Directed Mutagenesis Studies of the Metal Binding Site at the
Subunit Interface of Escherichia coli Inorganic
Pyrophosphatase*
Irina S.
Efimova
,
Anu
Salminen§,
Pekka
Pohjanjoki§,
Jukka
Lapinniemi§,
Natalia N.
Magretova
,
Barry S.
Cooperman¶,
Adrian
Goldman§
,
Reijo
Lahti§**, and
Alexander A.
Baykov
**
From the
A. N. Belozersky Institute of
Physico-Chemical Biology and School of Chemistry, Moscow State
University, Moscow 119899, Russia, the § Department of
Biochemistry, University of Turku, FIN-20014 Turku, Finland, the
¶ Department of Chemistry, University of Pennsylvania,
Pennsylvania 19104-6323, and the
Centre for Biotechnology,
University of Turku and Åbo Akademi University,
FIN-20251 Turku, Finland
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ABSTRACT |
Recent crystallographic studies on
Escherichia coli inorganic pyrophosphatase (E-PPase) have
identified three Mg2+ ions/enzyme hexamer in water-filled
cavities formed by Asn24, Ala25, and
Asp26 at the trimer-trimer interface (Kankare, J.,
Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and
Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we
show that D26S and D26N substitutions decrease the stoichiometry of
tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol
monomer and increase hexamer stability in acidic medium.
Mg2+ markedly decelerates the dissociation of enzyme
hexamer into trimers at pH 5.0 and accelerates hexamer formation from
trimers at pH 7.2 with wild type E-PPase and the N24D variant, in
contrast to the D26S and D26N variants, when little or no effect is
seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the
same magnitude for wild type E-PPase and the three variants. The
affinity of the intertrimer site for Mg2+ at pH 7.2 is
intermediate between those of two Mg2+ binding sites found
in the E-PPase active site. It is concluded that the metal ion binding
site found at the trimer-trimer interface of E-PPase is a high affinity
site whose occupancy by Mg2+ greatly stabilizes the enzyme
hexamer but has little effect on catalysis.
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INTRODUCTION |
Inorganic pyrophosphatase (EC 3.6.1.1;
PPase1) belongs to a group of
enzymes catalyzing phosphoryl transfer from phosphoric acid anhydrides
to water, a principal reaction of cellular energetics. Soluble PPases
hydrolyze PPi to Pi with release of energy as
heat and provide in this way a thermodynamic pull for many biosynthetic reactions (1). PPases are essential in bacteria (2), yeast (3), and
plants (4).
Escherichia coli PPase (E-PPase) is homohexameric, like many
other prokaryotic PPases (5-8). The active site, present in each
20-kDa monomeric unit, contains 14 polar amino acid residues that are
completely conserved in all known soluble PPases, despite only moderate
sequence similarity in the rest of the molecule (9). The overall
folding motifs are very similar in E-PPase (10, 11) and Thermus
thermophilus PPase (8) as well as in the core part of the larger
(32 kDa per subunit) PPase of Saccharomyces cerevisiae (12,
13).
The E-PPase hexamer is arranged as a dimer of trimers (11, 14-16). The
principal trimer-trimer interaction involves a three-center ionic,
hydrogen-bonding interaction among the residues
His140-Asp143-His136' (11, 14).
Another important trimer-trimer interaction occurs among
Asn24, Ala25, and Asp26 (Fig.
1). The cavity formed by
Asn24-Asp26 and
Asn24'-Asp26' contains several water molecules
one of which is replaced by Mg2+ when the crystals are
soaked in a decimolar concentration of a magnesium salt (15, 17). As a
result of the Mg2+ binding, the side chains of the
Asn24 residues reorient, and the trimers move toward each
other by about 0.4 Å.

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Fig. 1.
Part of the trimer-trimer interface in
E-PPase, containing an Mg2+ ion (black
sphere) surrounded by six water molecules (15).
Hydrogen bonds are shown as lines. The two monomers that
contribute to the interface are distinguished by the primed
and unprimed labels.
