Probing Essential Water in Yeast Pyrophosphatase by Directed Mutagenesis and Fluoride Inhibition Measurements*

Pekka PohjanjokiDagger , Igor P. Fabrichniy§, Vladimir N. Kasho, Barry S. Cooperman||, Adrian GoldmanDagger **, Alexander A. Baykov§DaggerDagger, and Reijo LahtiDagger §§

From the Dagger  Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland, the § A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia, the  Center for Ulcer Research and Education, Department of Medicine, University of California, Los Angeles, California 90073, the || Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and the ** Institute of Biotechnology, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland

Received for publication, August 14, 2000, and in revised form, October 6, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg2+-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr93 and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PPi hydrolysis and synthesis, formation of enzyme-bound PPi at equilibrium, phosphate-water oxygen exchange, and Mg2+ binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp117. The effects of the mutations on PPi hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PPi synthesis (as characterized by the net rate constant for PPi release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme·PPi intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme·PPi intermediates is proposed.



    INTRODUCTION
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EXPERIMENTAL PROCEDURES
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Inorganic pyrophosphatase (EC 3.6.1.1; PPase)1 catalyzes reversible phosphoryl transfer from pyrophosphate (PPi) to water, a metabolically important reaction chemically similar to that catalyzed by numerous ATPases and GTPases. Yeast PPase is a homodimer containing 286 amino acid residues/monomer (1) and requiring three or four divalent metal ions for catalysis, with Mg2+ conferring the highest activity (2-5). Two divalent metal ions (M1 and M2) per active site have been identified in the "resting" enzyme by x-ray crystallography and four metal ions (M1-M4) and two phosphates (P1 and P2) in the product complex of Y-PPase (6, 7).

PPi hydrolysis by PPase occurs via direct attack of water without formation of a phosphorylated enzyme intermediate (8). Modeling the transition state of the chemical step from the structure of the enzyme-product complex Y-PPase·Mn2(MnPi)2 has led to two models that differ in the identity of the water nucleophile placed between metal ions M1 and M2 in the model of Heikinheimo et al. (6) or in the vicinity of Tyr93 in the model of Harutyunyan et al. (7, 9). Although the former model requires an additional "relaxation" step, in which a new water molecule displaces Pi oxygen from the position between M1 and M2, it has an advantage of providing an efficient mechanism for nucleophile activation through combined action of the two metal ions and an adjacent Asp117 residue (see Fig. 1).

In aqueous solution and even in crystalline state, proteins are surrounded with water shells, making identification of function-related water molecules a difficult task. Use of fluoride, a potent and most specific inhibitor of cytoplasmic pyrophosphatase, provides a convenient approach to detect such water molecules because molecules of HF and H2O are isoelectronic and of similar size, as are the anions derived therefrom. Fluoride inhibition of yeast PPase during PPi hydrolysis and synthesis involves a rapid and slow phases (10), which refer to F- binding to enzyme-Pi and enzyme-PPi intermediates, respectively (Scheme I). The rapid binding decelerates PPi hydrolysis 10-fold at pH 7.2, whereas the slow binding arrests it completely. These characteristics of the inhibition are consistent with fluoride replacing an essential metal-bound water molecule/OH- ion, acting as nucleophile in the PPi hydrolysis step (11).



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Scheme I.   Interactions between Y-PPase and fluoride during catalysis. F, F-; M, Mg2+; P, Pi; PP, PPi (11).

In the present work, we employed fluoride inhibition in combination with site-directed mutagenesis to identify the fluoride binding site and, hence, the essential water molecule in the active site of PPase. Because fluoride inhibition of Y-PPase is closely associated with Mg2+ binding (10, 12), the effects of mutating five amino acid residues forming the M1 and M2 sites (Glu48, Asp115, Asp120, Asp117, and Asp152) on fluoride inhibition were studied. A Y93F variant was also included in this list, because in the PPase mechanism suggested by Harutyunyan et al. (9) a water molecule associated with Tyr93 is assumed to be the nucleophile and, hence, might be replaced by F-. X-ray crystallographic analysis of three relevant variants, D117E Y-PPase (13) and D65N and D70N Escherichia coli PPase (Asp65 and Asp70 in E. coli PPase correspond to Asp115 and Asp120 in Y-PPase, respectively) (14), indicated no marked structural changes induced by the mutations.


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The expression and purification of wild type Y-PPase and its active site variants from overproducing E. coli XL2blueb strain transformed with suitable plasmids were carried out as described by Heikinheimo et al. (15).

