Directed Mutagenesis Studies of the Metal Binding Site at the Subunit Interface of Escherichia coli Inorganic Pyrophosphatase*

Irina S. EfimovaDagger , Anu Salminen§, Pekka Pohjanjoki§, Jukka Lapinniemi§, Natalia N. MagretovaDagger , Barry S. Cooperman, Adrian Goldman§parallel , Reijo Lahti§**, and Alexander A. BaykovDagger **

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-20014 Turku, Finland, the  Department of Chemistry, University of Pennsylvania, Pennsylvania 19104-6323, and the parallel  Centre for Biotechnology, University of Turku and Åbo Akademi University, FIN-20251 Turku, Finland

    ABSTRACT
Top
Abstract
Introduction
References

Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase) have identified three Mg2+ ions/enzyme hexamer in water-filled cavities formed by Asn24, Ala25, and Asp26 at the trimer-trimer interface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium. Mg2+ markedly decelerates the dissociation of enzyme hexamer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active site. It is concluded that the metal ion binding site found at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg2+ greatly stabilizes the enzyme hexamer but has little effect on catalysis.

    INTRODUCTION
Top
Abstract
Introduction
References

Inorganic pyrophosphatase (EC 3.6.1.1; PPase1) belongs to a group of enzymes catalyzing phosphoryl transfer from phosphoric acid anhydrides to water, a principal reaction of cellular energetics. Soluble PPases hydrolyze PPi to Pi with release of energy as heat and provide in this way a thermodynamic pull for many biosynthetic reactions (1). PPases are essential in bacteria (2), yeast (3), and plants (4).

Escherichia coli PPase (E-PPase) is homohexameric, like many other prokaryotic PPases (5-8). The active site, present in each 20-kDa monomeric unit, contains 14 polar amino acid residues that are completely conserved in all known soluble PPases, despite only moderate sequence similarity in the rest of the molecule (9). The overall folding motifs are very similar in E-PPase (10, 11) and Thermus thermophilus PPase (8) as well as in the core part of the larger (32 kDa per subunit) PPase of Saccharomyces cerevisiae (12, 13).

The E-PPase hexamer is arranged as a dimer of trimers (11, 14-16). The principal trimer-trimer interaction involves a three-center ionic, hydrogen-bonding interaction among the residues His140-Asp143-His136' (11, 14). Another important trimer-trimer interaction occurs among Asn24, Ala25, and Asp26 (Fig. 1). The cavity formed by Asn24-Asp26 and Asn24'-Asp26' contains several water molecules one of which is replaced by Mg2+ when the crystals are soaked in a decimolar concentration of a magnesium salt (15, 17). As a result of the Mg2+ binding, the side chains of the Asn24 residues reorient, and the trimers move toward each other by about 0.4 Å.


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Fig. 1.   Part of the trimer-trimer interface in E-PPase, containing an Mg2+ ion (black sphere) surrounded by six water molecules (15). Hydrogen bonds are shown as lines. The two monomers that contribute to the interface are distinguished by the primed and unprimed labels.

Metal ions, including Mg2+, are rare but not uncommon at subunit interfaces (18-21). Brinen et al. (22) were able to engineer a metal binding site at the interface formed by trypsin and its protein inhibitor ecotin. However, in all known instances the intersubunit metal ion is coordinated directly to protein residues except for E-PPase where it is connected through water molecules (15, 17). On the other hand, water-filled cavities often occur at protein interfaces (23) and in protein interiors (24). Because even large molecules can rapidly penetrate deep into proteins and bind to seemingly inaccessible cavities (25), it is not unlikely that, in vivo, protein cavities can contain metal ions, especially Mg2+, the most abundant divalent cation in the cell. Understanding the effects of these cavities on protein properties may therefore be important in understanding how proteins function in vivo as well as in designing interacting proteins and ligands that can disrupt protein-protein interactions.

The present work addresses two major questions: (a) Does the intertrimer cavity in E-PPase bind Mg2+ strongly enough to be occupied appreciably at its physiological concentration? (b) What is the role of this unusual binding site in hexamer stability and catalytic activity?

