The Electrophilic and Leaving Group Phosphates in the Catalytic
Mechanism of Yeast Pyrophosphatase*
Anton B.
Zyryanov
,
Pekka
Pohjanjoki§,
Vladimir N.
Kasho¶,
Alexander S.
Shestakov
,
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-20500 Turku, Finland, the
¶ Center for Ulcer Research and Education, Department of Medicine,
University of California, Los Angeles, California 90073, and the
Institute of Biotechnology, University of Helsinki, P. O. Box
56, FIN-00014 Helsinki, Finland
Received for publication, January 16, 2001, and in revised form, February 14, 2001
 |
ABSTRACT |
Binding of pyrophosphate or two phosphate
molecules to the pyrophosphatase (PPase) active site occurs at two
subsites, P1 and P2. Mutations at P2 subsite residues (Y93F and K56R)
caused a much greater decrease in phosphate binding affinity of yeast PPase in the presence of Mn2+ or Co2+
than mutations at P1 subsite residues (R78K and K193R). Phosphate binding was estimated in these experiments from the inhibition of ATP
hydrolysis at a sub-Km concentration of ATP. Tight phosphate binding required four Mn2+ ions/active site.
These data identify P2 as the high affinity subsite and P1 as the low
affinity subsite, the difference in the affinities being at least
250-fold. The time course of five "isotopomers" of phosphate that
have from zero to four 18O during
[18O]Pi-[16O]H2O
oxygen exchange indicated that the phosphate containing added water is
released after the leaving group phosphate during pyrophosphate
hydrolysis. These findings provide support for the structure-based
mechanism in which pyrophosphate hydrolysis involves water attack on
the phosphorus atom located at the P2 subsite of PPase.
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INTRODUCTION |
Inorganic pyrophosphatase (EC 3.6.1.1;
PPase)1is a ubiquitous enzyme
catalyzing interconversion of PPi and Pi.
Soluble PPase provides a thermodynamic pull for biosynthetic reactions
by removing PPi formed when nucleoside 5'-triphosphates are
converted to the corresponding monophosphates (1). The PPase reaction
involves, in the direction of hydrolysis, PPi binding,
isomerization of the resulting complex, PPi hydrolysis, and
the stepwise release of two Pi molecules (Scheme
I) (2). The hydrolysis step proceeds via
direct attack of water on PPi without formation of a
covalent intermediate (3). Numerous mechanistic studies of PPase have been carried out (for reviews, see Refs. 4 and 5), making this enzyme
the best characterized among the catalysts of phosphoryl transfer from
various polyphosphates (including ATP and GTP) to water.
PPi-Pi
equilibration by PPase. E, enzyme; M, divalent metal
ion; PP, PPi; P, Pi; n = 1 or
2.
Scheme I.
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X-ray crystallographic studies of PPase complexed with phosphate have
identified two Pi binding subsites, P1 and P2, within the
active site (6, 7) (Fig. 1). In addition,
an activated water molecule, placed between two metal ions in the
vicinity of P2, was considered the only logical candidate for
nucleophile (6). This supposition was confirmed by the structure of the F--inhibited complex (8), which showed a fluoride ion
replacing that water molecule. Solution confirmation of this
interpretation has been harder to achieve, however. The oxygen exchange
measurements done in the presence of Mg2+ as the activator
have shown unequivocally that the Pi containing the
electrophilic phosphorus is released first after PPi
hydrolysis (9). In terms of the structure-based mechanism (6, 8, 10),
this would mean that Pi is first released from P2, where it
is more buried than at P1. Resolving this issue requires knowledge of
the relative affinities of P1 and P2 for Pi because it is
logical to expect faster release from a weaker binding subsite. Earlier attempts to compare the affinities of P1 and P2 used three types of
data (5), each of which has been subject to criticism. First, preferential binding of sulfate, an analog of Pi, occurred
at P1 in the PPase crystals grown in the presence of sulfate (11, 12).
However, the crystallization medium in these studies contained no metal
ions, the major Pi ligands at the P2 subsite (Fig. 1). Second, x-ray data indicate more extensive hydrogen bonding between the
enzyme and Pi at P1 than at P2 (Fig. 1), which was thought to provide greater binding strength at P1. We will show below that this
is not the case for Mn2+ and Co2+ as cofactors.
