J Biol Chem, Vol. 274, Issue 6, 3294-3299, February 5, 1999

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

Irina S. Efimova, Anu Salminen^§, Pekka Pohjanjoki^§, Jukka Lapinniemi^§, Natalia N. Magretova, Barry S. Cooperman^¶, Adrian Goldman^§, Reijo Lahti^§**, and Alexander A. Baykov^**

From the  A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia, the ^§ Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland, the ^¶ Department of Chemistry, University of Pennsylvania, Pennsylvania 19104-6323, and the  Centre for Biotechnology, University of Turku and Åbo Akademi University, FIN-20251 Turku, Finland

	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 PP_i to P_i 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.

View larger version (54K): [in this window] [in a new window]   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(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 A₂₈₀^1% of 11.8 (29).

Methods

Initial rates of PP_i 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 Mg₂PP_i, 20 mM Mg²⁺, 0.05 M Tris/HCl (pH 7.2), and 40 µM EGTA. The reaction was initiated by adding a suitable aliquot of enzyme solution and carried out for 3-4 min at 25 °C. No appreciable interconversion between the hexamer and less active trimer was observed during the assay, as evidenced by nearly linear product formation curves.

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

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

Calculations and Data Analysis

Hexamer-Trimer Equilibration-- Equations 1 and 2, derived from Scheme I, describe time courses of activity (A) resulting from hexamer (E₆) dissociation into trimers (E₃) and the reverse reaction, as well as the equilibrium activity (at t = , d_H/dt = 0) as functions of enzyme concentration. A_H and A_T are specific activities of the hexamer and trimer, respectively, _H is the fraction of enzyme in hexameric form at time t, [E]_t is total enzyme concentration, expressed in monomers, k_a and k_d 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).

(Eq. 1)

(Eq. 2)

The pH dependence of PPase activity at trimer-hexamer equilibrium is described by Equations 1 and 2 with d_H/dt = 0 and the apparent K_d = k_d/k_a given by Equation 3, where K_d ' is the pH-independent dissociation constant for the hexamer, K_a is the microscopic dissociation constant for H⁺ binding to the trimer, and m is the number of protons bound per hexamer.

(Eq. 3)

The effect of Mg²⁺ on k_a 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 k_a,0 for trimers without metal ions and increases by the same factor p with each added metal ion. Here  = 1/(1 + K_in'/[M]) is the probability of finding a metal ion (M) in one of the three metal ion binding sites/trimer, and K_in' 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).

(Eq. 4)

View larger version (18K): [in this window] [in a new window]   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 (K_M1 and K_M2) and for Mg²⁺ binding to the subunit interface site (K_in) (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 (K_in = ). Scheme II assumes that the binding reactions in the active site and in the intertrimer site are mutually independent.

(Eq. 5)

Spectral Titration-- The dependence of A₂₄₅, the change in protein absorbance at 245 nm, on Mg²⁺ concentration was fitted with Equation 6, where is the change in the extinction coefficient at saturating [M]. Equation 6 implies K_M2 K_M1.

(Eq. 6)

Mg₂PP_i Hydrolysis-- Values of the apparent catalytic constant k_h and the apparent Michaelis constant K_m,h were obtained from rate versus [Mg₂PP_i] dependences measured at fixed Mg²⁺ concentrations. Values of k_h/K_m,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 k_h greatly exceeds the rate constant for Mg₂PP_i release from the enzyme, as has been shown elsewhere for wild type E-PPase (33). The values of K_M1, K_M2, and K_in used in this fitting were those estimated by equilibrium dialysis (see above).

(Eq. 7)

The concentrations of free Mg²⁺ and Mg₂PP_i at pH 7.2 were calculated using the dissociation constants of 0.112 and 2.84 mM for the MgPP_i and Mg₂PP_i 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, s_20,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. s_20,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 s_20,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 s_20,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 s_20,w values compared with WT-PPase.