    EXPERIMENTAL PROCEDURES

Enzymes

Wild type and wild type-free variant PPases were expressed using the overproducing E. coli strains HB101 (26) and MC1061/YPPAI(Delta ppa) (27), respectively. The enzymes were purified by chromatography on Fast Flow DEAE-Sepharose (Amersham Pharmacia Biotech), heat treatment at 65 °C, and gel filtration on a column (2.6 × 60 cm) of Superdex 200 (Amersham Pharmacia Biotech) (27). The final preparations were homogeneous according to SDS and native polyacrylamide gel electrophoresis and sedimentation velocity measurements. The enzyme concentration was estimated on the basis of a subunit molecular mass of 20 kDa (28) and an A2801% of 11.8 (29).

Methods

Initial rates of PPi hydrolysis were estimated from continuous recordings of phosphate production obtained with an automatic phosphate analyzer (30). The assay medium of 25 ml total volume contained, except as noted, 20 µM Mg2PPi, 20 mM Mg2+, 0.05 M Tris/HCl (pH 7.2), and 40 µM EGTA. The reaction was initiated by adding a suitable aliquot of enzyme solution and carried out for 3-4 min at 25 °C. No appreciable interconversion between the hexamer and less active trimer was observed during the assay, as evidenced by nearly linear product formation curves.

Differential spectra of PPase induced by Mg2+ were recorded with a computer-controlled LKB Ultrospec Plus spectrophotometer in a 1-cm cuvette containing 0.7 ml of enzyme solution. After the base line stabilized, stock MgCl2 solution was added in 0.7-µl increments, and the spectra were recorded.

Equilibrium microdialysis (31) and analytical ultracentrifugation (32) were performed as described.

Calculations and Data Analysis

Hexamer-Trimer Equilibration-- Equations 1 and 2, derived from Scheme I, describe time courses of activity (A) resulting from hexamer (E6) dissociation into trimers (E3) and the reverse reaction, as well as the equilibrium activity (at t = infinity , dalpha H/dt = 0) as functions of enzyme concentration. AH and AT are specific activities of the hexamer and trimer, respectively, alpha H is the fraction of enzyme in hexameric form at time t, [E]t is total enzyme concentration, expressed in monomers, ka and kd are the apparent rate constants for hexamer formation and breakdown, respectively, measured at fixed H+ and Mg2+ concentrations. Equations 1 and 2 were fit to data simultaneously with SCIENTIST (MicroMath).
2E<SUB>3</SUB> <LIM><OP><ARROW>⇄</ARROW></OP><LL>k<SUB>d</SUB></LL><UL>k<SUB>a</SUB></UL></LIM> E<SUB>6</SUB>
<UP><SC>Scheme</SC> I. Hexamer-trimer equilibration</UP>
A=A<SUB><UP>T</UP></SUB>+(A<SUB><UP>H</UP></SUB>−A<SUB><UP>T</UP></SUB>)&agr;<SUB><UP>H</UP></SUB> (Eq. 1)
<FR><NU>d&agr;<SUB><UP>H</UP></SUB></NU><DE>dt</DE></FR>=<FR><NU>2</NU><DE>3</DE></FR>k<SUB>a</SUB>[E]<SUB>t</SUB>(1−&agr;<SUB><UP>H</UP></SUB>)<SUP>2</SUP>−k<SUB>d</SUB>&agr;<SUB><UP>H</UP></SUB> (Eq. 2)

The pH dependence of PPase activity at trimer-hexamer equilibrium is described by Equations 1 and 2 with dalpha H/dt = 0 and the apparent Kd = kd/ka given by Equation 3, where Kd ' is the pH-independent dissociation constant for the hexamer, Ka is the microscopic dissociation constant for H+ binding to the trimer, and m is the number of protons bound per hexamer.
K<SUB>d</SUB>=K<SUB>d</SUB>′<FENCE>1+<FR><NU>[<UP>H<SUP>+</SUP></UP>]</NU><DE>K<SUB>a</SUB></DE></FR></FENCE><SUP>m</SUP> (Eq. 3)