Third, protection of Arg78, located at P1, against chemical
modification upon binding of 1 mol of Pi/mol of subunit in
the presence of Mn2+ was interpreted as showing that P1
binds first (13). However, because of the close proximity of P1 and P2,
the protection could equally result from binding to P2. We will show
below that P2 does in fact exhibit a far greater affinity for
Pi in the presence of Mn2+ and
Co2+.

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Fig. 1.
Coordination of two phosphate ions in the
active site of Y-PPase (6). Black circles (M1-M4) are
metal ions, the gray circle (W1) is a water oxygen, and the
two black molecules (P1 and P2) are phosphates. Hydrogen
bonds and electrostatic interactions are shown by dashed
lines. The metal ions are further coordinated by one (M2 and M3),
two (M4), or three (M1) amino acid side chains (not shown).
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We addressed the correct assignment of the phosphate binding subsites
by employing site-directed mutagenesis of Pi ligands together with Pi binding and Pi-water oxygen
exchange measurements in the presence of Mn2+ and
Co2+. By comparison with Mg2+, these cations
induce a much greater difference in the binding affinities of P1 and
P2, as evidenced by the fact that only one Pi binding
site/subunit was observed over a wide range of Pi
concentrations (13, 14). In the presence of Mg2+, the
affinities of P1 and P2 for MgPi differ only 1-9-fold (9, 15, 16); for comparison, macroscopic binding constants for two sites
with equal microscopic constants differ 4-fold (17). The results
reported below demonstrate that the relative affinities of P1 and P2
and the order in which these sites release Pi during PPi hydrolysis depend on the nature of the metal ion
cofactor used and provide support for the mechanism currently proposed for this reaction.
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MATERIALS AND METHODS |
The expression and purification of wild type Y-PPase and its
active site variants from overproducing Escherichia coli
XL2blueb strain transformed with suitable plasmids were
carried out as described by Heikinheimo et al. (18). Enzyme
concentration was calculated on the basis of a subunit molecular mass
of 32 kDa (19) and an
A
equal to 14.5 (20).
Disodium ATP (Sigma Chemical Co.) was freed from Pi and
PPi by crystallization from water-ethanol (21). Stock
solutions of ATP were standardized by measuring light absorbance at 260 nm (
= 15, 400 M
1
cm
1) (22). 18O-enriched potassium
phosphate (96.4%) was prepared according to Hackney et al.
(23).
ATP hydrolysis was assayed luminometrically. The assay mixture of a
0.2-1-ml volume contained 1 µM ATP, 83 mM
TES/KOH (pH 7.2), 17 mM KCl, and varied amounts of
potassium phosphate and MnCl2. Assays done with
Co2+ employed a 100 mM MOPS/KOH buffer (without
KCl) because TES was reported to bind Co2+ appreciably
(24). Bovine serum albumin (0.01 mg/ml) was also added for enzyme
stability in the assays done with R78K-PPase. The reaction was
initiated by adding PPase. Aliquots (20 µl) of the assay mixture were
withdrawn at 2-3-min intervals over 20-25 min and added to 0.18 ml of
0.1 M Tris acetate buffer (pH 7.75) containing
luciferin/luciferase reagent (Sigma ATP assay mix), 10 mM
magnesium acetate, 2 mM EDTA, 1 mg/ml bovine serum albumin, and 1 mM dithiothreitol. The luminescence was then measured
with an LKB model 1250 luminometer. The concentration of the
luciferin/luciferase reagent was sufficient to produce a 100-mV signal
for samples containing 1 µM ATP.
