Probing Essential Water in Yeast Pyrophosphatase by Directed
Mutagenesis and Fluoride Inhibition Measurements*
Pekka
Pohjanjoki
,
Igor P.
Fabrichniy§,
Vladimir N.
Kasho¶,
Barry S.
Cooperman
,
Adrian
Goldman
**,
Alexander A.
Baykov§
, and
Reijo
Lahti
§§
From the
Department of Biochemistry, University of
Turku, FIN-20014 Turku, Finland, the § A. N. Belozersky
Institute of Physico-Chemical Biology and School of Chemistry, Moscow
State University, Moscow 119899, Russia, the ¶ Center for Ulcer
Research and Education, Department of Medicine, University of
California, Los Angeles, California 90073, the
Department of
Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania
19104, and the ** Institute of Biotechnology, University of Helsinki,
P.O. Box 56, FIN-00014 Helsinki, Finland
Received for publication, August 14, 2000, and in revised form, October 6, 2000
 |
ABSTRACT |
The pattern of yeast pyrophosphatase (Y-PPase)
inhibition by fluoride suggests that it replaces active site
Mg2+-bound nucleophilic water, for which two
different locations were proposed previously. To localize the bound
fluoride, we investigate here the effects of mutating Tyr93
and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PPi hydrolysis and synthesis,
formation of enzyme-bound PPi at equilibrium,
phosphate-water oxygen exchange, and Mg2+ binding). D117E
substitution had the largest effect on fluoride binding and made the
P-O bond cleavage step rate-limiting in the catalytic cycle, consistent
with the mechanism in which the nucleophile is coordinated by two metal
ions and Asp117. The effects of the mutations on
PPi hydrolysis (as characterized by the catalytic constant
and the net rate constant for P-O bond cleavage) were in general larger
than on PPi synthesis (as characterized by the net rate
constant for PPi release from active site). The effects of
fluoride on the Y-PPase variants confirmed that PPase catalysis
involves two enzyme·PPi intermediates, which bind
fluoride with greatly different rates (Baykov, A. A., Fabrichniy,
I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000)
Biochemistry 39, 11939-11947). A mechanism for the
structural changes underlying the interconversion of the
enzyme·PPi intermediates is proposed.
 |
INTRODUCTION |
Inorganic pyrophosphatase (EC 3.6.1.1;
PPase)1 catalyzes reversible
phosphoryl transfer from pyrophosphate (PPi) to water, a
metabolically important reaction chemically similar to that catalyzed
by numerous ATPases and GTPases. Yeast PPase is a homodimer containing
286 amino acid residues/monomer (1) and requiring three or four
divalent metal ions for catalysis, with Mg2+ conferring the
highest activity (2-5). Two divalent metal ions (M1 and M2) per
active site have been identified in the "resting" enzyme by x-ray
crystallography and four metal ions (M1-M4) and two phosphates (P1 and
P2) in the product complex of Y-PPase (6, 7).
PPi hydrolysis by PPase occurs via direct attack
of water without formation of a phosphorylated enzyme intermediate (8). Modeling the transition state of the chemical step from the structure of the enzyme-product complex
Y-PPase·Mn2(MnPi)2 has led to two models that differ in the identity of the water nucleophile placed between metal ions M1 and M2 in the model of Heikinheimo et
al. (6) or in the vicinity of Tyr93 in the model of
Harutyunyan et al. (7, 9). Although the former model
requires an additional "relaxation" step, in which a new water
molecule displaces Pi oxygen from the position
between M1 and M2, it has an advantage of providing an efficient
mechanism for nucleophile activation through combined action of the two metal ions and an adjacent Asp117 residue (see Fig. 1).
In aqueous solution and even in crystalline state, proteins are
surrounded with water shells, making identification of function-related water molecules a difficult task. Use of fluoride, a potent and most
specific inhibitor of cytoplasmic pyrophosphatase, provides a
convenient approach to detect such water molecules because molecules of
HF and H2O are isoelectronic and of similar size, as are
the anions derived therefrom. Fluoride inhibition of yeast PPase during PPi hydrolysis and synthesis involves a rapid and slow
phases (10), which refer to F
binding to
enzyme-Pi and enzyme-PPi intermediates,
respectively (Scheme I). The rapid
binding decelerates PPi hydrolysis 10-fold at pH 7.2, whereas the slow binding arrests it completely. These characteristics
of the inhibition are consistent with fluoride replacing an essential
metal-bound water molecule/OH
ion, acting as nucleophile
in the PPi hydrolysis step (11).
