HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 18, 15465-15471, May 3, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/18/15465    most recent M200101200v1 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 Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Salminen, A. Articles by Lahti, R. Search for Related Content PubMed PubMed Citation Articles by Salminen, A. Articles by Lahti, R. Social Bookmarking                      What's this?

Modulation of Dimer Stability in Yeast Pyrophosphatase by Mutations at the Subunit Interface and Ligand Binding to the Active Site^*

Anu Salminen^§, Alexey N. Parfenyev^§¶, Krista Salli, Irina S. Efimova^¶, Natalia N. Magretova^¶, Adrian Goldman, Alexander A. Baykov^¶**, and Reijo Lahti

From the  Department of Biochemistry, University of Turku, FIN-20500 Turku, Finland, ^¶ A. N.Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia, and  Institute of Biotechnology, University of Helsinki, P. O. Box 56, FIN-00014 Helsinki, Finland

Received for publication, January 4, 2002, and in revised form, February 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 µM, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH  8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys⁸³), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg²⁺ cofactor, P_i product, and the PP_i analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k_cat remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys⁸³ residue in the dimer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inorganic pyrophosphatase (EC 3.6.1.1; PPase)¹ is an essential enzyme that catalyzes the interchange between pyrophosphate and phosphate (1, 2). Due to its relatively simple structure and high catalytic efficiency (k_cat/K_m = ~10⁹ M¹ s¹), PPase has become a paradigm for mechanistic and structural studies of enzymatic phosphoryl transfer from phosphoric acid anhydrides to water (3, 4). Two nonhomologous families of soluble PPase have been identified to date. Yeast (Saccharomyces cerevisiae) PPase (Y-PPase) is a member of family I, which is fairly widespread in all types of organisms (5). Family I PPases are homohexamers of ~20-kDa subunits in prokaryotes and homodimers of ~32-kDa subunits in eukaryotes with highly conserved active sites and mechanisms of action (3, 4). Family II PPases have been discovered more recently (6, 7). All the established and putative members of this family belong to bacteria but are homodimers of ~33-kDa subunits (8), in contrast to bacterial PPases of family I. Although family II members have yet to be characterized in detail, available data suggest that the active sites of family I and family II PPases are quite similar, presenting a remarkable example of convergent enzyme evolution (9, 10).

The extensively studied Y-PPase enzyme exists as a very tight dimer in a wide range of conditions. The active site and subunit interface are separated by about 5 Å (11-13) and do not share common amino acid residues. All intersubunit interactions involve side chain atoms (Fig. 1). Core intersubunit contact is formed by a three-layer stacking of the aromatic rings of Trp⁵², His⁸⁷, His^87', and Trp^52', with His⁸⁷ and His^87' forming the central layer (' represents residues of the second subunit). Trp²⁷⁹ and Trp^279' pack perpendicular and on the outside of this three-layer stack. Polar contacts between subunits include hydrogen bonds His⁸⁷His^87', Arg⁵¹ side chainAsp^277' main chain oxygen, and a symmetrical Arg^51'Asp²⁷⁷ interaction. The interface is essentially conserved in other fungal and animal PPases, except for a His⁸⁷ to Lys replacement in four of the nine known sequences (5). Active monomeric Y-PPase was previously obtained upon covalent immobilization on Sepharose beads, followed by denaturation with guanidine hydrochloride and renaturation of the protein (14).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Stereo view of the intersubunit contact in Y-PPase (13). One subunit is drawn in dark gray and the other is drawn in light gray. The hydrogen bond is represented by the dashed line. The Cys83 residues shown are outside the contact region.

Here we describe the effects of Arg⁵¹, Trp⁵², His⁸⁷, and Trp²⁷⁹ substitutions on the quaternary structure and activity of Y-PPase. Our results indicate that mutation of Trp⁵² has the most significant effect on dimerization and that active site ligands enhance dimer stability.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Enzymes-- The production and purification of wild-type and variant Y-PPase from overproducing Escherichia coli XL2blue^b strains transformed with suitable plasmids were performed as described by Heikinheimo et al. (15), except that the Stratagene QuikChange mutagenesis kit was used. Enzyme concentration was calculated on the basis of the subunit molecular mass of 32.0 kDa (16) and the specific absorbance A equal to 14.5 for wild-type PPase (17). Substitution of each Trp residue with Ser decreased A by 1.7 (18).