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Metal ions, including Mg2+, are rare but not uncommon at
subunit interfaces (18-21). Brinen et al. (22) were able to
engineer a metal binding site at the interface formed by trypsin and
its protein inhibitor ecotin. However, in all known instances the intersubunit metal ion is coordinated directly to protein residues except for E-PPase where it is connected through water molecules (15,
17). On the other hand, water-filled cavities often occur at protein
interfaces (23) and in protein interiors (24). Because even large
molecules can rapidly penetrate deep into proteins and bind to
seemingly inaccessible cavities (25), it is not unlikely that, in
vivo, protein cavities can contain metal ions, especially
Mg2+, the most abundant divalent cation in the cell.
Understanding the effects of these cavities on protein properties may
therefore be important in understanding how proteins function in
vivo as well as in designing interacting proteins and ligands that
can disrupt protein-protein interactions.
The present work addresses two major questions: (a) Does the
intertrimer cavity in E-PPase bind Mg2+ strongly enough to
be occupied appreciably at its physiological concentration?
(b) What is the role of this unusual binding site in hexamer
stability and catalytic activity?
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EXPERIMENTAL PROCEDURES |
Enzymes
Wild type and wild type-free variant PPases were expressed using
the overproducing E. coli strains HB101 (26) and
MC1061/YPPAI(
ppa) (27), respectively. The
enzymes were purified by chromatography on Fast Flow DEAE-Sepharose
(Amersham Pharmacia Biotech), heat treatment at 65 °C, and gel
filtration on a column (2.6 × 60 cm) of Superdex 200 (Amersham
Pharmacia Biotech) (27). The final preparations were homogeneous
according to SDS and native polyacrylamide gel electrophoresis and
sedimentation velocity measurements. The enzyme concentration was
estimated on the basis of a subunit molecular mass of 20 kDa (28) and
an A2801% of 11.8 (29).
Methods
Initial rates of PPi hydrolysis were estimated from
continuous recordings of phosphate production obtained with an
automatic phosphate analyzer (30). The assay medium of 25 ml total
volume contained, except as noted, 20 µM
Mg2PPi, 20 mM Mg2+,
0.05 M Tris/HCl (pH 7.2), and 40 µM EGTA. The
reaction was initiated by adding a suitable aliquot of enzyme solution
and carried out for 3-4 min at 25 °C. No appreciable
interconversion between the hexamer and less active trimer was observed
during the assay, as evidenced by nearly linear product formation curves.
Differential spectra of PPase induced by Mg2+ were recorded
with a computer-controlled LKB Ultrospec Plus spectrophotometer in a
1-cm cuvette containing 0.7 ml of enzyme solution. After the base line
stabilized, stock MgCl2 solution was added in 0.7-µl increments, and the spectra were recorded.
Equilibrium microdialysis (31) and analytical ultracentrifugation (32)
were performed as described.
Calculations and Data Analysis
Hexamer-Trimer Equilibration--
Equations 1 and 2, derived
from Scheme I, describe time courses of activity (A)
resulting from hexamer (E6) dissociation into trimers (E3) and the reverse reaction, as well
as the equilibrium activity (at t =
,
d
H/dt = 0) as functions of
enzyme concentration. AH and
AT are specific activities of the hexamer and
trimer, respectively,
H is the fraction of enzyme in
hexameric form at time t,
[E]t is total enzyme concentration,
expressed in monomers, ka and
kd are the apparent rate constants for hexamer
formation and breakdown, respectively, measured at fixed H+
and Mg2+ concentrations. Equations 1 and 2 were fit to data
simultaneously with SCIENTIST (MicroMath).
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(Eq. 1)
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(Eq. 2)
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The pH dependence of PPase activity at trimer-hexamer
equilibrium is described by Equations 1 and 2 with
d
H/dt = 0 and the apparent
Kd = kd/ka given by Equation 3, where Kd ' is the pH-independent dissociation
constant for the hexamer, Ka is the microscopic
dissociation constant for H+ binding to the trimer, and
m is the number of protons bound per hexamer.
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(Eq. 3)
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The effect of Mg2+ on ka at
fixed pH is described by Equation 4, derived for the simplified model
shown in Fig. 2. The model assumes that
(a) a hexamer is formed from two trimers containing in total
from zero to three metal ions bound at the trimer-trimer interface and
(b) the association rate constant is equal to
ka,0 for trimers without metal ions and
increases by the same factor p with each added metal ion.