In the present work, we employed fluoride inhibition in combination
with site-directed mutagenesis to identify the fluoride binding site
and, hence, the essential water molecule in the active site of PPase.
Because fluoride inhibition of Y-PPase is closely associated with
Mg2+ binding (10, 12), the effects of mutating five amino
acid residues forming the M1 and M2 sites (Glu48,
Asp115, Asp120, Asp117, and
Asp152) on fluoride inhibition were studied. A Y93F variant
was also included in this list, because in the PPase mechanism
suggested by Harutyunyan et al. (9) a water molecule
associated with Tyr93 is assumed to be the nucleophile and,
hence, might be replaced by F
. X-ray crystallographic
analysis of three relevant variants, D117E Y-PPase (13) and D65N and
D70N Escherichia coli PPase (Asp65 and
Asp70 in E. coli PPase correspond to
Asp115 and Asp120 in Y-PPase, respectively)
(14), indicated no marked structural changes induced by the mutations.
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EXPERIMENTAL PROCEDURES |
The expression and purification of wild type Y-PPase and its
active site variants from overproducing E. coli
XL2blueb strain transformed with suitable plasmids were
carried out as described by Heikinheimo et al. (15).
PPi hydrolysis was measured continuously with an automatic
Pi analyzer (16). The assay medium contained, except as
noted, 0.32 mM (at pH 7.2) or 0.28 mM (at pH
8.5) total PPi (corresponding to 0.2 mM
Mg2PPi complex), 5.5 mM
MgCl2 (corresponding to 5 mM free
Mg2+), 0-20 mM F
(added as NaF)
and buffer. The following pH buffers were used (0.1 M ionic
strength, 50 mM K+): 83 mM TES/KOH,
17 mM KCl, 50 µM EGTA (pH 7.2), or 90 mM TAPS/KOH, 5 µM EGTA (pH 8.5). Enzyme-bound
PPi formation was assayed luminometrically with
ATP-sulfurylase and luciferase (17, 18) using 60-260 µM
Y-PPase concentration. PPi synthesis in solution was
measured continuously by the same coupled enzyme assay (11).
Pi-H2O oxygen exchange was measured by gas
chromatography/mass spectrometry (19). The assay medium used to measure
fluoride effects on PPase in the presence of Pi contained
13.4 mM MgCl2 (5 mM free
Mg2+), 20 mM total Pi, 55 mM TES/KOH buffer, 11 mM KCl, and 33 µM EGTA. Mg2+ binding was assayed by
equilibrium microdialysis in combination with atomic absorption
spectroscopy to measure Mg content in the dialysis chambers (20). All
experiments were performed at 25 °C.
Effects of fluoride on PPi hydrolysis were analyzed in
terms of Scheme II, a simplified version
of Scheme I. KF1 is the dissociation
constant governing rapid fluoride binding in the presence of
PPi, ki, app and
kr are the second-order and first-order
rate constants for slow fluoride binding and release, respectively.
Equations 1 and 2 describe product (P) formation curves at a
fixed fluoride concentration, where
is the fraction of enzyme that
has not yet undergone slow conversion into
Einactive, and v0, app
is the initial velocity of product formation.
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(Eq. 1)
|
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(Eq. 2)
|
Fitting [P] as a function of both time and [F] was
accomplished by making the following substitutions
(v0 and v0' are the initial velocities of product formation observed at [F] equal to zero
and infinity, respectively).
|
(Eq. 3)
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(Eq. 4)
|
Equations 1-4 were simultaneously fit, with the program
SCIENTIST (MicroMath), to sets of product formation curves, each
represented by 90-200 pairs of [P] and t values. The
calculated and measured curves agreed within 3%.
Values of the dissociation constants KM1
and KM2 for Mg2+ binding to
two sites on Y-PPase were estimated by fitting equilibrium dialysis
data to Equation 5, where n measures the number of
Mg2+ ions bound per monomer (21).
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(Eq. 5)
|
 |
RESULTS |
Mg2+ Binding--
The effects of conservative
mutations of Tyr93 and five amino acid ligands to
Mg2+ (Fig. 1) on the binding
of two activating metal ions in Y-PPase active site were studied by
equilibrium dialysis at pH 7.2 (Fig. 2).