The effect of Mg2+ on ka at fixed pH is described by Equation 4, derived for the simplified model shown in Fig. 2. The model assumes that (a) a hexamer is formed from two trimers containing in total from zero to three metal ions bound at the trimer-trimer interface and (b) the association rate constant is equal to ka,0 for trimers without metal ions and increases by the same factor p with each added metal ion. Here gamma  = 1/(1 + Kin'/[M]) is the probability of finding a metal ion (M) in one of the three metal ion binding sites/trimer, and Kin' is the respective metal binding constant for the trimer. Statistical factors of 12 (6 + 6) and 4 (2 + 2) were used when more than one metal ion was present in two interacting trimers, taking into account that the combination of two metal-bound monomers within a single subunit-subunit interface is not allowed (Fig. 2).
k<SUB>a</SUB>=k<SUB>a,0</SUB>[(1−&ggr;)<SUP>6</SUP>+6p&ggr;(1−&ggr;)<SUP>5</SUP>+12p<SUP>2</SUP>&ggr;<SUP>2</SUP>(1−&ggr;)<SUP>4</SUP>+4p<SUP>3</SUP>&ggr;<SUP>3</SUP>(1−&ggr;)<SUP>3</SUP>] (Eq. 4)


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Fig. 2.   Various modes of hexamer formation from trimers in the presence of Mg2+. Monomers with Mg2+ bound at the interface half-site are shaded. The respective rate association constants are shown below. The numerical factors in rate constant expressions refer to statistical weights; for instance, the factor of 6 indicates that there are six similar combinations of two trimers with same distribution of metal ions among them.

Equilibrium Dialysis-- Values of the dissociation constants for the successive binding of two Mg2+ ions to the E-PPase active site (KM1 and KM2) and for Mg2+ binding to the subunit interface site (Kin) (Scheme II) were estimated by fitting Equation 5 to measured values of n, the number of Mg2+ ions bound per monomer, as a function of free Mg2+ concentration. The first term in Equation 5 describes binding to the active site, and the second binding to the intertrimer site. The second term was ignored for variants that exhibited no binding to the intertrimer site (Kin = infinity ). Scheme II assumes that the binding reactions in the active site and in the intertrimer site are mutually independent.
<AR><R><C></C><C></C><C><UP>M</UP><SUB>3</SUB>E<UP>PP</UP></C><C></C><C><UP>M</UP><SUB>4</SUB>E<UP>PP</UP></C></R><R><C></C><C></C><C>↑ k<SUB>1</SUB><SUP>(1)</SUP></C><C></C><C>↑ k<SUB>1</SUB><SUP>(2)</SUP></C></R><R><C>E</C><C><LIM><OP><ARROW>⇄</ARROW></OP><UL>K<SUB><UP>M</UP>1</SUB></UL></LIM></C><C><UP>M</UP>E</C><C><LIM><OP><ARROW>⇄</ARROW></OP><UL>K<SUB><UP>M</UP>2</SUB></UL></LIM></C><C><UP>M</UP><SUB>2</SUB>E</C></R><R><C>⇅ K<SUB><UP>in</UP></SUB></C><C></C><C>⇅ K<SUB><UP>in</UP></SUB></C><C></C><C>⇅ K<SUB><UP>in</UP></SUB></C></R><R><C>E(<UP>M</UP>)</C><C><LIM><OP><ARROW>⇄</ARROW></OP><UL>K<SUB><UP>M</UP>1</SUB></UL></LIM></C><C><UP>M</UP>E(<UP>M</UP>)</C><C><LIM><OP><ARROW>⇄</ARROW></OP><UL>K<SUB><UP>M</UP>2</SUB></UL></LIM></C><C><UP>M</UP><SUB>2</SUB>E(<UP>M</UP>)</C></R><R><C></C><C></C><C>↓ k<SUB>1</SUB><SUP>(3)</SUP></C><C></C><C>↓ k<SUB>1</SUB><SUP>(4)</SUP></C></R><R><C></C><C></C><C><UP>M</UP><SUB>3</SUB>E(<UP>M</UP>)<UP>PP</UP></C><C></C><C><UP>M</UP><SUB>4</SUB>E(<UP>M</UP>)<UP>PP</UP></C></R></AR>
<UP><SC>Scheme</SC> II. Metal and substrate binding to PPase</UP>
&eegr;=<FR><NU>K<SUB><UP>M</UP>2</SUB>[<UP>M</UP>]+2[<UP>M</UP>]<SUP>2</SUP></NU><DE>K<SUB><UP>M</UP>1</SUB>K<SUB><UP>M</UP>2</SUB>+K<SUB><UP>M</UP>2</SUB>[<UP>M</UP>]+[<UP>M</UP>]<SUP>2</SUP></DE></FR>+<FR><NU>0.5[<UP>M</UP>]</NU><DE>K<SUB><UP>in</UP></SUB>+[<UP>M</UP>]</DE></FR> (Eq. 5)

