild-type enzyme, the cooperativity of metal ions did not show
significant alteration(16) . These results suggest, but do not
provide conclusive proof, that divalent metal ion cooperativity is
inter- rather than intrasubunit. The results of the present study may
lead to a better understanding of the basis of the cooperativity
phenomenon for metal ion binding to FBPase at the molecular level when
the three-dimensional structure of the Gly-122 mutant becomes
available.