Interestingly, every substitution suppressed Mg2+ binding
to the low affinity site, the lowest (1.8-fold) effect seen with Y93F,
and the highest (147-fold) with E48D substitutions (Table
I). Binding of Mg2+ to the
high affinity site was markedly suppressed in both Asp120
variants (34- and 16-fold effects with D120E and D120N, respectively) and less markedly (4.0-fold effect) in the D152E variant. These data
are consistent with M1 being the high affinity site and M2 being the
low affinity site, as measured by equilibrium dialysis.

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Fig. 1.
Part of Y-PPase active site structure in
Y-PPase·Mn2(MnPi)2 complex as
determined by x-ray crystallography (6). Larger black
circles, two of which are labeled M1 and M2,
are metal ions, smaller black circles are water oxygens, and
two black molecules are phosphates. Hydrogen bonds are shown
by dashed lines.
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Fig. 2.
Scatchard plots for Mg2+ binding
to wild type and variant Y-PPase. Lines were obtained
with Equation 5 using parameter values found in Table I. Experimental
conditions were pH 7.2, 0.5-2000 µM Mg2+,
and 50-500 µM PPase.
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Importantly, all the variants tested still exhibited appreciable
affinity for Mg2+, such that both sites were almost
saturated at 5 mM Mg2+ concentrations used in
the fluoride inhibition studies described below. The E48D variant,
which exhibited the highest KM2 value
(5.0 mM), is expected to be 50% saturated at the M2 site in the absence of substrate, but keeping in mind that substrate strengthens metal ion binding to the corresponding E-PPase E20D variant
(22) and WT-E-PPase (23), at least, 90% saturation is expected in the
presence of 200 µM Mg2PPi used in
fluoride inhibition studies. This rules out a possibility that the
effects of the mutations on fluoride inhibition reported below result from incomplete metal binding. Fluoride added at a 10 mM
concentration suppressed binding of M1 and stimulated binding of M2
approximately 2-fold in WT-Y-PPase (Table I).
Catalytic Properties--
Values of the catalytic constant for
PPi hydrolysis (kh), rate of
PPi synthesis (vs), rate of
Pi-H2O oxygen exchange
(vex), the distribution of five Pi
"isotopomers" during the exchange (as characterized by the
partition coefficient Pc = k4/(k5 + k4) (19), and the amount of enzyme-bound
PPi at equilibrium with medium Pi
(fepp) were measured for six variants at pH 7.2 and 8.5 (Table II). Such measurements
were not performed for the D120N variant, which displayed very low
hydrolytic activity (<0.005 s
1). These data allowed
evaluation of two important parameters: k3' = vex(1
0.75Pc)/fepp(1
Pc)[E]t and
kpp, off = vs/fepp[E]t,
the net rate constant for PPi release, i.e. back
conversion of EM4PP to EM2 (21). Equations 6
(11) and 7 (21) relate k3' and
kpp, off to the rate constants shown in Scheme
I (KAB = kA/kB). In fact,
k3' is the lower limit for
k3 (corresponding to KAB
=
), and kpp, off is the lower limit for both
k2 and kB. For WT-PPase,
KAB is equal to 25-65, and the values of
k3' and k3 differ by less
than 4% at pH
7.2 (11), but the values of
KAB for the variant PPases are unknown. The
advantage of using k3' and
kpp, off instead of
vex/[E]t and
vs/[E]t is that the
former parameters are independent of the degree of enzyme saturation
with Pi in corresponding measurements.
|
(Eq. 6)
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(Eq. 7)
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Fluoride Inhibition of PPi Hydrolysis--
According
to the inhibition pattern, all the variants could be classified into
three groups. Typical examples are shown in Fig.
3. Group I variants (E48D, Y93F, D115E,
and D117E) resembled WT-Y-PPase (11) by exhibiting a virtually instant
decrease in the initial slope of the Pi production curve,
followed by a slower inactivation step. The group II variant (D120E at
pH 7.2) exhibited only slow inactivation, without the instant decrease
in the initial slope, and the group III variant (D152E) exhibited only
instant inactivation. At pH 8.5, D120E-PPase also behaved like a group III member, whereas other variants did not change their behavior. It
should be noted that the Pi productions curves look very
similar for group I and group II variants (Fig. 3), and they could be only distinguished based on the fitting results shown below; the best-fit value of KF1 tended to
approach infinity for D120E-PPase, clearly indicating no change in
v0,app in the presence of fluoride. Values for
the rate constants ki and
kr describing the slow phase of the inhibition
and for the equilibrium constant KF1
describing the fast step of the inhibition (Scheme II) were
obtained by simultaneous fitting of Equations 1-4 to series of
product formation curves measured at different fluoride concentrations. The most drastic effects were observed on ki
with the D117E and D152E variants (the latter exhibited no slow
inactivation step at any pH value), on kr
with the D115E and D120E variants, and on
KF1 with the D117E variant (Table
III).