Spectral Titration-- The dependence of Delta A245, the change in protein absorbance at 245 nm, on Mg2+ concentration was fitted with Equation 6, where Delta epsilon is the change in the extinction coefficient at saturating [M]. Equation 6 implies KM2 >> KM1.
&Dgr;A<SUB>245</SUB>=<FR><NU>&Dgr;ϵ[E]<SUB>t</SUB>[<UP>M</UP>]</NU><DE>K<SUB><UP>M</UP>2</SUB>+[<UP>M</UP>]</DE></FR> (Eq. 6)

Mg2PPi Hydrolysis-- Values of the apparent catalytic constant kh and the apparent Michaelis constant Km,h were obtained from rate versus [Mg2PPi] dependences measured at fixed Mg2+ concentrations. Values of kh/Km,h as a function of [Mg2+] were fitted to Equation 7, allowing an estimation of k1(1), k1(2), k1(3), and k1(4), the rate constants for substrate binding to the ME, M2E, ME(M), and M2E(M) species (M in parentheses denotes the Mg2+ ion that is bound at the subunit interface), respectively (Scheme II). The assumption in deriving Equation 7 is that kh greatly exceeds the rate constant for Mg2PPi release from the enzyme, as has been shown elsewhere for wild type E-PPase (33). The values of KM1, KM2, and Kin used in this fitting were those estimated by equilibrium dialysis (see above).
 <FR><NU>k<SUB><UP>h</UP></SUB></NU><DE>K<SUB>m,<UP>h</UP></SUB></DE></FR>=<FR><NU>k<SUB>1</SUB><SUP>(1)</SUP>K<SUB><UP>M</UP>2</SUB>K<SUB><UP>in</UP></SUB>[<UP>M</UP>]+k<SUB>1</SUB><SUP>(2)</SUP>K<SUB><UP>in</UP></SUB>[<UP>M</UP>]<SUP>2</SUP>+k<SUB>1</SUB><SUP>(3)</SUP>K<SUB><UP>M</UP>2</SUB>[<UP>M</UP>]<SUP>2</SUP>+k<SUB>1</SUB><SUP>(4)</SUP>[<UP>M</UP>]<SUP>3</SUP></NU><DE>(K<SUB><UP>in</UP></SUB>+[<UP>M</UP>])(K<SUB><UP>M</UP>1</SUB>K<SUB><UP>M</UP>2</SUB>+K<SUB><UP>M</UP>2</SUB>[<UP>M</UP>]+[<UP>M</UP>]<SUP>2</SUP>)</DE></FR> (Eq. 7)

The concentrations of free Mg2+ and Mg2PPi at pH 7.2 were calculated using the dissociation constants of 0.112 and 2.84 mM for the MgPPi and Mg2PPi complexes, respectively (34).

    RESULTS

pH Effects on Quaternary Structure and Enzyme Activity-- The effects of substitutions at both Asn24 and Asp26 on quaternary structure and enzyme activity were studied as a function of pH. Direct evidence for shifts in the hexamer-trimer equilibrium was obtained from sedimentation data (Table I). At pH 7.2, s20,w for WT-PPase, and the three variants studied here (N24D, D26N, D26S) fell in the range 6.3-6.7 S, characteristic of a hexamer (28, 32). Lowering the pH to 5.0 had differential effects. s20,w decreased to 4.3 for both WT-PPase and N24D-PPase, decreased only to 5.2 for D26S-PPase, and was unchanged for D26N-PPase. Further lowering the pH to 3.8 led to s20,w values of 3.3-4.0 for all four PPases. Because the molecular mass of WT-PPase at pH 5.0 estimated by sedimentation equilibrium measurements (20 °C, 20 µM initial enzyme concentration) was 63 ± 1 kDa, these results indicate that WT-PPase and N24D-PPase are fully in the trimeric form by pH 5.0 but that lower pH is required for full conversion to trimer of the D26S and D26N variants. That such dissociation is reversible is shown by the increase in s20,w back to 6.7 ± 0.2 S when the pH of a WT-PPase sample is raised from 5.0 to 7.2. At pH 3.8, the variant PPases may undergo further dissociation to dimers or monomers as suggested by lower s20,w values compared with WT-PPase.