PPi hydrolysis was measured continuously with an automatic Pi analyzer (16). The assay medium contained, except as noted, 0.32 mM (at pH 7.2) or 0.28 mM (at pH 8.5) total PPi (corresponding to 0.2 mM Mg2PPi complex), 5.5 mM MgCl2 (corresponding to 5 mM free Mg2+), 0-20 mM F- (added as NaF) and buffer. The following pH buffers were used (0.1 M ionic strength, 50 mM K+): 83 mM TES/KOH, 17 mM KCl, 50 µM EGTA (pH 7.2), or 90 mM TAPS/KOH, 5 µM EGTA (pH 8.5). Enzyme-bound PPi formation was assayed luminometrically with ATP-sulfurylase and luciferase (17, 18) using 60-260 µM Y-PPase concentration. PPi synthesis in solution was measured continuously by the same coupled enzyme assay (11). Pi-H2O oxygen exchange was measured by gas chromatography/mass spectrometry (19). The assay medium used to measure fluoride effects on PPase in the presence of Pi contained 13.4 mM MgCl2 (5 mM free Mg2+), 20 mM total Pi, 55 mM TES/KOH buffer, 11 mM KCl, and 33 µM EGTA. Mg2+ binding was assayed by equilibrium microdialysis in combination with atomic absorption spectroscopy to measure Mg content in the dialysis chambers (20). All experiments were performed at 25 °C.

Effects of fluoride on PPi hydrolysis were analyzed in terms of Scheme II, a simplified version of Scheme I. KF1 is the dissociation constant governing rapid fluoride binding in the presence of PPi, ki, app and kr are the second-order and first-order rate constants for slow fluoride binding and release, respectively.



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Scheme II.   A minimal kinetic scheme of Y-PPase inactivation by fluoride during catalysis.

Equations 1 and 2 describe product (P) formation curves at a fixed fluoride concentration, where alpha  is the fraction of enzyme that has not yet undergone slow conversion into Einactive, and v0, app is the initial velocity of product formation.
<FR><NU>d&agr;</NU><DE>dt</DE></FR>=k<SUB>r</SUB>(1−&agr;)−k<SUB>i, <UP>app</UP></SUB>[<UP>F</UP>]&agr; (Eq. 1)

<FR><NU>d[<UP>P</UP>]</NU><DE>dt</DE></FR>=v<SUB>0, <UP>app</UP></SUB>&agr; (Eq. 2)

Fitting [P] as a function of both time and [F] was accomplished by making the following substitutions (v0 and v0' are the initial velocities of product formation observed at [F] equal to zero and infinity, respectively).
v<SUB>0, <UP>app</UP></SUB>=v′<SUB>0</SUB>+<FR><NU>v<SUB>0</SUB>−v′<SUB>0</SUB></NU><DE>1+[<UP>F</UP>]/K<SUB>F1</SUB></DE></FR> (Eq. 3)

k<SUB>i, <UP>app</UP></SUB>=<FR><NU>k<SUB>i</SUB></NU><DE>1+[<UP>F</UP>]/K<SUB>F1</SUB></DE></FR> (Eq. 4)
Equations 1-4 were simultaneously fit, with the program SCIENTIST (MicroMath), to sets of product formation curves, each represented by 90-200 pairs of [P] and t values. The calculated and measured curves agreed within 3%.

Values of the dissociation constants KM1 and KM2 for Mg2+ binding to two sites on Y-PPase were estimated by fitting equilibrium dialysis data to Equation 5, where n measures the number of Mg2+ ions bound per monomer (21).
n=<FR><NU>1+2[<UP>Mg</UP><SUP>2+</SUP>]/K<SUB>M2</SUB></NU><DE>1+K<SUB>M1</SUB>/[<UP>Mg</UP><SUP>2+</SUP>]+[<UP>Mg</UP><SUP>2+</SUP>]/K<SUB>M2</SUB></DE></FR> (Eq. 5)



    RESULTS
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Mg2+ Binding-- The effects of conservative mutations of Tyr93 and five amino acid ligands to Mg2+ (Fig. 1) on the binding of two activating metal ions in Y-PPase active site were studied by equilibrium dialysis at pH 7.2 (Fig. 2). Interestingly, every substitution suppressed Mg2+ binding to the low affinity site, the lowest (1.8-fold) effect seen with Y93F, and the highest (147-fold) with E48D substitutions (Table I). Binding of Mg2+ to the high affinity site was markedly suppressed in both Asp120 variants (34- and 16-fold effects with D120E and D120N, respectively) and less markedly (4.0-fold effect) in the D152E variant. These data are consistent with M1 being the high affinity site and M2 being the low affinity site, as measured by equilibrium dialysis.