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Fig. 3.
The effects of NaF on the progress curves for
PPi hydrolysis catalyzed by four variant Y-PPases at pH
7.2. F concentrations (in mmol/liter) are indicated
at the curves. Curves were normalized to 0.008 µM (E48D),
0.27 µM (D117E), 2.9 µM (D120E), or 3.5 µM (D152E) enzyme concentration.
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Fluoride Effect on Enzyme-bound PPi Formation--
The
Y-PPase active site is preformed to bind PPi. Although the
concentration of PPi present at equilibrium with 20 mM Pi and 5 mM Mg2+ at
pH 7.2 is <1 µM, i.e. the ratio
[Pi]/[PPi] > 20,000 (5, 24, 25), the
fraction of enzyme containing bound PPi
(fepp = ([EM4PP*] + [EM4PP])/[E]t) in these conditions is as high
as 16% (Table II), in agreement with previous estimates (5, 26, 27).
This remarkable feature of the active site was completely retained in
D117E and D120E variants, partially retained in the D48E, Y93F, and
D115E variants and almost completely lost in the D152E variant (Table
II and Fig. 4).

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Fig. 4.
The effects of 2 mM
F on the amount of PPi bound to Y-PPase
variants (60-260 µM)
pre-equilibrated with 5 mM free Mg2+ and 20 mM total Pi at pH 7.2. Aliquots (20 µl)
of the reaction mixture were withdrawn at the indicated time intervals,
quenched with 4 µl of 5 M trifluoroacetic acid, and
assayed for PPi (18). The two panels refer to different
time scales. Lines were obtained with Equations 8 and 9
using parameter values found in Table IV.
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As shown in Fig. 4, addition of 2 mM fluoride to E48D,
Y93F, and D115E variants at pH 7.2 caused a rapid decrease in
enzyme-bound PPi, not resolved in time (F
binding to enzyme-Pi complex in Scheme I), followed by a
slow increase to a higher level (F
binding to
enzyme-PPi complex) (Fig. 4), like in wild type Y-PPase (11). The time course of the slow increase could be described by
Equations 8 and 9 (11), where
f eppfast refers to the amount of
enzyme-bound PPi after completion of the fast phase of the
interaction with F
(i.e. the zero time point
for the slow phase), and
feppslow is the amount of
the enzyme-bound PPi accumulated at time t
during the slow phase. Fluoride binding was assumed to be a
second-order reaction (see also below).
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(Eq. 8)
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(Eq. 9)
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For the D117E and D120E variants, the initial decrease in
fepp could be resolved in time. Moreover, no
slow increase in fepp followed after its rapid
drop in the D120E variant. Values of ki, app, kr, and
feppfast for the
D117E variant were obtained with Equations 8 and 9 using the points
collected at t
2 min on the curve shown in Fig. 4. The curve for the D120E variant and the initial part of the
curve for the D117E variant (t
2 min) could be
described by Equations 10 and 11 for a reversible binding, where
r is the percentage of fraction of enzyme-Pi
complex containing bound fluoride and the primed rate constants refer
to F
binding to and release from enzyme-Pi
complex. As the enzyme-Pi complex binds two fluoride ions
(see below), Equation 11 contains the second power of fluoride
concentration. The effect of F
on PPi bound
to D152E-PPase was quite small, if there was any.
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(Eq. 10)
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(Eq. 11)
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The ratio of
ki, app/feppfast,
the true rate constant for F
binding to
enzyme-PPi complex (ki, app
refers to total enzyme), is decreased only in the D117E variant by
comparison with wild type Y-PPase (Table
IV).