Here
= 1/(1 + Kin'/[M]) is the probability
of finding a metal ion (M) in one of the three metal ion binding
sites/trimer, and Kin' is the respective metal
binding constant for the trimer. Statistical factors of 12 (6 + 6) and 4 (2 + 2) were used when more than one metal ion was present in two
interacting trimers, taking into account that the combination of two
metal-bound monomers within a single subunit-subunit interface is not
allowed (Fig. 2).
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(Eq. 4)
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Fig. 2.
Various modes of hexamer formation from
trimers in the presence of Mg2+. Monomers
with Mg2+ bound at the interface half-site are
shaded. The respective rate association constants are shown
below. The numerical factors in rate constant expressions
refer to statistical weights; for instance, the factor of 6 indicates
that there are six similar combinations of two trimers with same
distribution of metal ions among them.
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Equilibrium Dialysis--
Values of the dissociation constants
for the successive binding of two Mg2+ ions to the E-PPase
active site (KM1 and KM2)
and for Mg2+ binding to the subunit interface site
(Kin) (Scheme II) were estimated by fitting
Equation 5 to measured values of n, the number of
Mg2+ ions bound per monomer, as a function of free
Mg2+ concentration. The first term in Equation 5 describes
binding to the active site, and the second binding to the intertrimer site. The second term was ignored for variants that exhibited no
binding to the intertrimer site (Kin =
).
Scheme II assumes that the binding reactions in the active site and in
the intertrimer site are mutually independent.
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(Eq. 5)
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Spectral Titration--
The dependence of
A245, the change in protein absorbance at 245 nm, on Mg2+ concentration was fitted with Equation 6, where

is the change in the extinction coefficient at saturating [M].
Equation 6 implies KM2
KM1.
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(Eq. 6)
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Mg2PPi Hydrolysis--
Values of the
apparent catalytic constant kh and the apparent
Michaelis constant Km,h were obtained from rate
versus [Mg2PPi] dependences
measured at fixed Mg2+ concentrations. Values of
kh/Km,h as a function of
[Mg2+] were fitted to Equation 7, allowing an estimation
of k1(1),
k1(2),
k1(3), and
k1(4), the rate constants for
substrate binding to the ME, M2E, ME(M), and
M2E(M) species (M in parentheses denotes the
Mg2+ ion that is bound at the subunit interface),
respectively (Scheme II). The assumption in deriving Equation 7 is that
kh greatly exceeds the rate constant for
Mg2PPi release from the enzyme, as has been
shown elsewhere for wild type E-PPase (33). The values of
KM1, KM2, and
Kin used in this fitting were those estimated by
equilibrium dialysis (see above).
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(Eq. 7)
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The concentrations of free Mg2+ and
Mg2PPi at pH 7.2 were calculated using the
dissociation constants of 0.112 and 2.84 mM for the
MgPPi and Mg2PPi complexes,
respectively (34).
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RESULTS |
pH Effects on Quaternary Structure and Enzyme Activity--
The
effects of substitutions at both Asn24 and
Asp26 on quaternary structure and enzyme activity were
studied as a function of pH. Direct evidence for shifts in the
hexamer-trimer equilibrium was obtained from sedimentation data (Table
I). At pH 7.2, s20,w for WT-PPase, and the three
variants studied here (N24D, D26N, D26S) fell in the range 6.3-6.7 S,
characteristic of a hexamer (28, 32). Lowering the pH to 5.0 had
differential effects. s20,w decreased
to 4.3 for both WT-PPase and N24D-PPase, decreased only to 5.2 for
D26S-PPase, and was unchanged for D26N-PPase. Further lowering the pH
to 3.8 led to s20,w values of 3.3-4.0 for all four PPases. Because the molecular mass of WT-PPase at
pH 5.0 estimated by sedimentation equilibrium measurements (20 °C,
20 µM initial enzyme concentration) was 63 ± 1 kDa,
these results indicate that WT-PPase and N24D-PPase are fully in the trimeric form by pH 5.0 but that lower pH is required for full conversion to trimer of the D26S and D26N variants. That such dissociation is reversible is shown by the increase in
s20,w back to 6.7 ± 0.2 S when
the pH of a WT-PPase sample is raised from 5.0 to 7.2. At pH 3.8, the
variant PPases may undergo further dissociation to dimers or monomers
as suggested by lower s20,w values
compared with WT-PPase.