Methods-- The initial rates of PP_i hydrolysis were measured using a continuous P_i assay (19). The assay medium contained 6 µM PP_i, 20 mM Mg²⁺, 0.15 M Tris/HCl, pH 7.2, and 40 µM EGTA, except where specified. The reaction was initiated by adding enzyme and continued for 3-4 min at 25 °C. Polyacrylamide gel electrophoresis under nondenaturing conditions was done with a 12.5% gel in a Sigma-Aldrich vertical electrophoresis unit. The gel buffer and the running buffer were 25 mM Tris-glycine, pH 9.3, 0.5 mM dithiothreitol (20). Electrophoresis in the presence of 0.55% dodecyl sulfate was performed with a 8-25% gradient gel, using the Phast System (Amersham Biosciences). Analytical ultracentrifugation was carried out in a Spinco E instrument (Beckman-Spinco), with scanning at 280 nm. Sedimentation velocity was measured at 48,000 rpm, and the sedimentation coefficient, s_20,w, was calculated using standard procedures (21). A partial specific volume of 0.730 cm³/g at 25 °C was calculated from the amino acid composition.

The following pH buffers were used for enzyme incubations: (a) 0.1 M citric acid/NaOH and 50 µM EGTA (pH 4.5); (b) 0.083 M TES/KOH, 0.017 M KCl, and 50 µM EGTA (pH 7.2); (c) 0.09 M TAPS/KOH and 5 µM EGTA (pH 8.5); and (d) 0.052 M TAPS/KOH and 0.048 M CAPS/KOH (pH 9.3). All measurements were performed at 25 °C.

Data Analysis-- Eqs. 1 and 2 (derived from Scheme I) describe time-courses of activity (A) resulting from dimer (D) conversion into monomer (M) and the reverse reaction, as well as the equilibrium activity (at t =  and d_D/dt = 0) as a function of enzyme concentration (22). A_D and A_M are the specific activities of the dimer and monomer, respectively; _D is the fraction of the dimer at time t; [E]_t is total enzyme subunit concentration; k_a and k_d are the apparent rate constants for association and dissociation, respectively. Eqs. 1 and 2 were simultaneously fit to data with the SCIENTIST program (MicroMath).

(Eq. 1)

(Eq. 2)

Monomer-dimer equilibrium. Scheme I.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Production of Y-PPase Variants-- The primary goal of this work was to redesign the subunit interface to yield nonassociating and stable monomer. Low probability substitutions (23) of dimer-forming residues were specifically selected (W/S, H/T, and R/S) to induce structural changes within this region of the protein. Three single variants (W52S, H87T, and W279S) and one double variant (H87T/W279S) were expressed and isolated in amounts ranging from 90 to 200 mg/liter culture medium. However, the R51S variant, as well as the R51G and R51L variants, could not be expressed, possibly as a consequence of replacing the buried and charged Arg⁵¹ side chain with uncharged side chains. A more conservative R51K replacement preserving the positive charge on the side chain resulted in yield improvement. Another major problem was the low stability of the W52S variant during short-term incubations in solution or long-term storage as a frozen solution, which ultimately resulted in a large scatter in data for this variant. This behavior was dithiothreitol-dependent, suggesting the involvement of SH groups. The problem was solved by replacement of the single Cys residue in Y-PPase (Cys⁸³) with Ser.

All preparations of the variant proteins used in this study were >95% homogeneous, as observed by SDS-PAGE analysis.

Effect of Substitutions on Quaternary Structure-- The first indication of altered quaternary structure in Y-PPase interface variants was evident upon native polyacrylamide gel electrophoresis, which revealed that they migrated faster than the wild-type enzyme and the noninterface variant, C83S (Fig. 2).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Native polyacrylamide gel electrophoresis of wild-type (WT) and variant Y-PPases. Protein samples were preincubated as described for Table I, except that the protein concentration was 10 µM, and the preincubation buffer used was 0.01 M Tris-glycine, pH 9.3. The gel was stained with Bio-Safe Coomassie (Bio-Rad).

Direct evidence for changes in the quaternary structure was additionally provided by sedimentation data (Table I). The W52S variant exhibited a lower s_20,w value (2.4 S) than wild-type PPase (4.0 S) at both pH 7.2 and pH 9.3, indicating monomeric protein as a result of the mutation. The other single-amino acid-substituted variants were dimers at pH 7.2 and mixtures of monomer and dimer at pH 9.3 and 5 µM enzyme, as indicated by the s_20,w values. The W52S substitution had the the most significant effect on the quaternary structure of the protein. A combination of two substitutions (H87T and W279S), each inadequate in yielding monomers at pH 7.2, also resulted in monomeric protein at both pH values.