Inactivation by Fluoride in the Presence of
Pi--
Upon the incubation with Pi and
F
at pH 7.2, the variant PPases were diluted
900-4500-fold into assay medium, and the initial velocity of
PPi hydrolysis was estimated from the slope of the Pi production curve 1 min after the dilution (the dead-time
of the Pi analyzer increased by comparison with Fig. 3
because of the Pi present in the enzyme solution). The
extensive dilution and the 1-min delay were expected to completely
reverse the fast fluoride binding step seen in Fig. 4 for the E48D,
Y93E, and D115E variants and to partially reverse the slower initial
binding step observed with the D120E and D117E variants. In agreement
with the data reported above, the preincubation with fluoride did not affect the activity of the D152E variant, slightly inactivated the
D115E and D120E variants and more deeply
inactivated the E48D, Y93E, and D117E variants (Figs. 5 and
6). Low degree of the inactivation seen
with the D115E variant is consistent with its high
kr value (Table IV). For all the variants, the
degree of the inactivation observed after 80 min was smaller than for
wild type Y-PPase (11), consistent with lower
ki, app or higher
kr value (Table IV). The values of these
parameters estimated with Equation 1 for the two variants exhibiting
>30% inactivation in Figs. 5 and 6 (Y93F and D117E) were similar to
those derived from Fig. 4 (Table IV). For the D117E variant, only
points with t > 5 min were used in this
estimation.

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Fig. 5.
Inactivation of Y-PPase variants during
incubation with 2 mM F in the presence of 20 mM Pi and 5 mM Mg2+ at
pH 7.2. The incubation was carried out in a total volume of 70 µl at 6-850 µM enzyme concentration. Aliquots of 2-12
µl were withdrawn at indicated time intervals and immediately added
to the activity assay containing 1 mM PPi, 0.2 mM MgCl2, 10 µM EGTA, and 0.05 M Tris-HCl, pH 8.5. The line for the Y93F variant was
obtained with Equation 1 using parameter values found in Table IV, the
line for wild type Y-PPase is from Baykov et al. (11), and
the other lines were drawn by eye.
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Fig. 6.
Inactivation of D117E-PPase during incubation
with F in the presence of 20 mM
Pi and 5 mM Mg2+ at pH 7.2. F concentrations in mmol/liter are shown at the curves.
Other conditions were as for Fig. 5, except that the activity
assay was started by adding PPi 1 min after enzyme was
added; this 1-min delay was introduced to completely reverse the first,
faster step of F binding. The lines are the
best fits of Equation 1.
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Increasing the F
concentration decreased D117E-PPase
inactivation (Fig. 6), a phenomenon previously observed with wild type Y-PPase and explained by binding of two F
ions to
enzyme-Pi complex (11). Such binding decreases the concentration of the enzyme·PPi complex (the true binding
species) proportionally to [F]2, and this is only partly
compensated by an increase in the fluoride binding rate, which is
proportional to [F].
Fluoride Inhibition of D117E-PPase during PPi
Synthesis--
Studies of PPi synthesis provided further
evidence for the ability of D117E-PPase to bind two F
ions in the presence of Pi (Fig.
7). The effect of [F] on the initial
velocity of PPi synthesis was only poorly described by Equation 3 (with v'0 = 0), implying binding of
only one F
ion but was well described by Equation 12,
implying binding of two F
ions (Fig. 7, right
panel).

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Fig. 7.
The effects of NaF on PPi
synthesis catalyzed by D117E-PPase. Left panel,
progress curves at indicated F concentrations (in
mmol/liter). Curves were normalized to 1.2 µM enzyme
concentration; actual enzyme concentrations were 1.2-2.3
µM. One unit of luminescence corresponds to 1 µM PPi at zero time (luciferase fully
active). Right panel, dependence of
v0 on NaF concentration. The dotted
and solid lines show the best fits of Equations 3 (with
v'0 = 0) and 12, respectively.
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(Eq. 12)
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The nonlinearity of the luminescence versus time curves
(Fig. 7, left panel) was similar at different [F] and
could be fully explained by fluoride-independent inactivation of
luciferase (11). This means that the slow fluoride binding step is not
evident over the 1-4.5-min time interval in the presence of
Pi, consistent with the data in Fig. 4, showing no
significant increase in fepp during this time interval.
 |
DISCUSSION |
Roles for the Six Residues in Metal Binding and
Catalysis--
There is a substantial increase in
KM2 with five of the six variants under
study (Table I), indicating the M2 site to be highly sensitive to
changes in the delicate network of interactions in the active site of
Y-PPase (Fig. 1). The most drastic effects on
KM2 are seen with the E48D and D120E
variants, which is consistent with the x-ray crystallographic data
showing Glu48 and Asp120 to be most closely
associated with M2 (Fig. 1). The M1 site is clearly much more resistant
to amino acid substitutions than the M2 site; a significant effect is
only seen with the D120E(N), D152E, and D117E variants. This is
compatible with changes seen between the EM2 and
EM2(MPi)2 complexes (6). In these structures, the M2 site showed itself to be more rigidly fixed than the M1 site, so
that the loops carrying Asp115 and Asp152 swing
in toward a rigid M2 binding site in the product complex structure.