The procedures used to measure enzyme-bound PPi formation
at equilibrium (25) and enzyme-catalyzed Pi-water oxygen
exchange (26) were as described previously. The media used in the
incubations for oxygen exchange and synthesis of enzyme-bound
PPi were prepared by mixing appropriate volumes of 100 mM potassium phosphate, 100 mM MOPS/KOH buffer
(pH 7.2), and 100 or 3 mM CoCl2, respectively. Although the solubility products for CoHPO4
(1.9·10
7 M2 (Ref.
27)) and MnHPO4 (1.4·10
13
M2 (Ref. (28)) were exceeded in many of the
incubations, no incipient precipitation occurred in the concentration
ranges of the metal ions and Pi used in this study. That
the precipitation in these systems is quite slow was confirmed by the
following data. First, no decrease in Pi concentration was
detected when solutions containing 1 mM Mn2+
plus 200 µM Pi or 20 mM
Mn2+ plus 20 µM Pi were incubated
for 30 min and centrifuged for 15 min at 4,000 × g.
Similarly, no change in Co2+ concentration, measured with
Arsenazo III (28), was detected at 0.5 mM Co2+
and 5 mM Pi after a 15-min incubation and
centrifugation as above. At higher concentrations of the metal ions and
Pi, the solutions became opalescent, and a decrease in
Pi or Co2+ concentration was observed after centrifugation.
All measurements were performed at 25 °C.
The concentrations of free Mn2+ and Co2+
ions and of their complexes with Pi were calculated using
dissociation constants of 3.6 and 9.8 mM,
respectively, which were measured with Arsenazo III (29) under
the conditions used in the present
work.2
ATP hydrolysis in the presence of Pi is described by Scheme
II, where kcat is
the catalytic constant and Kp is the
dissociation constant of the enzyme·Pi complex. Y-PPase
converts ATP into ADP and Pi, and further hydrolysis of ADP
to yield AMP and Pi proceeds at a negligible rate (30).
ATP hydrolysis in the presence of
Pi. Subscript t refers to the sum of free and
metal-bound phosphate.
Scheme II.
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The rate of ATP hydrolysis obeyed Equation 1, where t is
time, Km is the Michaelis constant, and
[E] is free enzyme concentration. In the absence of
Pi (added externally or formed from ATP), [E]
could be equated to the total enzyme concentration, [E]t, because the Michaelis constant for ATP
is in excess of 100 µM, a value much greater than its
concentration in the reaction medium. In the presence of
Pi, [E] could be calculated from Equation 2,
where [P]t is the total Pi concentration in
the reaction medium. Equation 2 was obtained by solving the equation
for Kp (Equation 3) together with Equations 4
and 5, describing mass balance for enzyme and inhibitor,
respectively.
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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It should be noted that [P]t in Equations 2 and 5
refers to the sum of the added and enzymatically generated
Pi and is therefore given by Equation 6, where
[P]0 and [ATP]0 are the initial
concentrations of Pi and ATP, respectively.
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(Eq. 6)
|
In theory, the value for Kp could be
obtained from the time course of ATP hydrolysis measured in the absence
of added Pi ([P]0 = 0), thus evaluating the
effect of Pi produced enzymatically. However, much more
accurate estimates for this parameter were obtained from fittings that
simultaneously employed the time courses measured in the absence and
presence of added Pi (Fig.
2). When such time courses were measured
at varied [E]t, it was treated as a variable
along with t and [P]0.

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Fig. 2.
Time courses of Mn2+-supported
ATP hydrolysis by wild type Y-PPase in the absence and presence of
1 µM phosphate. Conditions:
0.3 µM enzyme, 10 mM Mn2+. The
lines show the best fits of Equations 1, 2, and 6.
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The rate of Pi-water oxygen exchange,
vex, was calculated as
4[P]tln(E0/E)/t,
where E0 and E are the average
18O enrichments of Pi before and after
incubation with Y-PPase, and t is the time of the
incubation. Values of the partition coefficient Pc were calculated using the program written by
Hackney (31).
All the fittings were performed using the program SCIENTIST (MicroMath).
 |
RESULTS |
Pi Inhibition of Mn2+-supported ATP
Hydrolysis--
Pi was found to be a very potent inhibitor
of wild type Y-PPase in the presence of Mn2+ (Fig. 2). The
inhibition constant calculated from the time courses of ATP hydrolysis,
as described under "Materials and Methods," decreased with
increasing [Mn2+], approaching a constant level of about
0.04 µM (Fig. 3). The mutations decreased the affinity of Y-PPase to Pi, the
effect being moderate with R78K and K193R variants and quite large with the K56R, and especially Y93F variants (Fig. 3).