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Table I
Sedimentation coefficients
Conditions were 2-4 °C, 10 µM enzyme, 48,000 rpm. The
buffer (0.1 M citric acid, NaOH, pH 3.8; 0.1 M
MES/NaOH, pH 5.0; 0.1 M Tris/HCl, pH 7.2) contained 1 mM MgCl2 and 40 µM EGTA.
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Earlier we had shown for other variants with weakened trimer-trimer
interaction (32) that trimers had lower activity than hexamers at low
(20 µM) substrate concentration as a result of a drastic
increase of Km,h
(103-104-fold), with little change in
kh. Trimers of WT-PPase and the three variants
investigated in this paper also had lower activity, and studies of the
effect of pH on enzyme activity, paralleling those on
s20,w, demonstrated conclusively that
rates and equilibria of hexamer-trimer interconversion could be
monitored by activity measurements. The activities presented in Fig.
3 represent equilibrium values, no
changes being observed on longer incubation at the final pH values.
Matching the s20,w values, they demonstrate that as pH is lowered from 7.2 to 5.0, WT- and N24D-PPase are fully converted to less active forms, D26S-PPase is partially converted, whereas D26N-PPase retains almost full activity, and, in
addition, lowering the pH further to 3.8 converts the latter two
variants virtually completely to their lower activity forms. Furthermore, WT-PPase activity increases with enzyme concentration at
pH 5.0 (Fig. 4, inset), as
expected for an equilibrium between an active hexamer and less active
trimer, and activity is a fully reversible property of pH (Fig. 4). The
N24D, D26S, and D26N variants, inactivated at pH 5.0, 4.8, and 3.9, respectively, could also be reactivated at pH 7.2, affording > 80% of original activity. Finally, in all cases, inactivation and
reactivation followed strict first- and second-order kinetics,
respectively.

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Fig. 3.
Specific activity of wild type and variant
PPases preincubated at different pH values. The enzyme (10 µM) was incubated at the pH indicated for 2 h (WT-,
D26S-, and N24D-PPases) or 2-72 h (D26N-PPase) at 0 °C in a medium
containing 0.1 M buffer, 1 mM Mg2+,
and 40 µM EGTA, and its residual activity was assayed at
pH 7.2, 25 °C. The buffers were sodium citrate (pH 3.9), MES/NaOH
(pH 4.8-6.8), or Tris/HCl (pH 7.2-8.5). The lines are
drawn to Equations 1-3 with m = 6 ( , WT-PPase),
m = 12 ( , N24D-PPase), or m = 2 ( , D26N-PPase; , D26S-PPase).
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Fig. 4.
Reversibility of WT-PPase inactivation at pH
5.0. Stock enzyme solution (800 µM) preequilibrated
at pH 8.0, 25 °C, was diluted to 20 µM with 0.1 M MES/NaOH buffer (pH 5.0, measured at 0 °C) and
incubated at 0 °C for 2 h, after which the pH was raised to 7.2 with 1 M Tris, and the incubation was continued. Aliquots
were withdrawn as a function of time, and PPase activity was assayed at
pH 7.2, 25 °C. The lines are drawn to Equations 1 and 2.
Inset, the specific activity of WT-PPase preequilibrated at
pH 5.0 as a function of enzyme concentration. The line is
drawn to Equations 1 and 2 with
kd/ka = 170 µM, AH = 135 s 1,
AT = 9.5 s 1, t = .
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The data in Fig. 3 could be fit quantitatively to a model (Equations
1-3) in which deprotonation of a group within trimers (which has a
much lower pKa in a hexamer) is required for
hexamer formation. Satisfactory fits were obtained when the number of
bound protons per hexamer (m) was equal to 6, 2, 2, or 12 for WT-, D26N-, D26S-, and N24D-PPase, respectively. From earlier work
(35), Kd' for WT-PPase is < 1 nM, allowing a lower limit estimate of
pKa of 6.7.
Mg2+ Effects on Rates and Equilibrium of Hexamer
Trimer Transition--
Mg2+ has been shown to stabilize
the hexameric structure of variant E-PPases with weakened trimer-trimer
interactions (32, 35). As estimated from time courses of activity loss
at pH 5.0 and restoration at pH 7.2, added Mg2+ markedly
decreased kd (Fig.