Consequently, variation at the M1 ligands Asp115 and
Asp152 is more readily tolerated than variation at the M2
ligands Asp120 and Glu48. However, it is
unclear why D115E and D152E substitutions have much larger effect on M2
than on M1 binding (Table I). One possible explanation is that the
substitutions effectively misposition M1 and Asp120 and so
prevent the rigid M2 binding site from forming properly. Consistent
with this notion is the fact that the changes seen at M2 for the D115E
and D152E substitutions, although large, are smaller than that seen for
directly mutating Asp120, as if the effects of the D115E
and D152E substitutions were to misalign Asp120. As
expected, mutating Tyr93, which is not involved in metal
binding (Fig. 1), had no effect on KM1
and only a minor effect on KM2 (Table I).
In general, the effects of the mutations on PPi hydrolysis
(as characterized by kh and
k3') are larger than the effects on PPi synthesis (as characterized by
kpp, off) (Table II). All the mutations, with
the only exception of E48D at pH 8.5, decrease
kh, whereas kpp, off is
increased in three variants (E48D, Y93F, and D115E) at pH 8.5. The
latter effect appears to result from increased
kB (see Equation 7), because the
kB step is rate-limiting in PPi
synthesis by wild type PPase (11). A similar stimulating effect of an
E20D substitution on PPi synthesis was earlier observed
with E. coli PPase (Glu20 in E. coli
PPase corresponds to Glu48 in Y-PPase) (22).
Combining Equation 6 with the expression for kh
(11), one obtains
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(Eq. 13)
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At pH 7.2, kA (840-2200 s
1),
k3' (1200 s
1),
k5 (800 s
1), and
k7 (960-3300 s
1) are nearly equal
for wild type Y-PPase (11), and (1
Pc) does not differ much from unity (Table II). Therefore, a mutation decreasing only k3' by a factor of n
would decrease kh by a factor approaching
(n + 3)/4. This criterion is met for most mutations, except
for D120E and D152E, at pH 7.2 (Table II). For the latter mutations,
the effect on kh is nearly equal to (D120E) or
exceeds (D152E) the effect on k3', which can
only result if, at least, one of kA,
k5, and k7 is also
decreased. The anomalous behavior of the D152E variant is preserved at
pH 8.5. Moreover, despite a decrease in k3',
kh is slightly increased in the E48D variant at
pH 8.5, suggesting an increase in kA,
k5, and/or k7. Thus, both
PPi hydrolysis (k3') and
Pi release are decelerated in the D152E variant,
PPi hydrolysis is decelerated, and Pi release
is accelerated in the E48D variant, and only PPi hydrolysis
is affected in the other four variants. According to Equation 6, the
effects on k3' may result from a decrease in
k3 or KAB. At present, we cannot distinguish between these alternatives. The invariance of
k5 in the Y93F, D115E, and D117E variants would
mean that the decrease in their Pc values (Table
II) results from a drop in k4. This effect is
the largest with the D117E variant (6-13-fold) but, nevertheless, is
much less than the effect on k3'.
The fluoride inhibition data support the notion that PPi
conversion to Pi becomes rate-limiting in some variants.
For D117E-PPase, values of ki in hydrolysis
(Table III) and
ki, app/feppfast
(Table IV) are the same. This is possible if EM4PP* and
EM4PP predominate during hydrolysis and are at equilibrium
with each other, which is equivalent to k3 being
the rate-determining step in hydrolysis. This is consistent with high
KF1 value for this variant (Table
III), indicating that the fraction of EM3P is quite low
during hydrolysis. That EM3P, when present, binds fluoride
well is indicated by its large effect on PPi synthesis by
D117E-PPase (Fig. 7) by comparison with wild type PPase (11). The
effect of the Y93F substitution on
KF1 is also larger at pH 7.2 (Table
III), where k3' is nearly rate-determining
(Table II), than at pH 8.5, where k3' is not such.
Interestingly, changes in pH had an opposite effect on
kh for all the variants, except for D115E, by
comparison with wild type Y-PPase (Table II). This appears to result
from a substantial alkaline shift in the bell-shaped pH profile for
kh (28). As a result, the pH 7.2 and 8.5 points
are on the descending limb of the pH rate profile for wild type Y-PPase
and on its ascending limb for most variants. By contrast, the D115E
substitution leaves the pH profile for kh nearly
unchanged (28). The shift may be explained by a change in the
pKa of the nucleophilic water (28, 29) or by
inhibition of the three-metal pathway in the variant PPases (21).