Wild-type PPase remained dimeric at pH values as low as 4.5, in both the absence and presence of Mg²⁺, as indicated by the unchanged s_20,w value (4.0 S).

Effects of Active Site Ligands on the Equilibrium and Rates of the Dimer-Monomer Interconversion in W279S-PPase-- Dimer-monomer interconversion was conveniently monitored by activity measurements because activities of the dimer and monomer were different, and they converted into each other slowly on the time scale of the enzyme assay. This approach was used previously in studies on E. coli PPase variants with weakened quaternary structure (22, 24, 25, 27); here, we use it to characterize the W279S variant, which exists as either a dimer or a monomer, depending on specific conditions (Table I). In accordance with the sedimentation data, the specific activity of W279S-PPase (measured with 6 µM substrate) increased with enzyme concentration in a stock solution preincubated at pH 9.3 (Fig. 3), as expected for a slow equilibrium between dimer and less active monomer. No such inactivation of W279S-PPase at low enzyme concentrations was observed upon preincubation at pH 7.2. In contrast, the specific activity of wild-type Y-PPase and its W52S/C83S variant remained constant (240 ± 15 and 29 ± 2 s¹, respectively) on preincubation with 1 mM Mg²⁺ at both pH 7.2 and pH 9.3 at the same range of enzyme concentrations (data not shown). The inactivation of the W279S variant observed at pH 9.3 was completely reversed by decreasing the pH to 7.2 (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Specific activity of W279S-PPase preincubated with active site ligands as a function of enzyme concentration. Preincubation was performed for 2-5 h at pH 9.3 in the presence of 0.5 mM dithiothreitol and 1 mg/ml bovine serum albumin. After preincubation, enzyme activity was assayed with 6 µM PPi, as described under "Experimental Procedures." Curve labels indicate the ligands present during preincubation (PNP, imidodiphosphate). The lines were obtained with Eqs. 1 and 2 (with dD/dt = 0) using parameter values specified in Table II. Monomer activity, AM, was fixed at 43 s1, as determined from the curves measured in the presence of Mg2+ only.

View larger version (9K): [in this window] [in a new window]   Fig. 4.   Reversibility of W279S-PPase inactivation at pH 9.3. Enzyme solution (16 nM) was pre-equilibrated at pH 9.3 in the presence of 1 mM Mg2+ as described in the Fig. 3 legend, and the pH was lowered to 7.2 with 0.5 M TES at a specific time (indicated by the arrow). Aliquots were withdrawn as a function of time, and PPase activity was assayed at pH 7.2. The line was obtained with Eqs. 1 and 2 using ka value specified in Table II.

The shift in the activity versus enzyme concentration profile to the left caused by the active site ligands Mg²⁺, P_i, and imidodiphosphate (a PP_i analog containing N instead of O at the bridge position) (Fig. 3) indicated that the ligands stabilize the dimer rather than the monomer. The value of the equilibrium dissociation constant for dimer (K_d = k_d/k_a) derived from these profiles with Eqs. 1 and 2 decreased by 4 orders of magnitude in the presence of Mg²⁺ and imidodiphosphate. The effects of the active site ligands on the rate of W279S-PPase dissociation (Fig. 5) paralleled their effects on K_d (Table II). Because imidodiphosphate is a tightly bound (K_m < 1 µM) and slowly converted (k_cat = 0.01 s¹) substrate for Y-PPases (28), enzyme concentration was limited to 0.6 µM in the experiments illustrated in Figs. 3 and 5 to ensure that at least 50% of imidodiphosphate remained intact at the end of the incubation.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effects of ligands on W279S-PPase dissociation into monomers at pH 9.3. Stock enzyme solution (22 µM) pre-equilibrated at pH 7.2 to convert essentially all enzyme into dimeric form was diluted to 0.2 µM with the pH 9.3 buffer containing the indicated ligands, 0.5 mM dithiothreitol, and 1 mg/ml bovine serum albumin. Aliquots were withdrawn as a function of time, and PPase activity was assayed at pH 7.2. The lines represent a reversible first-order reaction with the kd and ka values specified in Table II.