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Fig. 1.   Part of Y-PPase active site structure in Y-PPase·Mn2(MnPi)2 complex as determined by x-ray crystallography (6). Larger black circles, two of which are labeled M1 and M2, are metal ions, smaller black circles are water oxygens, and two black molecules are phosphates. Hydrogen bonds are shown by dashed lines.



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Fig. 2.   Scatchard plots for Mg2+ binding to wild type and variant Y-PPase. Lines were obtained with Equation 5 using parameter values found in Table I. Experimental conditions were pH 7.2, 0.5-2000 µM Mg2+, and 50-500 µM PPase.


                              
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Table I
Dissociation constants for Mg2+ binding at pH 7.2

Importantly, all the variants tested still exhibited appreciable affinity for Mg2+, such that both sites were almost saturated at 5 mM Mg2+ concentrations used in the fluoride inhibition studies described below. The E48D variant, which exhibited the highest KM2 value (5.0 mM), is expected to be 50% saturated at the M2 site in the absence of substrate, but keeping in mind that substrate strengthens metal ion binding to the corresponding E-PPase E20D variant (22) and WT-E-PPase (23), at least, 90% saturation is expected in the presence of 200 µM Mg2PPi used in fluoride inhibition studies. This rules out a possibility that the effects of the mutations on fluoride inhibition reported below result from incomplete metal binding. Fluoride added at a 10 mM concentration suppressed binding of M1 and stimulated binding of M2 approximately 2-fold in WT-Y-PPase (Table I).

Catalytic Properties-- Values of the catalytic constant for PPi hydrolysis (kh), rate of PPi synthesis (vs), rate of Pi-H2O oxygen exchange (vex), the distribution of five Pi "isotopomers" during the exchange (as characterized by the partition coefficient Pc = k4/(k5 + k4) (19), and the amount of enzyme-bound PPi at equilibrium with medium Pi (fepp) were measured for six variants at pH 7.2 and 8.5 (Table II). Such measurements were not performed for the D120N variant, which displayed very low hydrolytic activity (<0.005 s-1). These data allowed evaluation of two important parameters: k3' = vex(1 - 0.75Pc)/fepp(1 - Pc)[E]t and kpp, off vs/fepp[E]t, the net rate constant for PPi release, i.e. back conversion of EM4PP to EM2 (21). Equations 6 (11) and 7 (21) relate k3' and kpp, off to the rate constants shown in Scheme I (KAB = kA/kB). In fact, k3' is the lower limit for k3 (corresponding to KAB =infinity ), and kpp, off is the lower limit for both k2 and kB. For WT-PPase, KAB is equal to 25-65, and the values of k3' and k3 differ by less than 4% at pH >=  7.2 (11), but the values of KAB for the variant PPases are unknown. The advantage of using k3' and kpp, off instead of vex/[E]t and vs/[E]t is that the former parameters are independent of the degree of enzyme saturation with Pi in corresponding measurements.
k′<SUB>3</SUB>=<FR><NU>k<SUB>3</SUB></NU><DE>1+1/K<SUB><UP>AB</UP></SUB></DE></FR> (Eq. 6)

k<SUB><UP>pp, off</UP></SUB>=<FR><NU>1</NU><DE>1/k<SUB><UP>B</UP></SUB>+1/k<SUB>2</SUB>+k<SUB><UP>A</UP></SUB>/k<SUB><UP>B</UP></SUB>k<SUB>2</SUB></DE></FR> (Eq. 7)


                              
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Table II
Catalytic properties of Y-PPase variants

Fluoride Inhibition of PPi Hydrolysis-- According to the inhibition pattern, all the variants could be classified into three groups. Typical examples are shown in Fig. 3. Group I variants (E48D, Y93F, D115E, and D117E) resembled WT-Y-PPase (11) by exhibiting a virtually instant decrease in the initial slope of the Pi production curve, followed by a slower inactivation step. The group II variant (D120E at pH 7.2) exhibited only slow inactivation, without the instant decrease in the initial slope, and the group III variant (D152E) exhibited only instant inactivation. At pH 8.5, D120E-PPase also behaved like a group III member, whereas other variants did not change their behavior. It should be noted that the Pi productions curves look very similar for group I and group II variants (Fig. 3), and they could be only distinguished based on the fitting results shown below; the best-fit value of KF1 tended to approach infinity for D120E-PPase, clearly indicating no change in v0,app in the presence of fluoride. Values for the rate constants ki and kr describing the slow phase of the inhibition and for the equilibrium constant KF1 describing the fast step of the inhibition (Scheme II) were obtained by simultaneous fitting of Equations 1-4 to series of product formation curves measured at different fluoride concentrations. The most drastic effects were observed on ki with the D117E and D152E variants (the latter exhibited no slow inactivation step at any pH value), on kr with the D115E and D120E variants, and on KF1 with the D117E variant (Table III).