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Fig. 3.
Values of Kp for
wild type and variant Y-PPase as a function of Mn2+
concentration. The lines are drawn according to
Equation 7, using parameter values given in Table I.
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The Mn2+ concentration dependences of
Kp shown in Fig. 3 could be well described by
Scheme III. This implies that two enzyme
species, EM2 and EM3,
bind MnPi with different affinities. Y-PPase is known to
have three binding sites for divalent metal ions in the absence of
substrate and Pi (9, 32, 33), but the affinities of two of
these sites are quite high (see next section), and they are always
saturated at the Mn2+ concentrations used in the present
study. Fitting of the data in Fig. 3 to Equation 7 derived for Scheme
III yielded the parameter values shown in Table
I. The fittings indicated that the
EM3P species is present in insignificant amounts
with wild type Y-PPase and its R78K variant over the Pi
concentration ranges used (0-5 and 0-7 µM,
respectively). In terms of Scheme III, the decrease in
Kp with increasing [Mn2+] is
caused by accumulation of MP (the actual binding form of Pi) and EM3 (a better binding form
by comparison with EM2).
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(Eq. 7)
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Values of kcat/Km, also
obtained from Equations 1, 2, and 6 using the time courses of ATP
hydrolysis (Fig. 2), either increased with [Mn2+] until a
constant level was attained (wild type, K56R) or passed through a
maximum at 5-10 mM Mn2+ and decreased slightly
(by 15-20%) at 20 mM [Mn2+] (R78K, Y93F,
K193R) (data not shown). The maximum values of kcat/Km observed for wild
type PPase and each variant are listed in Table I.
Phosphate and metal ion binding to
Y-PPase.
Scheme III.
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Mn2+ Binding in the Presence of
Pi--
Equilibrium dialysis measurements of
Mn2+ binding in the presence of Pi provided
direct support for Scheme III. In these experiments, Y-PPase was
equilibrated with 50 µM MnCl2 at varied
concentrations of Pi, and the manganese content of the two
chambers separated by a dialysis membrane was measured by atomic
absorption spectroscopy. As shown in Fig.
4, the stoichiometry of Mn2+
binding approached 2 in the absence of Pi, indicating
nearly full occupancy of the two high affinity sites, consistent with earlier data (13, 14, 34), and was above 3 at the highest Pi concentration. The theoretical curve obtained using
Equation 8 with K
= 0.041 µM,
K
=
,
KM3 = 1.7 mM (Table I) and [M] and
[MP] given by Equations 9 and 10 is in satisfactory agreement with
the measured data. Parameter KM2 (equal to 20 µM3) in
Equation 8 is the dissociation constant characterizing the equilibrium
EM = EM2 (not shown in Scheme
III).

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Fig. 4.
Mn2+ binding to wild type Y-PPase
in the presence of Pi, as measured by equilibrium
dialysis. Total metal concentration was fixed at 50 µM. The line is drawn according to Equations
8-10, using parameter values derived from Pi inhibition
measurements (Table I).
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(Eq. 8)
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(Eq. 9)
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(Eq. 10)
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Pi Inhibition of Co2+-supported ATP
Hydrolysis--
Pi binding by Y-PPase and its variants in
the presence of 0.5 mM Co2+ was also estimated
from the inhibition of ATP hydrolysis. The binding seemed to be
somewhat less strong (Table II), but the effects of the mutations closely paralleled those obtained with Mn2+ (Fig. 3). The effects on
kcat/Km were also very
similar with Mn2+ (Table I) and Co2+ (Table
II).
Formation of Enzyme-bound PPi in the Presence of
Co2+--
The Y-PPase active site contains two
Pi binding subsites. Pi binding to both of
these subsites can be accessed by measuring the fraction of the enzyme
containing bound PPi (fepp) as a
function of Pi concentration in solution (9) because
PPi synthesis requires occupancy of both subsites. Values
of fepp measured in the presence of 0.5 mM free Co2+ ion exhibited a hyperbolic
dependence on [P]t (Fig. 5),
allowing calculation of the limiting value of
fepp at infinite [P]t (0.18 ± 0.03) and the dissociation constant for Pi binding
(3 ± 1 mM). The latter value exceeds
Kp (Table II) by a factor of 240 and therefore
characterizes binding of the second Pi molecule.