Fluoride Binding Site--
The slow inhibition step results in the
incorporation of one fluoride ion per substrate molecule, which remains
intact and tightly bound to PPase (30), suggesting that fluoride
replaces the nucleophilic water. Substitutions mispositioning its
ligands are thus expected to affect fluoride binding. Computer
modelling of the enzyme-PPi complex from the x-ray
structure of Y-PPase and E-PPase suggested that the nucleophile is
either Wat1, located between metal ions M1 and M2 and further liganded
by Asp117 (Fig. 1 and Ref. 6) or the water molecule
associated with M2 and Tyr93 (7). The results presented in
Tables III and IV clearly favor Wat1 as the nucleophile, because they
show that the D117E substitution does have the most drastic effect on
F
binding, as characterized by ki,
KF1, KF2, and
ki, app/feppfast.
Furthermore, a strong effect of the D120E substitution on
F
binding is consistent with Asp120 being
important for exact placing M1 and M2 and, hence, Wat1 (Fig. 1). The
substitutions of Asp115 and Asp152, which are
in the first coordination sphere of M1, and of Glu48, which
is in the second coordination sphere of M2, apparently have smaller
effects on Wat1 position, which correlates with much greater activity
of the D115E, D152E and D48E variants by comparison with the D120E or
D120N variants (Table II; see also Ref. 28). The Y93F substitution has
only a moderate effect on fluoride inhibition, despite a more drastic
nature of this substitution (the hydroxy group interacting with water
is eliminated) by comparison with the D117E substitution (the side
chain is elongated). For the same reason, similar effects of the two
substitutions on different catalytic properties in Y-PPase (Table II;
see also Ref. 28) and of the corresponding substitutions in E-PPase
(29, 31) also point to a greater importance of Asp117 by
comparison with Tyr93.
Several other lines of evidence support the identification of Wat1 as
the water molecule substituted by fluoride. First, Wat1 is replaced
with a Glu117 carboxylate oxygen in D117E-PPase (13),
showing the largest decrease in fluoride binding. Second, as measured
by differential UV spectroscopy, fluoride binding to Y-PPase in the
absence of substrates requires metal ions M1 and M2 (10), the ligands
to Wat1. Third, F
ion becomes entrapped together with two
Mg2+ ions in the stable
enzyme-F
-PPi complex (12). Fourth,
F
inhibition of E-PPase, in which the carboxylate of
Asp67 (equivalent of Asp117 in Y-PPase) is
moved away from M1 and M2 by about 1 Å by comparison with Y-PPase (7),
exhibits only a fast step. Moreover, a D67N substitution results in
appearance of the slow inhibition step in E-PPase (31), which is
similar in this respect to Y-PPase. In a series of D/N substitutions in
the active site of E-PPase, the D67N substitution had the largest
effect on fluoride binding (31). Fifth, other dimetal
hydrolases, including enolase (32, 33), aminopeptidase (34),
purple acid phosphatase (35), and urease (36), presumably generating a
hydroxide ion in the dimetal active site, are similarly inhibited by fluoride.
Mechanism of Fluoride Inhibition and Catalysis--
According to
Scheme I, derived from studies of wild type Y-PPase (11), fluoride
binds with different rates and stoichiometry to two enzyme
intermediates. Effects of fluoride on Y-PPase variants provide further
support to Scheme I. Thus, five of the six variants exhibit a biphasic
transition in the amount of enzyme-bound PPi upon addition
of fluoride (Fig. 4; compare values for fepp and feppfast in Tables II
and IV), the rapid drop and slow rise corresponding to fluoride binding
to EM3P and EM4PP*, respectively. These data are inconsistent with a consecutive binding model, in which rapid preassociation of a single enzyme form (EM4PP* in our case)
with the inhibitor is followed by a slow conformational change, because such a model predicts an increase in the amount of enzyme-bound PPi during both rapid and slow phases. Besides,
the involvement of two different intermediates is supported by the
observation that the D120E (at pH 8.5) and D152E substitutions cancel
the slow inhibition phase without significantly affecting the fast phase. These substitutions appear to greatly reduce the steady-state concentration of EM3P, which binds fluoride rapidly. The
ability of EM3P to bind two F
ions, another
important feature of Scheme I, is confirmed by the inactivation
patterns seen in Figs. 6 and 7 for the D117E variant.
