The Electrophilic and Leaving Group Phosphates in the Catalytic Mechanism of Yeast Pyrophosphatase*

Anton B. ZyryanovDagger , Pekka Pohjanjoki§, Vladimir N. Kasho, Alexander S. ShestakovDagger , Adrian Goldman||, Reijo Lahti§**, and Alexander A. BaykovDagger DaggerDagger

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


E<UP>M<SUB>2</SUB></UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>2</SUB></LL><UL>k<SUB>1</SUB></UL></LIM> E<UP>M<SUB>4</SUB>PP*</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB><UP>B</UP></SUB></LL><UL>k<SUB><UP>A</UP></SUB></UL></LIM> E<UP>M<SUB>4</SUB>PP</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>4</SUB></LL><UL>k<SUB>3</SUB></UL></LIM> E<UP>M<SUB>4</SUB>P<SUB>2</SUB></UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>6</SUB></LL><UL>k<SUB>5</SUB></UL></LIM> E<UP>M<SUB>3</SUB>P</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>8</SUB></LL><UL>k<SUB>7</SUB></UL></LIM> E<UP>M<SUB>2</SUB></UP>

PPi-Pi equilibration by PPase. E, enzyme; M, divalent metal ion; PP, PPi; P, Pi; n = 1 or 2.

Scheme I.  

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+.


View larger version (20K):
[in this window]
[in a new window]
 
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).

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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>280</SUB><SUP>1%</SUP></UP> 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 (epsilon  = 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).


<AR><R><C>E+<UP>ATP</UP> <LIM><OP><ARROW>⇌</ARROW></OP><UL>   </UL></LIM> E<UP>·ATP</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>cat</UP></SUB></UL></LIM> E+<UP>ADP</UP>+(<UP>P</UP>)<SUB><UP>t</UP></SUB>
</C></R><R><C><UP>⥮K<SUB>p</SUB>
</UP></C></R><R><C><UP>E·</UP>(<UP>P</UP>)<SUB><UP>t</UP></SUB></C></R></AR>

ATP hydrolysis in the presence of Pi. Subscript t refers to the sum of free and metal-bound phosphate.

Scheme II.  

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.
<UP>−</UP><FR><NU><UP>d</UP>[<UP>ATP</UP>]</NU><DE><UP>d</UP>t</DE></FR>=<FR><NU>k<SUB><UP>cat</UP></SUB></NU><DE>K<SUB>m</SUB></DE></FR>[<UP>ATP</UP>][E] (Eq. 1)

  [E]=0.5<FENCE>[E]<SUB><UP>t</UP></SUB>−[<UP>P</UP>]<SUB><UP>t</UP></SUB>−K<SUB><UP>p</UP></SUB>+<RAD><RCD>([E]<SUB><UP>t</UP></SUB>−[<UP>P</UP>]<SUB>t</SUB>−K<SUB><UP>p</UP></SUB>)<SUP>2</SUP>+4K<SUB><UP>p</UP></SUB>[E]<SUB><UP>t</UP></SUB></RCD></RAD></FENCE> (Eq. 2)

[<UP>P</UP>][E]/[E · <UP>P</UP>]=K<SUB><UP>p</UP></SUB> (Eq. 3)

[E]+[E · <UP>P</UP>]=[E]<SUB><UP>t</UP></SUB> (Eq. 4)

[<UP>P</UP>]+[E · <UP>P</UP>]=[<UP>P</UP>]<SUB><UP>t</UP></SUB> (Eq. 5)

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.
[<UP>P</UP>]<SUB><UP>t</UP></SUB>=[<UP>P</UP>]<SUB>0</SUB>+[<UP>ATP</UP>]<SUB>0</SUB>−[<UP>ATP</UP>] (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.


View larger version (16K):
[in this window]
[in a new window]
 
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.

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (17K):
[in this window]
[in a new window]
 
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.