Fitting Eqs. 1 and 2 with k_a = k_d/K_d (K_d values specified in Table II) to the time-courses shown in Fig. 5 allowed the estimation of k_d. The value of k_a at pH 7.2 was obtained by fitting Eqs. 1 and 2 to the time-course of the association reaction (Fig. 4), which was essentially irreversible (i.e. k_d could be set to 0) because the enzyme was predominantly monomeric at the start of the reaction and dimeric at the end of the reaction.

A similar analysis was performed for W279S-PPase at a wide range of Mg²⁺ concentrations for yielding k_d and k_a dependences, as shown in Fig. 6. The dependence of k_d may be described by Scheme II, which implies an effect of two metal ions bound sequentially with dissociation constants of K and K. The shape of the profile indicates that k_d2 is the lowest of the three individual dissociation rate constants shown in Scheme II. Fitting the data of Fig. 6 to Eq. 3 allowed the evaluation of all parameters in Scheme II: K = 19 ± 8 µM, K > 20,000 µM, k_d1 = 2.1 ± 0.3 min¹, k_d2 = 0.12 ± 0.08 min¹, and k_d3 > 1 min¹.

(Eq. 3)

The less significant effect of [Mg²⁺] on k_a (Fig. 6) is described by Eq. 4, which indicates one metal binding site/subunit. The corresponding dissociation constant, K_MX, and the values of the rate constants k_a1 for free monomer and k_a2 for its magnesium complex were found to be 1600 ± 600 µM, 4.1 ± 0.1 µM¹ min¹, and 6.9 ± 0.2 µM¹ min¹, respectively.

(Eq. 4)

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Rate constants for W279S-PPase dissociation into monomers at pH 9.3 () and reassociation into dimers at pH 7.2 () as a function of Mg2+ concentration. Experiments were performed as described in the Fig. 4 legend. Lines were obtained with Eqs. 3 and 4, using the parameter values specified under "Results."

Dissociation of dimeric W279S-PPase in the presence of metal ions. Scheme II.

Sensitivity to SH Reagents-- Additional evidence for the different quaternary structures of the W279S and W52S variants at pH 7.2 was obtained by comparing the effects of SH reagents on different enzyme forms. In dimeric Y-PPase, the single Cys residue/subunit, located at ~5 Å from the subunit interface, is shielded by C-terminal residues 280-284 (13), which protect the residue from modification by bulky reagents (29). Consistent with this structure, N-ethylmaleimide had a minimal effect on the activity of wild-type Y-PPase and its dimeric W279S variant at pH 7.2, as confirmed by the s_20,w values, but inactivated the monomeric W52S variant (Fig. 7). p-HMBS, a more reactive SH reagent, inactivated the dimeric PPases appreciably, but again, the effect on the monomeric W52S variant was much more significant. The Cys residue was not appreciably modified by N-ethylmaleimide in the dimeric PPases but was nearly completely modified in monomeric W52S-PPase at pH 7.2, as confirmed by a greater inactivating effect of p-HMBS on the N-ethylmaleimide-treated wild-type PPase and W279S-PPase compared with W52S-PPase (Fig. 7). The monomeric double variant (W52S/C83S) lacking Cys was not inactivated by N-ethylmaleimide.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Sensitivity of wild-type and variant PPases to inactivation by SH reagents at pH 7.2 and pH 9.3. Enzymes were incubated for 20-30 min without SH reagents () or with 0.1 mM p-HMBS (), 1 mM N-ethylmaleimide (), or N-ethylmaleimide followed by p-HMBS () and assayed for activity as described under "Experimental Procedures." Incubation conditions: 5 µM enzyme, 1 mM Mg2+. Where available, values of s20,w are presented above the bars.

At pH 9.3, both p-HMBS and N-ethylmaleimide modified wild-type PPase significantly, causing partial dissociation into monomers, as indicated by the decrease in s_20,w (Fig. 7). Again, the W279S variant that is predominantly monomeric at this pH value displayed significantly greater reactivity to these reagents and decreased s_20,w values upon the modification.