The fluoride binding data obtained for the E48D and D115E variants
confirm the occurrence of two PPi-containing intermediates (EM4PP* and EM4PP in Scheme I) in PPase
catalysis. For both variants, ki values (Table
III) are greater than
ki, app/feppfast
values (Table IV), despite the fact that the former parameter refers to
the total enzyme, of which only a fraction contains bound
PPi during steady-state hydrolysis, and therefore
underestimates the rate constant for fluoride binding. Importantly, the
ki, app/feppfast
values are true rate constants for fluoride binding with the sum of
EM4PP* and EM4PP. Like with wild type Y-PPase,
this difference can be only explained by existence of two
PPi-containing intermediates that react with fluoride at
substantially different rates (11). Independent evidence for two
PPi-containing intermediates was obtained in
presteady-state measurements of PPi
hydrolysis.2
The nature of the structural changes accompanying the
EM4PP* to EM4PP transition still remains to be
determined. An attractive hypothesis presented below is based on the
observation that the metals M1 and M2 are bridged by two water
molecules in EM2 (6, 9) and by only one water molecule in
EM4P2 (6) and EM4PP.3
We suggest that the two-water bridge is
preserved upon initial substrate binding (i.e. in
EM4PP*), but one water molecule is expelled during the
transition to EM4PP (Scheme
III), consistent with a decrease in the
pKa of the essential basic group, attributable to
Wat1, from >7.0 to 5.8 (21). Recent model studies of Kaminskaia
et al. (37) have indicated no substantial increase in
nucleophilicity for a bridging water versus terminally bound water. Thus, the M1-Wat1-M2 structure apparently helps to favorably position Wat1 with respect to the attacked Pi residue.
Scheme III differs from Scheme I in that Scheme I shows fluoride
interaction with EM4PP* as one bimolecular reaction,
whereas in Scheme III this interaction involves rapidly reversible
binding of fluoride to EM4PP*, with a dissociation constant
of K'F, and slow conversion of the resulting
unstable intermediate, with a rate constant of k'A, to a form having a fluoride ion
between M1 and M2. However, the two mechanisms are kinetically
equivalent, provided that the bottom left species in Scheme III is
stoichiometrically insignificant, i.e.
K'F
[F]. In terms of Scheme III,
ki is equal to
k'A/K'F. Given the
chemistry of fluoride, the M1-F-M2 complex is likely to be very stable,
making the reverse reaction, which requires insertion of a water
molecule, very slow. For this reason, it is not shown in Scheme III. On
the other hand, the bound PPi cannot hydrolyze and leave as
Pi. This explains why fluoride stabilizes the
enzyme-substrate intermediate to an extent allowing its isolation by
gel filtration (30). At neutral pH, the bridging water molecule exists
predominantly as OH
in EM4PP and, presumably,
EM4P2 (21), which explains their very slow, if
any, binding of the negatively charged fluoride ion. The proposed
structure of EM4PP* is consistent with the results of
recent x-ray crystallographic studies of the fluoride-inhibited PPi complex of Y-PPase, showing a single electron density
between M1 and M2.3 By contrast, two water molecules are
expected to be present between M1 and M2 in EM3P because
fluoride binding to this species is relatively weak and proceeds in a
rapidly reversible manner with a stoichiometry of two per active site.
The unsurpassed stability of the F-H-F hydrogen bond (38) would favor
the M1-F-H-F-M2 structure, despite the low pKa value
(3.2) for hydrofluoric acid.
 |
FOOTNOTES |
*
This work was supported by Academy of Finland Grants 35736 and 47513 and Russian Foundation for Basic Research Grants 00-04-48310 and 00-15-97907.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.

To whom correspondence may be addressed. Tel.:
95-939-5541; Fax: 95-939-3181; E-mail: baykov@genebee.msu.su.
§§
To whom correspondence may be addressed. Tel.: 358-02-333-6845;
Fax: 358-02-3336860; E-mail: reijo.lahti@utu.fi.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M007360200
2
P. Halonen et al., manuscript in preparation.
3
P. Heikinheimo, V. Tuominen, A.-K. Ahonen, A. Teplyakov, B. S. Cooperman, A. A. Baykov, R. Lahti, and A. Goldman,
submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
PPase, inorganic
pyrophosphatase;
E-PPase, E. coli PPase;
PPi, pyrophosphate;
WT, wild type;
Y-PPase, yeast (Saccharomyces
cerevisiae) PPase;
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.
 |
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