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).
K<SUB><UP>p</UP></SUB>=<FR><NU>K<SUP><UP>M3</UP></SUP><SUB><UP>p</UP></SUB>K<SUP><UP>M4</UP></SUP><SUB><UP>p</UP></SUB></NU><DE>K<SUP><UP>M3</UP></SUP><SUB><UP>p</UP></SUB>+K<SUP><UP>M4</UP></SUP><SUB><UP>p</UP></SUB>K<SUB><UP>M3 </UP></SUB><UP>/</UP>[<UP>M</UP>]</DE></FR><FENCE>1+<FR><NU>K<SUB><UP>M3</UP></SUB></NU><DE>[<UP>M</UP>])</DE></FR></FENCE><FENCE>1+<FR><NU>K<SUB><UP>MP</UP></SUB></NU><DE>[<UP>M</UP>])</DE></FR></FENCE> (Eq. 7)
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.


<AR><R><C>  E<UP>M<SUB>2</SUB></UP>  <LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB><UP>p</UP></SUB><SUP><UP>M3</UP></SUP></UL></LIM> E<UP>M<SUB>3</SUB>P
</UP></C></R><R><C><UP>K<SUB>M3</SUB>⥮              ⥮     P</UP> <LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB><UP>MP</UP></SUB></UL></LIM><UP> MP
</UP></C></R><R><C><UP>  EM<SUB>3</SUB></UP>  <LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB><UP>p</UP></SUB><SUP><UP>M4</UP></SUP></UL></LIM> E<UP>M<SUB>4</SUB>P</UP></C></R></AR>

Phosphate and metal ion binding to Y-PPase.

Scheme III.  

                              
View this table:
[in this window]
[in a new window]
 
Table I
Parameters of Scheme III for Pi binding and kcat/Km values in the presence of Mn2+

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<UP><SUB>p</SUB><SUP>M4</SUP></UP> = 0.041 µM, K<UP><SUB>p</SUB><SUP>M3</SUP></UP> infinity , 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).


View larger version (13K):
[in this window]
[in a new window]
 
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).


   n=<FR><NU>K<SUP><UP>M4</UP></SUP><SUB><UP>p</UP></SUB>K<SUB><UP>M2</UP></SUB>K<SUB><UP>M3</UP></SUB>+2K<SUP><UP>M4</UP></SUP><SUB><UP>p</UP></SUB>K<SUB><UP>M3</UP></SUB>[<UP>M</UP>]+3K<SUP><UP>M4</UP></SUP><SUB><UP>p</UP></SUB>[<UP>M</UP>]<SUP>2</SUP>+4[<UP>M</UP>]<SUP><UP>2</UP></SUP>[<UP>MP</UP>]</NU><DE>K<SUP><UP>M4</UP></SUP><SUB><UP>p</UP></SUB>K<SUB><UP>M2</UP></SUB>K<SUB><UP>M3</UP></SUB>+K<SUP><UP>M4</UP></SUP><SUB><UP>p</UP></SUB>K<SUB><UP>M3</UP></SUB>[<UP>M</UP>]+K<SUP><UP>M4</UP></SUP><SUB><UP>p</UP></SUB>[<UP>M</UP>]<SUP>2</SUP>+[<UP>M</UP>]<SUP><UP>2</UP></SUP>[<UP>MP</UP>]</DE></FR> (Eq. 8)

[<UP>M</UP>]=0.5<FENCE>[<UP>M</UP>]<SUB><UP>t</UP></SUB>−[<UP>P</UP>]<SUB><UP>t</UP></SUB>−K<SUB><UP>MP</UP></SUB>+<RAD><RCD>([<UP>M</UP>]<SUB><UP>t</UP></SUB>−[<UP>P</UP>]<SUB><UP>t</UP></SUB>−K<SUB><UP>MP</UP></SUB>)<SUP>2</SUP>+4K<SUB><UP>MP</UP></SUB>[<UP>M</UP>]<SUB><UP>t</UP></SUB></RCD></RAD></FENCE> (Eq. 9)

[<UP>MP</UP>]=[<UP>M</UP>]<SUB><UP>t</UP></SUB>−[<UP>M</UP>] (Eq. 10)

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).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Parameters of Scheme II measured in the presence of 0.5 mM Co2+

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.


View larger version (10K):
[in this window]
[in a new window]
 
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."

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.

                              
View this table:
[in this window]
[in a new window]
 
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.