5) and increased ka (Fig.
6) for WT- and N24D-PPases. The sigmoidal
dependence of ka on [Mg2+] was
described satisfactorily by Equation 4, yielding fitted values of
Kin', ka,0, and
p (Table II) which were
similar for both PPases. The Kin ' values may
reflect the dissociation constant from a half-of-interface site (for
WT-PPase, residues 24-26 from one subunit).

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Fig. 5.
The rate constant for wild type and variant
PPase inactivation at pH 5.0 as a function of Mg2+
concentration. The enzyme stored at pH 7.2 at a
concentration of 125 µM was diluted to 1 µM
with 0.1 M MES/NaOH buffer (pH 5.0, 0 ° C) containing
varying amounts of MgCl2, and the time course of PPase
activity was measured. Values of kd were
estimated with Equations 1 and 2. , WT-PPase; ,
N24D-PPase; , D26N-PPase; , D26S-PPase.
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Fig. 6.
The rate constant for wild type and variant
PPase reactivation at pH 7.2 as a function of Mg2+
concentration. The enzyme inactivated at pH 5.0 (see Fig. 3)
was diluted to 0.4 µM with 0.1 M Tris/HCl
buffer (pH 7.2, 25 ° C) containing varying amounts of
Mg2+, and the time course of PPase activity was measured.
Values of ka were estimated with Equations 1 and
2. The lines are drawn to Equation 4. , WT-PPase; ,
N24D-PPase; , D26S-PPase.
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Table II
Parameters describing Mg2+ effects on trimer reactivation at pH
7.2, 25 °CM, (0.1 Tris/HCl, 40 µM EGTA).
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Hence, Mg2+ ions stabilize the hexamer by changing both
kd and ka. In full
agreement with these data, the activity versus pH profile
for WT-PPase (Fig. 3) shifted to lower pH values at higher Mg2+ concentrations, as inferred from the observation that
WT-PPase retained 80% activity at pH 5 in the presence of 140 mM MgCl2.
In contrast, for D26S- and D26N-PPase, the already low value of
kd in the absence of Mg2+ was
somewhat increased by the addition of Mg2+ (Fig. 5), most
likely because of nonspecific binding. Similarly, ka for D26S-PPase in the absence of
Mg2+ is 5-10 times larger than the corresponding values
for WT- and N24D-PPases and rises only slightly as
[Mg2+] is raised (Fig. 6).
Mg2+ Binding to Hexameric Variant
PPases--
Mg2+ binding to WT- and the variant E-PPases
was measured by equilibrium dialysis, providing the macroscopic binding
constants for all three types of the metal binding sites present in
E-PPase, M1 and M2 at the active site and Min at the
trimer-trimer interface, and by differential spectroscopy (16), which,
as shown recently,2 provides
the binding constant for M2.
The dialysis data indicated a decrease in the stoichiometry of tightly
binding sites by approximately 0.5 mol/mol monomer in the D26S and D26N
variants, but not in the N24D variant, when compared with WT-PPase
(Fig. 7). These data are thus consistent with a loss of Min upon Asp26 substitution and
also suggest that Min displays high affinity for
Mg2+. These conclusions were supported by quantitative
analysis of the binding data. The binding affinity of M1 was only
slightly affected by the mutations, whereas the affinity of
Min was increased 3-fold after N24D substitution, which
introduced a negatively charged ligand into the intertrimer cavity.
Accordingly, in the N24D variant sites M1 and Min have
similar affinity for Mg2+, whereas in WT-PPase the order in
which the three sites become occupied at increasing Mg2+
concentration is M1, Min, M2 (Table
III).

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Fig. 7.
Mg2+ binding to
E-PPase and its variants as measured by equilibrium dialysis. Data
for WT-PPase are from Velichko et al. (36). Lines
are drawn to Equation 5, using parameter values found in Table III
(Kin was set to infinity for the D26N and D26S
variants); n measures the number of Mg2+ ions
bound per monomer. Experimental conditions: 83 mM TES/KOH
(pH 7.2), 17 mM KCl, 50 µM EGTA, 0.35-0.70
mM [PPase], 0.02-3 mM [Mg2+].