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 K_m,h (10³-10⁴-fold), with little change in k_h. 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 s_20,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 s_20,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.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Specific activity of wild type and variant PPases preincubated at different pH values. The enzyme (10 µM) was incubated at the pH indicated for 2 h (WT-, D26S-, and N24D-PPases) or 2-72 h (D26N-PPase) at 0 °C in a medium containing 0.1 M buffer, 1 mM Mg2+, and 40 µM EGTA, and its residual activity was assayed at pH 7.2, 25 °C. The buffers were sodium citrate (pH 3.9), MES/NaOH (pH 4.8-6.8), or Tris/HCl (pH 7.2-8.5). The lines are drawn to Equations 1-3 with m = 6 (, WT-PPase), m = 12 (, N24D-PPase), or m = 2 (, D26N-PPase; , D26S-PPase).

View larger version (25K): [in this window] [in a new window]   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 s1, AT = 9.5 s1, t = .

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 pK_a 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), K_d' for WT-PPase is < 1 nM, allowing a lower limit estimate of pK_a of 6.7.

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

View larger version (19K): [in this window] [in a new window]   Fig. 5.   The rate constant for wild type and variant PPase inactivation at pH 5.0 as a function of Mg2+ concentration. The enzyme stored at pH 7.2 at a concentration of 125 µM was diluted to 1 µM with 0.1 M MES/NaOH buffer (pH 5.0, 0 ° C) containing varying amounts of MgCl2, and the time course of PPase activity was measured. Values of kd were estimated with Equations 1 and 2. , WT-PPase; , N24D-PPase; , D26N-PPase; , D26S-PPase.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   The rate constant for wild type and variant PPase reactivation at pH 7.2 as a function of Mg2+ concentration. The enzyme inactivated at pH 5.0 (see Fig. 3) was diluted to 0.4 µM with 0.1 M Tris/HCl buffer (pH 7.2, 25 ° C) containing varying amounts of Mg2+, and the time course of PPase activity was measured. Values of ka were estimated with Equations 1 and 2. The lines are drawn to Equation 4. , WT-PPase; , N24D-PPase; , D26S-PPase.

Hence, Mg²⁺ ions stabilize the hexamer by changing both k_d and k_a. 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 k_d in the absence of Mg²⁺ was somewhat increased by the addition of Mg²⁺ (Fig. 5), most likely because of nonspecific binding. Similarly, k_a 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 M_in 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 M_in upon Asp²⁶ substitution and also suggest that M_in 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 M_in 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 M_in have similar affinity for Mg²⁺, whereas in WT-PPase the order in which the three sites become occupied at increasing Mg²⁺ concentration is M1, M_in, M2 (Table III).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Mg2+ binding to E-PPase and its variants as measured by equilibrium dialysis. Data for WT-PPase are from Velichko et al. (36). Lines are drawn to Equation 5, using parameter values found in Table III (Kin was set to infinity for the D26N and D26S variants); n measures the number of Mg2+ ions bound per monomer. Experimental conditions: 83 mM TES/KOH (pH 7.2), 17 mM KCl, 50 µM EGTA, 0.35-0.70 mM [PPase], 0.02-3 mM [Mg2+]. , WT-PPase; , N24D-PPase; , D26N-PPase; , D26S-PPase.

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 PP_i Hydrolysis-- As measured by k_h, k_h/K_m,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 k_h and k_h/K_m,h (Fig. 8) were generally quite similar for WT, N24D-, D26N-, and D26S-PPases, with the exception that k_h/K_m,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.

View larger version (20K): [in this window] [in a new window]   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 s1 and Ka = 0.26 mM for WT-PPase. The lines in the lower graph are drawn to Equation 7, using parameter values found in Table IV. For clarity, the lines for D26S-PPase and D26N-PPase are shown as broken and dotted lines, respectively. Experimental conditions: 87 mM TES/KOH (pH 7.2), 17 mM KCl, 50 µM EGTA. , WT-PPase; , N24D-PPase; , D26N-PPase; , D26S-PPase.

	DISCUSSION

Equilibrium dialysis measurements have revealed three types of metal binding sites in E-PPase (33), which we now designate as sites M1, M_in, 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 M_in (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 M_in 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 k_a 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 pK_a 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 pK_a 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; PP_i, 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.

	REFERENCES

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