View larger version (19K):
[in this window]
[in a new window]
 
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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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).
P<SUB><UP>c</UP></SUB>=<FR><NU>1</NU><DE>1+k<SUB>7</SUB>(k<SUB>4</SUB>+k<SUB>5</SUB>)/(k<SUB>4</SUB>k<SUB>6</SUB>[<UP>P</UP>]<SUB><UP>t</UP></SUB>)</DE></FR> (Eq. 11)
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.

Dagger Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Kornberg, A. (1962) in Horizons in Biochemistry (Kasha, M. , and Pullman, B., eds) , p. 251, Academic Press, New York
2. Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947[CrossRef][Medline] [Order article via Infotrieve]
3. Gonzalez, M. A., Webb, M. R., Welsh, K. M., and Cooperman, B. S. (1984) Biochemistry 23, 797-801[Medline] [Order article via Infotrieve]
4. Cooperman, B. S., Baykov, A. A., and Lahti, R. (1992) Trends Biochem. Sci. 17, 262-266[CrossRef][Medline] [Order article via Infotrieve]
5. Baykov, A. A., Cooperman, B. S., Goldman, A., and Lahti, R. (1999) Progr. Mol. Subcell. Biochem. 23, 127-150
6. Heikinheimo, P., Lehtonen, J., Baykov, A., Lahti, R., Cooperman, B., and Goldman, A. (1996) Structure 4, 1491-1508[Medline] [Order article via Infotrieve]
7. Harutyunyan, E. H., Kuranova, I. P., Vainshtein, B. K., Höhne, W. E., Lamzin, V. S., Dauter, Z., Teplyakov, A. V., and Wilson, K. S. (1996) Eur. J. Biochem. 239, 220-228[Abstract]
8. Heikinheimo, P., Tuominen, V., Ahonen, A.-K., Teplyakov, A., Cooperman, B. S., Baykov, A. A., Lahti, R., and Goldman, A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3121-3126[Abstract/Free Full Text]
9. Springs, B., Welsh, K. M., and Cooperman, B. S. (1981) Biochemistry 20, 6384-6391[Medline] [Order article via Infotrieve]
10. Harutyunyan, E. H., Oganessyan, V. Y., Oganessyan, N. N., Avaeva, S. M., Nazarova, T. I., Vorobyeva, N. N., Kurilova, S. A., Huber, R., and Mather, T. (1997) Biochemistry 36, 7754-7760[CrossRef][Medline] [Order article via Infotrieve]
11. Teplyakov, A., Obmolova, G., Wilson, K. S., Ishii, K., Kaji, H., Samejima, T., and Kuranova, I. (1994) Protein Sci. 3, 1098-1107[Abstract/Free Full Text]
12. Avaeva, S., Kurilova, S., Nazarova, T., Rodina, E., Vorobyeva, N., Sklyankina, V., Grigorjeva, O., Harutyunyan, E., Oganessyan, V., Wilson, K., Dauter, Z., Huber, R., and Mather, T. (1997) FEBS Lett. 410, 502-508[CrossRef][Medline] [Order article via Infotrieve]
13. Cooperman, B. S., Panackal, A., Springs, B., and Hamm, D. J. (1981) Biochemistry 20, 6051-6060[Medline] [Order article via Infotrieve]
14. Baykov, A. A., Shestakov, A. S., Pavlov, A. R., Smirnova, I. N., Larionov, V. N., and Avaeva, S. M. (1989) Biokhimiya 54, 796-803
15. Kasho, V. N., and Baykov, A. A. (1989) Biochem. Biophys. Res. Commun. 161, 475-480[Medline] [Order article via Infotrieve]
16. Smirnova, I. N., Shestakov, A. S., Dubnova, E. B., and Baykov, A. A. (1989) Eur. J. Biochem. 182, 451-456[Abstract]
17. Dixon, M., and Webb, E. C. (1979) Enzymes , pp. 144-145, Longman Group Ltd., London
18. Heikinheimo, P., Pohjanjoki, P., Helminen, A., Tasanen, M., Cooperman, B. S., Goldman, A., Baykov, A. A., and Lahti, R. (1996) Eur. J. Biochem. 239, 138-143[Abstract]