Michaelis-Menten Parameters for Dimer and Monomer-- Only minor changes in k_cat and K_m values were observed in the dimeric variant PPases, compared with wild-type protein (Table III), indicating no significant alterations in the active site. The monomeric forms of all variant PPases exhibited markedly increased K_m values, whereas k_cat values decreased significantly in only two variants (W52S and W279S).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Contribution of Different Residues to Dimerization-- X-ray crystallography has led to the identification of four critical residues at the subunit interface of Y-PPase, specifically Arg⁵¹, Trp⁵², His⁸⁷, and Trp²⁷⁹ (Fig. 1). Sedimentation, gel electrophoresis, and activity measurements reveal that the W52S substitution has a greater effect on dimer stability than the R51K, H87T, and W279S substitutions. Among other factors, the nature of a substitution largely influences the extent of the effects on protein structure and function. With this in mind, one can conclude that Trp⁵² contributes more to dimer formation than Trp²⁷⁹ (identical substitution) and perhaps His⁸⁷ (drastic substitution), whereas the role of Arg⁵¹ may be underestimated (charge-preserving substitution). The importance of Trp⁵² may be explained by its participation in the three-layer stacking of aromatic rings with His^87', His⁸⁷, and Trp^52' (Fig. 1). Our inability to express the R51S, R51G, and R51L variants suggests that the Arg⁵¹ residue, whose side chain is charged and buried, is important not only in dimer formation but also in maintaining overall structure.

Factors Affecting Dimer Stability-- Whereas wild-type Y-PPase exists as dimer in a wide range of conditions, the W279S variant is a mixture of dimer and monomer, facilitating analyses of the effects of various stimuli on the dimer  monomer equilibrium. The results of these analyses indicated that substrate (imidodiphosphate), product (P_i), and Mg²⁺ stimulate dimerization (Figs. 3 and 6), whereas modification of Cys⁸³ and increasing pH have an opposite effect (Fig. 7).

The effects of Mg²⁺ on dimer stability are mainly a result of its effect on k_d (however, it should be taken into account that the k_d and k_a values were obtained at different pH conditions) and are reversed at high Mg²⁺ concentrations (Fig. 6). Four metal binding sites/subunit have been identified in the phosphate complex of dimeric wild-type Y-PPase by x-ray crystallography (11). Three of them bind Mg²⁺ in the absence of phosphate (30, 31), with dissociation constants of 2.3, 15, and >5000 µM at pH 9.3 (31). A comparison of these values with K of 19 ± 8 µM and K of > 20,000 µM in Scheme II indicates that the decrease in k_d and reversal of this effect with increasing Mg²⁺ concentrations (Fig. 6) are associated with the binding of the second and third Mg²⁺ ions, respectively. It should be noted that the destabilizing effect of Mg²⁺ on the dimer is insignificant at physiological concentrations of the ion (~1 mM). The Mg²⁺ concentration dependence of k_a in Fig. 6 yielded an Mg²⁺ binding constant of 1600 ± 600 µM for monomeric Y-PPase at pH 7.2. Our recent kinetic analysis of the activating effect of Mg²⁺ on monomeric W279S-PPase² suggests that this constant refers to binding of the second Mg²⁺ ion and thus implies an 80-fold decrease in affinity for monomer, compared with dimer. Accordingly, Mg²⁺ stimulates dimer formation via tighter binding. Because the active site and subunit interface are separated in space, the effect of Mg²⁺ implies a conformational difference between monomer and dimer and between dimers with vacant and occupied M2 sites. The same considerations apply to imidodiphosphate and P_i binding.

In addition to confirming that the W52S variant is monomeric, Cys⁸³ modification helped to identify the conformational differences between dimer and monomer. The increased reactivity of Cys⁸³ in monomer clearly indicates that the C-terminal segment that normally shields Cys⁸³ in dimer (Fig. 8) becomes more mobile or possibly adopts a completely different conformation, making the SH group accessible to modifying agents. This displacement is not a specific effect of the W279S substitution because (a) the SH group is inaccessible to N-ethylmaleimide in both dimeric wild-type PPase and dimeric W279S-PPase at pH 7.2 (Fig. 7), and (b) two different mutations (W279S and W52S) similarly increase the reactivity of the SH group in monomer (Fig. 7). At pH 9.3, the reactivity of dimer to the SH reagents is increased, which may mean that the dimer represents an equilibrium mixture of several conformations (32), with the more reactive conformations becoming more populated at increasing pH values or in variants with weakened subunit interactions.

View larger version (109K): [in this window] [in a new window]   Fig. 8.   Arrangement of Cys83, the C-terminal segment (residues 280-284), and subunit interface residues in wild-type Y-PPase (13).