                              
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Table I
Sedimentation coefficients
Conditions were 2-4 °C, 10 µM enzyme, 48,000 rpm. The buffer (0.1 M citric acid, NaOH, pH 3.8; 0.1 M MES/NaOH, pH 5.0; 0.1 M Tris/HCl, pH 7.2) contained 1 mM MgCl2 and 40 µM EGTA.

Earlier we had shown for other variants with weakened trimer-trimer interaction (32) that trimers had lower activity than hexamers at low (20 µM) substrate concentration as a result of a drastic increase of Km,h (103-104-fold), with little change in kh. Trimers of WT-PPase and the three variants investigated in this paper also had lower activity, and studies of the effect of pH on enzyme activity, paralleling those on s20,w, demonstrated conclusively that rates and equilibria of hexamer-trimer interconversion could be monitored by activity measurements. The activities presented in Fig. 3 represent equilibrium values, no changes being observed on longer incubation at the final pH values. Matching the s20,w values, they demonstrate that as pH is lowered from 7.2 to 5.0, WT- and N24D-PPase are fully converted to less active forms, D26S-PPase is partially converted, whereas D26N-PPase retains almost full activity, and, in addition, lowering the pH further to 3.8 converts the latter two variants virtually completely to their lower activity forms. Furthermore, WT-PPase activity increases with enzyme concentration at pH 5.0 (Fig. 4, inset), as expected for an equilibrium between an active hexamer and less active trimer, and activity is a fully reversible property of pH (Fig. 4). The N24D, D26S, and D26N variants, inactivated at pH 5.0, 4.8, and 3.9, respectively, could also be reactivated at pH 7.2, affording > 80% of original activity. Finally, in all cases, inactivation and reactivation followed strict first- and second-order kinetics, respectively.


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Fig. 3.   Specific activity of wild type and variant PPases preincubated at different pH values. The enzyme (10 µM) was incubated at the pH indicated for 2 h (WT-, D26S-, and N24D-PPases) or 2-72 h (D26N-PPase) at 0 °C in a medium containing 0.1 M buffer, 1 mM Mg2+, and 40 µM EGTA, and its residual activity was assayed at pH 7.2, 25 °C. The buffers were sodium citrate (pH 3.9), MES/NaOH (pH 4.8-6.8), or Tris/HCl (pH 7.2-8.5). The lines are drawn to Equations 1-3 with m = 6 (bullet , WT-PPase), m = 12 (open circle , N24D-PPase), or m = 2 (, D26N-PPase; triangle , D26S-PPase).


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Fig. 4.   Reversibility of WT-PPase inactivation at pH 5.0. Stock enzyme solution (800 µM) preequilibrated at pH 8.0, 25 °C, was diluted to 20 µM with 0.1 M MES/NaOH buffer (pH 5.0, measured at 0 °C) and incubated at 0 °C for 2 h, after which the pH was raised to 7.2 with 1 M Tris, and the incubation was continued. Aliquots were withdrawn as a function of time, and PPase activity was assayed at pH 7.2, 25 °C. The lines are drawn to Equations 1 and 2. Inset, the specific activity of WT-PPase preequilibrated at pH 5.0 as a function of enzyme concentration. The line is drawn to Equations 1 and 2 with kd/ka = 170 µM, AH = 135 s-1, AT = 9.5 s-1, t = infinity .

The data in Fig. 3 could be fit quantitatively to a model (Equations 1-3) in which deprotonation of a group within trimers (which has a much lower pKa in a hexamer) is required for hexamer formation. Satisfactory fits were obtained when the number of bound protons per hexamer (m) was equal to 6, 2, 2, or 12 for WT-, D26N-, D26S-, and N24D-PPase, respectively. From earlier work (35), Kd' for WT-PPase is < 1 nM, allowing a lower limit estimate of pKa of 6.7.