19. Cohen, S., Sterner, R., Keim, P., and Heinrikson, R. (1978) J. Biol. Chem. 253, 889-893[Abstract]
20. Kunitz, M. (1952) J. Gen. Physiol. 35, 423-450[Free Full Text]
21. Berger, L. (1956) Biochim. Biophys. Acta 20, 23-26[Medline] [Order article via Infotrieve]
22. Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M. (1986) Data for Biochemical Research , Clarendon Press, Oxford
23. Hackney, D. D., Stempel, K. E., and Boyer, P. D. (1980) Methods Enzymol. 64, 60-83[Medline] [Order article via Infotrieve]
24. Vanni, A., and Gastaldi, D. (1986) Ann. Chim. 76, 375-385
25. Fabrichniy, I. P., Kasho, V. N., Hyytiä, T., Salminen, T., Halonen, P., Dudarenkov, V. Yu., Heikinheimo, P., Chernyak, V. Ya., Goldman, A., Lahti, R., Cooperman, B. S., and Baykov, A. A. (1997) Biochemistry 36, 7746-7753[CrossRef][Medline] [Order article via Infotrieve]
26. Baykov, A. A., Hyytiä, T., Volk, S. E., Kasho, V. N., Vener, A. V., Goldman, A., Lahti, R., and Cooperman, B. S. (1996) Biochemistry 35, 4655-4661[CrossRef][Medline] [Order article via Infotrieve]
27. Goloshchapov, M. V., Martynenko, B. V., and Filatova, T. N. (1966) Inorg. Chem. (Russian) 11, 935-937
28. Chukhlantsev, V. G., and Alyamovskaya, K. V. (1961) Izv. Vyssh. Uchebn. Zaved. SSSR Khim. Khim. Technol. 5, 707-709
29. Rowatt, E., and Williams, R. J. P. (1989) Biochem. J. 259, 295-298[Medline] [Order article via Infotrieve]
30. Schlesinger, M. J., and Coon, M. J. (1960) Biochim. Biophys. Acta 41, 30-36[Medline] [Order article via Infotrieve]
31. Hackney, D. D. (1980) J. Biol. Chem. 255, 5320-5328[Abstract/Free Full Text]
32. Baykov, A. A., and Shestakov, A. S. (1992) Eur. J. Biochem. 206, 463-470[Abstract]
33. Belogurov, G. A., Fabrichniy, I. P., Pohjanjoki, P., Kasho, V. N., Lehtihuhta, E., Turkina, M. V., Cooperman, B. S., Goldman, A., Baykov, A. A., and Lahti, R. (2000) Biochemistry 39, 13931-13938[CrossRef][Medline] [Order article via Infotrieve]
34. Knight, W. B., Dunaway-Mariano, D., Ransom, S. C., and Villafranca, J. J. (1984) J. Biol. Chem. 259, 2886-2895[Abstract/Free Full Text]
35. Cohn, M. (1958) J. Biol. Chem. 230, 369-379[Free Full Text]
36. Janson, C. A., Degani, C., and Boyer, P. D. (1979) J. Biol. Chem. 254, 3743-3749[Abstract]
37. Welsh, K. M., Jacobyansky, A., Springs, B., and Cooperman, B. S. (1983) Biochemistry 22, 2243-2248[Medline] [Order article via Infotrieve]
38. Tuominen, V., Heikinheimo, P., Kajander, T., Torkkel, T., Hyytiä, T., Käpylä, J., Lahti, R., Cooperman, B. S., and Goldman, A. (1998) J. Mol. Biol. 284, 1565-1580[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.



This Article
Abstract
Full Text (PDF)
All Versions of this Article:
276/21/17629    most recent
M100343200v1
Purchase Article
View Shopping Cart
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Copyright Permissions
Google Scholar
Articles by Zyryanov, A. B.
Articles by Baykov, A. A.
Articles citing this Article
PubMed
PubMed Citation
Articles by Zyryanov, A. B.
Articles by Baykov, A. A.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   Biochemistry and Molecular Biology Education 
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.