The effect of pH on dimer stability (Table II) may be associated with His⁸⁷. In dimeric Y-PPase, two His⁸⁷ residues of the different subunits are linked by a hydrogen bond, implying that one of them is protonated. Loss of this proton at a high pH value should destabilize subunit interactions (see Table II for details).

Role of the Quaternary Structure in Catalysis-- The conformational change resulting from monomer to dimer conversion is accompanied by an alteration in the Michaelis-Menten parameters (Table III). In three variants (R51K, H87T, and W279S) that exist as both dimers and monomers, dimerization only slightly increases k_cat (by a factor of 1.05-1.4) but markedly decreases K_m (by a factor of 5-9). The data measured for wild-type Y-PPase and its other variants that exist in only one oligomeric form are in accordance with this trend. The difference in the K_m values between dimer and monomer explains the major effect of enzyme concentration on the specific activity of W279S-PPase measured at low (6 µM) substrate concentration (Fig. 3). Consistent with this explanation, a much lower inactivation was observed at low concentrations of W279S-PPase, when activity was assayed with 1000 µM substrate (data not shown).

Thus, dimerization fine-tunes the active site of Y-PPase, allowing tighter binding of the metal cofactor and substrate (as verified by the Michaelis constant). Conversely, intersubunit interactions increase in strength upon active site adjustment caused by metal cofactor, product, or substrate analog. MgPP_i binding is sufficient for complete active site organization, as indicated by similar k_cat values for dimer and monomer. In this respect, the family I member, Y-PPase, principally differs from family II PPases with similar subunit size and quaternary structure, which exhibit an ~40-fold increase in k_cat upon dimerization (8). This may be a consequence of the two-domain structure of family II PPases with active site location at the domain interface (9, 10). In this case, dissociation into monomers may cause domain flexibility that cannot be overcome by substrate binding. In contrast, Y-PPase is a one-domain protein, like all family I PPases.

Another interesting comparison may be made with E. coli PPase, which, like Y-PPase, belongs to family I. The active sites of the two PPases are nearly identical, but the subunit size is much smaller for E. coli PPase (20 versus 32 kDa). Accordingly, the E. coli enzyme is inactive as a monomer but fully functional as a hexamer, similar to other prokaryotic PPases of family I (27). For E. coli PPase, k_cat values are similar in dimer, trimer, and hexamer, whereas K_m values decrease progressively (10⁴-fold) from dimer to hexamer (22, 27). This comparison suggests that the critical mass required to form a well-ordered active site in homo-oligomeric PPases is inversely proportional to monomer mass, i.e. larger monomers lead to smaller homo-oligomers. This information is crucial in the design of other polypeptides with enzymatic activity de novo.

The structure of the subunit interface is highly conserved in other fungal and animal family I PPases (5) but is significantly different in prokaryotic PPases of both families (5, 9, 10, 26). This allows the design of selective inhibitors of PPases of families I and II that would interfere with oligomerization in pathogenic prokaryotes. In this context, it is important to note that subunit contacts are weaker in prokaryotic than in eukaryotic PPases.

	ACKNOWLEDGEMENTS

We thank P. V. Kalmykov, K. Mikalahti, and T. Vehmas for technical help.

	FOOTNOTES

^* This work was supported by Russian Foundation for Basic Research Grants 00-04-48310, 00-15-97907, and 01-04-06111 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.

^§ Both authors contributed equally to this work.

^** To whom correspondence may be addressed. Tel.: 7-095-939-5541; Fax: 7-095-939-3181; E-mail: baykov@genebee.msu.su.

To whom correspondence may be addressed. Tel.: 358-2-333-6845; Fax: 358-2-333-6860; E-mail: reijo.lahti@utu.fi.

Published, JBC Papers in Press, February 19, 2002, DOI 10.1074/jbc.M200101200

² A. N. Parfenyev, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PPase, pyrophosphatase; CAPS, 3-(cyclohexylamino)propanesulfonic acid; p-HMBS, p-hydroxymercurobenzosulfonate; Y-PPase, yeast (Saccharomyces cerevisiae) pyrophosphatase; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This Article Abstract Full Text (PDF) All Versions of this Article: 277/18/15465    most recent M200101200v1 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 Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Salminen, A. Articles by Lahti, R. Search for Related Content PubMed PubMed Citation Articles by Salminen, A. Articles by Lahti, R. Social Bookmarking