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Fig. 5.
Formation of enzyme-bound PPi by
wild type Y-PPase in the presence of 0.5 mM
Co2+. Enzyme concentration was 30 µM.
The line shows the best fit to a simple hyperbolic
saturation function with parameter values given under
"Results."
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Pi-Water Oxygen Exchange in the Presence of
Co2+--
Y-PPase catalyzes rapid exchange of oxygen
between Pi and water in the presence of Mg2+
and other metal ions that activate PPi hydrolysis (30,
35-37). This exchange results from a dynamic reversal of the steps
characterized by k3 and
k4 in Scheme I (36, 37): one oxygen originally
present in Pi is released as water when the two bound
Pi molecules dehydrate forming PPi
(k4 step) and is subsequently replaced by an
oxygen from water when the PPi is converted back into
Pi (k3 step). The exchange is
conveniently followed by mass spectrometry, starting from
[18O]Pi and
[16O]H2O (23).
Table III shows two examples of the
observed distributions of the five Pi species that have
from 0 to 4 exchanged oxygens in the Co2+-supported
reaction catalyzed by wild type Y-PPase. Such distributions are
characterized by two parameters: the exchange rate
vex and the partition coefficient
Pc, which equals the probability of bound
phosphate conversion into EM4PP versus its
release into solution in Scheme I (31). As shown in Table III and Fig.
6A, Pc
exhibited a strong dependence on [P]t in the
Co2+-supported reaction, changing from less than 0.3 at low
[P] to ~0.9 at its saturating concentration. By contrast,
Pc is independent of [P]t in the
Mg2+-supported reaction (9, 15, 31).
vex/[E]t increased with [P]t, reaching a maximum at about 1.5 mM
Pi, and dropped slightly at higher [P]t (Fig.
6B). By dividing
vex/[E]t by
4Pc/(4
3Pc) (the
average number of the exchanged oxygens in each Pi molecule leaving the enzyme), one obtains the rate of release of the phosphate that contains exchanged oxygens. This parameter dropped hyperbolically with increasing Pi concentration (Fig. 6B),
yielding a dissociation constant of 1.2 ± 0.4 mM, a
value not much different from that for the binding of the second
Pi molecule, as derived above from fepp measurements.
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Table III
Distribution of [18O]Pi isotopomers during
Pi-water oxygen exchange by wild type Y-PPase in the
presence of 0.5 mM Co2+
The 18O isotopomers containing from four to zero 18O
atoms are designated as P18O4, P18O3,
P18O2, P18O1, and
P18O0. The initial distribution of the 18O
isotopomers in this order was 91.19, 7.31, 0.35, 0.06, and 1.09%. The
enzyme concentration was 0.62 µM (0.125 mM
Pi) or 6.2 µM (5 mM Pi). The
observed distributions shown were measured after a 10-min incubation.
The best fit theoretical distributions are shown in the upper row for
each Pi concentration. Two more theoretical distributions,
calculated assuming the same average 18O enrichment in the
final Pi, show that the final distribution is highly sensitive
to Pc value.
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Fig. 6.
Pi-water oxygen exchange
catalyzed by wild type Y-PPase in the presence of 0.5 mM
Co2+ as a function of Pi concentration.
A, Pc values. The line
shows the best fit of Equation 11 multiplied by 0.88, the limiting
value of Pc at infinite phosphate concentration.
B, exchange rate. Open circles,
vex/[E]t; closed
circles, vex(4 3Pc)/4Pc[E]t.
The line shows the best fit of the equation
vex(4 3Pc)/4Pc[E]t = a + b/(1 + [P]t/Kd), with the following best fit
values of the parameters: a, 0.52 ± 0.11 s 1; b, 2.04 ± 0.09;
Kd, 1.2 ± 0.4 mM.