, WT-PPase; , N24D-PPase; , D26N-PPase; , D26S-PPase.
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As measured by spectral titrations conducted at pH 8.5 (the effect of
Mg2+ binding on absorbance was quite small at pH 7.2 used
in the dialysis experiments; see Ref. 16) the affinity at site M2
appeared unaffected by the substitutions (Table III). Moreover, the
values obtained for the three variants are essentially identical to
those measured by dialysis at pH 7.2. The one apparent inconsistency in
the data concerns the considerably higher value obtained by dialysis
for WT-PPase at pH 7.2. However, the error in this value is quite high
because it greatly exceeds the enzyme concentration used in these measurements.
Kinetics of PPi Hydrolysis--
As measured by
kh,
kh/Km,h, and
k1, substitution at the subunit interface has
only minor effects on PPase catalytic function determined at pH 7.2 as
a function of [Mg2+]. Values of kh
and kh/Km,h (Fig.
8) were generally quite similar for WT,
N24D-, D26N-, and D26S-PPases, with the exception that
kh/Km,h was somewhat
lower for N24D-PPase. Values of k1
were also determined as a function of [Mg2+] and
interpreted according to Scheme II (Table IV)
. For WT-PPase and N24D-PPase, binding to
the interface site has at most a small effect on
k1 (i.e.
k1(1) is approximately equal to
k1(3)), but binding to M2 lowers
k1 (k1(3) > k1(4)). Consistent with these
results, the two variants lacking a high affinity Mg2+
interface site, D26N- and D26S-PPases, have
k1(1) values similar to that for
WT-PPase, and their k1(2) values
(reflecting M2 site occupancy) are both lower than their k1(1) values and similar to
k1(4) for WT-PPase It should be
noted that k1(2) could not be
estimated for WT- and N24D-PPase because the M2E species
was stoichiometrically insignificant for these variants.

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Fig. 8.
Dependences of kh
and
kh/Km,h
for Mg2PPi
hydrolysis on Mg2+ concentration. The
line in the upper graph shows the best fit to the
equation kh = kh,
lim[M]/(Ka + [M]) with
kh,lim = 157 s 1 and
Ka = 0.26 mM for WT-PPase. The
lines in the lower graph are drawn to Equation 7,
using parameter values found in Table IV. For clarity, the lines for
D26S-PPase and D26N-PPase are shown as broken and
dotted lines, respectively. Experimental conditions: 87 mM TES/KOH (pH 7.2), 17 mM KCl, 50 µM EGTA. , WT-PPase; , N24D-PPase; , D26N-PPase;
, D26S-PPase.
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Table IV
Rate constants for substrate binding
Results were estimated with Equation 7, using the metal binding
constants obtained by equilibrium dialysis (Table III); for WT-PPase,
KM2 was also an adjustable parameter, whose best fit
value was found to be 3.5 mM. Conditions were: 25 °C, pH
7.2 (83 mM TES/KOH, 17 mM KCl, 50 µM EGTA).
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 |
DISCUSSION |
Equilibrium dialysis measurements have revealed three types of
metal binding sites in E-PPase (33), which we now designate as sites
M1, Min, and M2, following the order in which they become occupied in WT-PPase at an increasing Mg2+ concentration at
pH 7.2 (Table III). We assumed previously that the three binding sites
are present in each subunit of homohexameric E-PPase (33). However,
crystallographic identification of an Mg2+ ion between
pairs of subunits (15, 17) together with the effects of
Asn24 and Asp26 mutations on metal binding and
hexamer stability reported above suggest a binding stoichiometry of 0.5 mol/mol monomer for one of the sites detected by equilibrium dialysis.
Two other lines of evidence support the idea that the site with
intermediate affinity is located at the subunit interface. First, x-ray
crystallographic data show that E-PPase crystals soaked in 140 mM MgCl2 contain Mg2+ only at sites
M1 and Min (15), whereas crystals grown at 250 mM MgCl2 contain Mg2+ at all three
sites (17). Also, the site that binds Mn2+ most tightly is
located in the active site and exhibits a 1 mol/mol binding
stoichiometry (11). Second, site Min is apparently absent in trimeric E-PPase prepared by substitution of both His136
and His140 in the trimer-trimer interface (36).