Mg2+ Effects on Rates and Equilibrium of Hexamer left-right-arrow  Trimer Transition-- Mg2+ has been shown to stabilize the hexameric structure of variant E-PPases with weakened trimer-trimer interactions (32, 35). As estimated from time courses of activity loss at pH 5.0 and restoration at pH 7.2, added Mg2+ markedly decreased kd (Fig. 5) and increased ka (Fig. 6) for WT- and N24D-PPases. The sigmoidal dependence of ka on [Mg2+] was described satisfactorily by Equation 4, yielding fitted values of Kin', ka,0, and p (Table II) which were similar for both PPases. The Kin ' values may reflect the dissociation constant from a half-of-interface site (for WT-PPase, residues 24-26 from one subunit).


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Fig. 5.   The rate constant for wild type and variant PPase inactivation at pH 5.0 as a function of Mg2+ concentration. The enzyme stored at pH 7.2 at a concentration of 125 µM was diluted to 1 µM with 0.1 M MES/NaOH buffer (pH 5.0, 0 ° C) containing varying amounts of MgCl2, and the time course of PPase activity was measured. Values of kd were estimated with Equations 1 and 2. bullet , WT-PPase; open circle , N24D-PPase; black-square, D26N-PPase; triangle , D26S-PPase.


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Fig. 6.   The rate constant for wild type and variant PPase reactivation at pH 7.2 as a function of Mg2+ concentration. The enzyme inactivated at pH 5.0 (see Fig. 3) was diluted to 0.4 µM with 0.1 M Tris/HCl buffer (pH 7.2, 25 ° C) containing varying amounts of Mg2+, and the time course of PPase activity was measured. Values of ka were estimated with Equations 1 and 2. The lines are drawn to Equation 4. bullet , WT-PPase; open circle , N24D-PPase; triangle , D26S-PPase.

                              
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Table II
Parameters describing Mg2+ effects on trimer reactivation at pH 7.2, 25 °CM, (0.1 Tris/HCl, 40 µM EGTA).

Hence, Mg2+ ions stabilize the hexamer by changing both kd and ka. In full agreement with these data, the activity versus pH profile for WT-PPase (Fig. 3) shifted to lower pH values at higher Mg2+ concentrations, as inferred from the observation that WT-PPase retained 80% activity at pH 5 in the presence of 140 mM MgCl2.

In contrast, for D26S- and D26N-PPase, the already low value of kd in the absence of Mg2+ was somewhat increased by the addition of Mg2+ (Fig. 5), most likely because of nonspecific binding. Similarly, ka for D26S-PPase in the absence of Mg2+ is 5-10 times larger than the corresponding values for WT- and N24D-PPases and rises only slightly as [Mg2+] is raised (Fig. 6).

Mg2+ Binding to Hexameric Variant PPases-- Mg2+ binding to WT- and the variant E-PPases was measured by equilibrium dialysis, providing the macroscopic binding constants for all three types of the metal binding sites present in E-PPase, M1 and M2 at the active site and Min at the trimer-trimer interface, and by differential spectroscopy (16), which, as shown recently,2 provides the binding constant for M2.

The dialysis data indicated a decrease in the stoichiometry of tightly binding sites by approximately 0.5 mol/mol monomer in the D26S and D26N variants, but not in the N24D variant, when compared with WT-PPase (Fig. 7). These data are thus consistent with a loss of Min upon Asp26 substitution and also suggest that Min displays high affinity for Mg2+. These conclusions were supported by quantitative analysis of the binding data. The binding affinity of M1 was only slightly affected by the mutations, whereas the affinity of Min was increased 3-fold after N24D substitution, which introduced a negatively charged ligand into the intertrimer cavity. Accordingly, in the N24D variant sites M1 and Min have similar affinity for Mg2+, whereas in WT-PPase the order in which the three sites become occupied at increasing Mg2+ concentration is M1, Min, M2 (Table III).