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DISCUSSION |
Five amino acid residues interact through their side chains with
Pi in the PPase active site (Fig. 1): three
(Arg78, Tyr192, and Lys193) at
subsite P1 and two (Lys56 and Tyr93) at subsite
P2. The remaining interactions are through four metal ions. We have
demonstrated previously that the Y-PPase variants used in the present
work retain 2-20% activity against PPi (18), indicating
that the active site remains catalytically competent. It is noteworthy
that the mutations used are highly conservative and do not change the
overall charge of the active site. The absence of large changes in
structure outside active site in the R78K variant has been demonstrated
by x-ray crystallography (38). In this variant, the positive charge on
the introduced lysine is displaced by about 3 Å compared with arginine.
The Pi binding capacity of four of these variants was
estimated here from the inhibition of ATP hydrolysis in the presence of
Mn2+ and Co2+ as cofactors. The advantage of
these cations over Mg2+ is 3-fold. First and most
important, the binding affinities of the subsites P1 and P2 differ much
more in the presence of Mn2+ or Co2+ (13, 14).
Second, these cations support hydrolysis of ATP (Mg2+ does
not), a substrate with a high Km value. At 1 µM ATP, more than 99% of Y-PPase is substrate-free in
the assay medium. Therefore, the true Pi binding constant
can be estimated directly from the effect of Pi on activity
at this fixed ATP concentration. Third, the structure shown in Fig. 1
was, in fact, determined for M = Mn2+, although
similar radii and coordination chemistry for Mn2+ and
Mg2+ suggest that this structure should be largely
preserved with Mg2+.
P2 Is the Tighter Binding Subsite for Phosphate in the Presence of
Mn2+ and Co2+--
The data in Fig. 3 and
Tables I and II indicate that the two mutations at subsite P2 have a
much greater effect on Pi binding to Y-PPase than the two
mutations at subsite P1. The measured binding clearly refers to the
tighter binding subsite because occupancy of any one of the two
subsites by Pi will suffice for inhibition. Furthermore,
the data in Fig. 5 demonstrate that the dissociation constant for the
weaker binding subsite is as high as 3 mM in the presence
of Co2+, 240 times larger than for the tighter binding site
(Table II). The weaker binding site was thus essentially empty during
Kp measurements, consistent with the earlier
equilibrium dialysis and Pi titration data revealing only
one Pi binding subsite in the presence of Mn2+
and Co2+ and up to 0.1 mM Pi (13,
14).
Other data support the identification of P2 as the tighter binding
subsite in the presence of Mn2+ and Co2+.
First, the tighter binding site requires four Mn2+ ions for
optimal Pi binding (Scheme III), as one would expect for P2
that has four metal ligands, rather than P1 that has only two (Fig. 1).
Second, Pi was observed only in P2 in the manganese structure of the R78K variant (38), whose Pi binding
affinity is similar to that of the wild type enzyme. Finally, mutations at the P2 subsite (K29R and Y55F) in E. coli PPase have also
been shown to have greater effects on Pi binding measured
in the presence of 0.1 mM Mn2+ than mutations
at the P1 subsite (R43K, Y141F, and
K142R).4
Interestingly, the effects of the mutations on ATP binding, as
characterized by kcat/Km
(Tables I and II), parallel those on Pi binding (Fig. 3 and
Table II). This indicates that ATP binding is dominated by interactions
at subsite P2. One of the P1 mutations (R78K) increases
kcat/Km for
Mn2+-supported ATP hydrolysis nearly 5-fold (Table I),
suggesting that the interaction of Arg78 with the
-phosphate of ATP is destabilizing in the enzyme-substrate complex.
P2 Contains the Electrophilic Phosphorus Atom--
One would
expect faster release of the product Pi from P1, where
binding is much weaker, insofar as this Pi faces solution, whereas the P2 Pi is buried at the bottom of the active
site (6, 7). On the other hand, the Pi containing the
electrophilic center is released first in the presence of
Mg2+ (9). Therefore, either the electrophilic center is at
P1, or the order of Pi release is reversed with
Mn2+ and Co2+. A choice between these
alternatives can be made on the basis of the oxygen exchange data.