Our results clearly show that [H+] and
[Mg2+] determine the quaternary structure of E-PPase and
agree with earlier studies showing that (a) low pH induces
PPase hexamer dissociation (6, 37) and (b) in variants with
weakened trimer-trimer interactions (H136Q-PPase, 32; E20D-PPase, 35)
added Mg2+ stabilizes hexamer formation at pH values
7.2. A simple model consistent with our results is that protonation
of one or more sites per monomer induces dissociation (Fig. 3).
According to this model, added Mg2+ favors the hexamer
because it competes with H+ for a common site (or sites),
in addition to stabilizing hexamer structure as such directly.
Consistent with this model are (a) the increased stability
of the D26S and D26N hexamers because substitution of Asp26
eliminates a potential protonation site, (b) the shift in
activity versus pH profiles to lower pH values with
increasing Mg2+ concentration, (c) the stability
of hexamers at higher pH even in the absence of added Mg2+,
and (d) the rise in ka for WT- and
N24D-PPase with increasing [Mg2+] at pH 7.2, when the
basic group(s) governing hexamer stability is(are) deprotonated (Fig.
6).
This model certainly suggests that Asp26 is at least one,
and perhaps the major, basic group controlling hexamer stability in WT-PPase. This would require Asp26 to have a markedly
elevated pKa of > 6.7, which would not be unexpected, given the extensive hydrogen bonding network at the subunit
interface site (see Fig. 1). The value of m equal to 6 for
WT-PPase suggests that all three Asp26 residues/trimer
become protonated on subunit dissociation. Following this logic, it is
tempting to speculate that the increase in m value to 12 for
the N24D variant reflects protonation of the Asp24 and
Asp26 residues in this variant. That m does not
become 0 in the D26S and D26N variants would imply the presence of
(an)other, lower pKa group(s) whose protonation
also destabilizes the hexamer. One possibility is His140,
as suggested previously (14, 32). Alternatively, other carboxyl side
chains, as yet unidentified, could be involved.
It remains unclear why protonation of Asp26 would
destabilize the hexamer, whereas mutation of Asp26 into Asn
or Ser does not. The simplest possibility is that the hydrogen bonding
network at the subunit interface site (Fig. 1) requires a hydrogen-bond
acceptor at position 26, a role that can be filled by a carboxylate
anion, by an amide, and by an alcohol, but not by a carboxylic acid.
High resolution structures of the variants described in this paper
could provide a clear test of the validity of our proposals.
In summary, we have shown that the metal binding site formed by
protein-bound water molecules at the trimer-trimer interface of E-PPase
is a high affinity site whose occupancy by Mg2+ stabilizes
the enzyme hexamer by neutralizing the negative charges and preventing
protonation of a site or sites within the binding cavity, without
having any substantial effect on catalysis.
 |
ACKNOWLEDGEMENTS |
We thank P. V. Kalmykov for help in
ultracentrifugation and Drs D. Bergen and V. A. Sklyankina for helpful suggestions.
 |
FOOTNOTES |
*
This work was supported by Russian Foundation for Basic
Research Grants 97-04-48487 and 96-15-97969, Russian State Project Bioengineering/Enzyme Engineering Grant 1-42, Academy of Finland Grants
1444, 35736, and 4310, and National Institutes of Health Grants
TW00407.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.
The first two authors contributed equally to this work.
**
To whom correspondence should be addressed. Tel.: 358-2-333-6845;
Fax: 358-2-333-6860; E-mail: reila{at}utu.fi or Tel.: 095-939-5541; Fax:
095-939-3181; E-mail: baykov{at}genebee.msu.su.
The abbreviations used are:
PPase, inorganic
pyrophosphatase; PPi, pyrophosphate; E-PPase, E. coli pyrophosphatase; WT, wild type; MES, 4-morpholineethanesulfonic acid; TES, 2-((2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino)ethanesulfonic
acid; TAPS, 3-((2-hydroxy-1,1-bis (hydroxymethyl)ethyl)amino)-1-propanesulfonic acid.
2
A. A. Baykov, T. Hyytiä, M. V. Turkina,
I. S. Efimova, V. N. Kasho, A. Goldman, B. S. Cooperman, and R. Lahti, manuscript in preparation.
 |
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