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Fig. 7.   Mg2+ binding to E-PPase and its variants as measured by equilibrium dialysis. Data for WT-PPase are from Velichko et al. (36). Lines are drawn to Equation 5, using parameter values found in Table III (Kin was set to infinity for the D26N and D26S variants); n measures the number of Mg2+ ions bound per monomer. Experimental conditions: 83 mM TES/KOH (pH 7.2), 17 mM KCl, 50 µM EGTA, 0.35-0.70 mM [PPase], 0.02-3 mM [Mg2+]. bullet , WT-PPase; open circle , N24D-PPase; , D26N-PPase; triangle , D26S-PPase.

                              
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Table III
Dissociation constants for Mg2+ binding

As measured by spectral titrations conducted at pH 8.5 (the effect of Mg2+ binding on absorbance was quite small at pH 7.2 used in the dialysis experiments; see Ref. 16) the affinity at site M2 appeared unaffected by the substitutions (Table III). Moreover, the values obtained for the three variants are essentially identical to those measured by dialysis at pH 7.2. The one apparent inconsistency in the data concerns the considerably higher value obtained by dialysis for WT-PPase at pH 7.2. However, the error in this value is quite high because it greatly exceeds the enzyme concentration used in these measurements.

Kinetics of PPi Hydrolysis-- As measured by kh, kh/Km,h, and k1, substitution at the subunit interface has only minor effects on PPase catalytic function determined at pH 7.2 as a function of [Mg2+]. Values of kh and kh/Km,h (Fig. 8) were generally quite similar for WT, N24D-, D26N-, and D26S-PPases, with the exception that kh/Km,h was somewhat lower for N24D-PPase. Values of k1 were also determined as a function of [Mg2+] and interpreted according to Scheme II (Table IV) . For WT-PPase and N24D-PPase, binding to the interface site has at most a small effect on k1 (i.e. k1(1) is approximately equal to k1(3)), but binding to M2 lowers k1 (k1(3) > k1(4)). Consistent with these results, the two variants lacking a high affinity Mg2+ interface site, D26N- and D26S-PPases, have k1(1) values similar to that for WT-PPase, and their k1(2) values (reflecting M2 site occupancy) are both lower than their k1(1) values and similar to k1(4) for WT-PPase It should be noted that k1(2) could not be estimated for WT- and N24D-PPase because the M2E species was stoichiometrically insignificant for these variants.


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Fig. 8.   Dependences of kh and kh/Km,h for Mg2PPi hydrolysis on Mg2+ concentration. The line in the upper graph shows the best fit to the equation kh = kh, lim[M]/(Ka + [M]) with kh,lim = 157 s-1 and Ka = 0.26 mM for WT-PPase. The lines in the lower graph are drawn to Equation 7, using parameter values found in Table IV. For clarity, the lines for D26S-PPase and D26N-PPase are shown as broken and dotted lines, respectively. Experimental conditions: 87 mM TES/KOH (pH 7.2), 17 mM KCl, 50 µM EGTA. bullet , WT-PPase; open circle , N24D-PPase; , D26N-PPase; triangle , D26S-PPase.

                              
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Table IV
Rate constants for substrate binding
Results were estimated with Equation 7, using the metal binding constants obtained by equilibrium dialysis (Table III); for WT-PPase, KM2 was also an adjustable parameter, whose best fit value was found to be 3.5 mM. Conditions were: 25 °C, pH 7.2 (83 mM TES/KOH, 17 mM KCl, 50 µM EGTA).


    DISCUSSION

Equilibrium dialysis measurements have revealed three types of metal binding sites in E-PPase (33), which we now designate as sites M1, Min, and M2, following the order in which they become occupied in WT-PPase at an increasing Mg2+ concentration at pH 7.2 (Table III). We assumed previously that the three binding sites are present in each subunit of homohexameric E-PPase (33). However, crystallographic identification of an Mg2+ ion between pairs of subunits (15, 17) together with the effects of Asn24 and Asp26 mutations on metal binding and hexamer stability reported above suggest a binding stoichiometry of 0.5 mol/mol monomer for one of the sites detected by equilibrium dialysis. Two other lines of evidence support the idea that the site with intermediate affinity is located at the subunit interface. First, x-ray crystallographic data show that E-PPase crystals soaked in 140 mM MgCl2 contain Mg2+ only at sites M1 and Min (15), whereas crystals grown at 250 mM MgCl2 contain Mg2+ at all three sites (17). Also, the site that binds Mn2+ most tightly is located in the active site and exhibits a 1 mol/mol binding stoichiometry (11). Second, site Min is apparently absent in trimeric E-PPase prepared by substitution of both His136 and His140 in the trimer-trimer interface (36).