The rationale for using this approach is as follows. Once bound to the
electrophilic center, each Pi molecule undergoes a series
of synthesis/hydrolysis cycles, each resulting in exchange of one of
the four oxygens. The longer the Pi molecule stays in the
active site, the more extensive is the exchange before it is released
into solution. The average number of exchange cycles depends on the
probability of Pi forming PPi versus
being released into solution, Pc. If the
electrophilic Pi molecule is released first,
Pc is equal to
k4/(k4 + k5) (9, 31) and is thus independent of
[P]t, as observed in the case of the
Mg2+-supported reaction (9, 15, 31). If, however, the
electrophilic Pi molecule is released second, the time it
stays in the active site would increase with increasing the occupancy
of the other subsite and hence would depend on [P]t
according to Equation 11 (9). At infinite [P]t,
Pi never leaves the enzyme, resulting in exchange of all of
its oxygens (Pc = 1). At [P]t
approaching 0, Pc would also approach 0, corresponding to zero exchange. A thorough theoretical analysis of the
Pi-water oxygen exchange catalyzed by PPase was done by
Hackney (31).
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(Eq. 11)
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Fig. 6A shows that Pc increases
from about 0 to 0.88 with [P]t in the
Co2+-supported reaction. Importantly, this effect is
observed at the Pi concentrations allowing appreciable
binding to both P2 and P1 (Fig. 5). This provides strong evidence that
the Pi molecule acquiring oxygen from water during
PPi hydrolysis is released second. This conclusion is
consistent with
vex/[E]t going through a maximum at increasing [P]t (Fig. 6B).
Indeed, the exchange rate depends on [EP] in this case,
and [EP] passes through a maximum when Pi
concentration is increased. Accordingly, the rate of the release of
phosphate-containing exchanged oxygens (equal to
vex(4
3Pc)/4Pc[E]t,
where 4Pc/(4
3Pc)
is the average number of the exchanged oxygens in each Pi
molecule (23)) decreases with [P]t (Fig. 6B).
In the Mg2+-supported reaction, Pc
is independent of [P]t, which is strong evidence that the
Pi molecule containing the oxygen that comes from water is
released first, and the exchange rate, which is proportional to
[EPP] in this case, always increases with
[P]t (9, 15, 31). Because slower release of
Pi is expected from the tighter binding site, the
electrophilic center is thus at P2.
As mentioned above, the independence of Pc from
[Pi] in the presence of Mg2+ provides strong
evidence that the Pi containing the electrophilic phosphorus is released first after PPi hydrolysis (9). This implies that the order in which the two subsites release Pi
is reversed in the presence of Mg2+, which may mean that
the Pi binding affinities of P1 and P2 are also reversed.
It should be noted that the upper limit for Pc
in Fig. 6A is less than the value of unity predicted by
Equation 7. A likely explanation is that the release of the two
Pi molecules formed is not a strictly ordered reaction in
the presence of Co2+; that is to say, a significant
fraction of Pi leaves P1 first. This explanation is
consistent with the observation that the value of
vex(4
3Pc)/4Pc[E]t
tends to reach a constant non-zero value with increasing Pi
concentration (Fig. 6B) rather than approach 0, as expected
for a strictly ordered release.
To sum up, these data provide strong support for the mechanism of
PPi hydrolysis (6, 8), involving water/hydroxide addition to the phosphorus atom located in the site P2 of PPase.
 |
FOOTNOTES |
*
This work was supported by Russian Foundation for Basic
Research Grants 00-04-48310 and 00-15-97907 and Academy of Finland Grants 35736 and 47513.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.: 358-2-333-6845; Fax:
358-2-333-6860; E-mail: reijo.lahti@utu.fi.

To whom correspondence may be addressed. Tel.:
7-095-939-5541; Fax: 7-095-939-3181; E-mail:
baykov@genebee.msu.su.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100343200
2
A. B. Zyryanov, manuscript in preparation.
4
Hyytiä, T., Halonen, P., Salminen, A.,
Goldman, A., Lahti, R., and Cooperman, B. S. (2001)
Biochemistry, in press.
3
P. Pohjanjoki, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
PPase, inorganic
pyrophosphatase;
Y-PPase, yeast (Saccharomyces cerevisiae)
inorganic pyrophosphatase;
TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid;
MOPS, 4-morpholinepropanesulfonic acid.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.