Our results clearly show that [H+] and [Mg2+] determine the quaternary structure of E-PPase and agree with earlier studies showing that (a) low pH induces PPase hexamer dissociation (6, 37) and (b) in variants with weakened trimer-trimer interactions (H136Q-PPase, 32; E20D-PPase, 35) added Mg2+ stabilizes hexamer formation at pH values >=  7.2. A simple model consistent with our results is that protonation of one or more sites per monomer induces dissociation (Fig. 3). According to this model, added Mg2+ favors the hexamer because it competes with H+ for a common site (or sites), in addition to stabilizing hexamer structure as such directly. Consistent with this model are (a) the increased stability of the D26S and D26N hexamers because substitution of Asp26 eliminates a potential protonation site, (b) the shift in activity versus pH profiles to lower pH values with increasing Mg2+ concentration, (c) the stability of hexamers at higher pH even in the absence of added Mg2+, and (d) the rise in ka for WT- and N24D-PPase with increasing [Mg2+] at pH 7.2, when the basic group(s) governing hexamer stability is(are) deprotonated (Fig. 6).

This model certainly suggests that Asp26 is at least one, and perhaps the major, basic group controlling hexamer stability in WT-PPase. This would require Asp26 to have a markedly elevated pKa of > 6.7, which would not be unexpected, given the extensive hydrogen bonding network at the subunit interface site (see Fig. 1). The value of m equal to 6 for WT-PPase suggests that all three Asp26 residues/trimer become protonated on subunit dissociation. Following this logic, it is tempting to speculate that the increase in m value to 12 for the N24D variant reflects protonation of the Asp24 and Asp26 residues in this variant. That m does not become 0 in the D26S and D26N variants would imply the presence of (an)other, lower pKa group(s) whose protonation also destabilizes the hexamer. One possibility is His140, as suggested previously (14, 32). Alternatively, other carboxyl side chains, as yet unidentified, could be involved.

It remains unclear why protonation of Asp26 would destabilize the hexamer, whereas mutation of Asp26 into Asn or Ser does not. The simplest possibility is that the hydrogen bonding network at the subunit interface site (Fig. 1) requires a hydrogen-bond acceptor at position 26, a role that can be filled by a carboxylate anion, by an amide, and by an alcohol, but not by a carboxylic acid. High resolution structures of the variants described in this paper could provide a clear test of the validity of our proposals.

In summary, we have shown that the metal binding site formed by protein-bound water molecules at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg2+ stabilizes the enzyme hexamer by neutralizing the negative charges and preventing protonation of a site or sites within the binding cavity, without having any substantial effect on catalysis.

    ACKNOWLEDGEMENTS

We thank P. V. Kalmykov for help in ultracentrifugation and Drs D. Bergen and V. A. Sklyankina for helpful suggestions.

    FOOTNOTES

* This work was supported by Russian Foundation for Basic Research Grants 97-04-48487 and 96-15-97969, Russian State Project Bioengineering/Enzyme Engineering Grant 1-42, Academy of Finland Grants 1444, 35736, and 4310, and National Institutes of Health Grants TW00407.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The first two authors contributed equally to this work.

** To whom correspondence should be addressed. Tel.: 358-2-333-6845; Fax: 358-2-333-6860; E-mail: reila{at}utu.fi or Tel.: 095-939-5541; Fax: 095-939-3181; E-mail: baykov{at}genebee.msu.su.

The abbreviations used are: PPase, inorganic pyrophosphatase; PPi, pyrophosphate; E-PPase, E. coli pyrophosphatase; WT, wild type; MES, 4-morpholineethanesulfonic acid; TES, 2-((2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino)ethanesulfonic acid; TAPS, 3-((2-hydroxy-1,1-bis (hydroxymethyl)ethyl)amino)-1-propanesulfonic acid.

2 A. A. Baykov, T. Hyytiä, M. V. Turkina, I. S. Efimova, V. N. Kasho, A. Goldman, B. S. Cooperman, and R. Lahti, manuscript in preparation.

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Abstract
Introduction
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