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Fig. 3.   The effects of NaF on the progress curves for PPi hydrolysis catalyzed by four variant Y-PPases at pH 7.2. F- concentrations (in mmol/liter) are indicated at the curves. Curves were normalized to 0.008 µM (E48D), 0.27 µM (D117E), 2.9 µM (D120E), or 3.5 µM (D152E) enzyme concentration.


                              
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Table III
Parameters for fluoride inhibition of PPi hydrolysis

Fluoride Effect on Enzyme-bound PPi Formation-- The Y-PPase active site is preformed to bind PPi. Although the concentration of PPi present at equilibrium with 20 mM Pi and 5 mM Mg2+ at pH 7.2 is <1 µM, i.e. the ratio [Pi]/[PPi] > 20,000 (5, 24, 25), the fraction of enzyme containing bound PPi (fepp = ([EM4PP*] + [EM4PP])/[E]t) in these conditions is as high as 16% (Table II), in agreement with previous estimates (5, 26, 27). This remarkable feature of the active site was completely retained in D117E and D120E variants, partially retained in the D48E, Y93F, and D115E variants and almost completely lost in the D152E variant (Table II and Fig. 4).



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Fig. 4.   The effects of 2 mM F- on the amount of PPi bound to Y-PPase variants (60-260 µM) pre-equilibrated with 5 mM free Mg2+ and 20 mM total Pi at pH 7.2. Aliquots (20 µl) of the reaction mixture were withdrawn at the indicated time intervals, quenched with 4 µl of 5 M trifluoroacetic acid, and assayed for PPi (18). The two panels refer to different time scales. Lines were obtained with Equations 8 and 9 using parameter values found in Table IV.

As shown in Fig. 4, addition of 2 mM fluoride to E48D, Y93F, and D115E variants at pH 7.2 caused a rapid decrease in enzyme-bound PPi, not resolved in time (F- binding to enzyme-Pi complex in Scheme I), followed by a slow increase to a higher level (F- binding to enzyme-PPi complex) (Fig. 4), like in wild type Y-PPase (11). The time course of the slow increase could be described by Equations 8 and 9 (11), where eppfast refers to the amount of enzyme-bound PPi after completion of the fast phase of the interaction with F- (i.e. the zero time point for the slow phase), and feppslow is the amount of the enzyme-bound PPi accumulated at time t during the slow phase. Fluoride binding was assumed to be a second-order reaction (see also below).
f<SUB><UP>epp</UP></SUB>=f<SUP><UP>slow</UP></SUP><SUB><UP>epp</UP></SUB>+f<SUP><UP>fast</UP></SUP><SUB><UP>epp</UP></SUB>(100−f<SUP><UP>slow</UP></SUP><SUB><UP>epp</UP></SUB>)/100 (Eq. 8)

<FR><NU>df<SUP><UP>slow</UP></SUP><SUB><UP>epp</UP></SUB></NU><DE>dt</DE></FR>=k<SUB>i, <UP>app</UP></SUB>[<UP>F</UP><SUP>−</SUP>](100−f<SUP><UP>slow</UP></SUP><SUB><UP>epp</UP></SUB>)−k<SUB>r</SUB>f<SUP><UP>slow</UP></SUP><SUB><UP>epp</UP></SUB> (Eq. 9)
For the D117E and D120E variants, the initial decrease in fepp could be resolved in time. Moreover, no slow increase in fepp followed after its rapid drop in the D120E variant. Values of ki, app, kr, and feppfast for the D117E variant were obtained with Equations 8 and 9 using the points collected at t >=  2 min on the curve shown in Fig. 4. The curve for the D120E variant and the initial part of the curve for the D117E variant (t <=  2 min) could be described by Equations 10 and 11 for a reversible binding, where r is the percentage of fraction of enzyme-Pi complex containing bound fluoride and the primed rate constants refer to F- binding to and release from enzyme-Pi complex. As the enzyme-Pi complex binds two fluoride ions (see below), Equation 11 contains the second power of fluoride concentration. The effect of F- on PPi bound to D152E-PPase was quite small, if there was any.
f<SUB><UP>epp</UP></SUB>=f<SUB><UP>epp, 0</UP></SUB>(100−r)/100 (Eq. 10)

<FR><NU>df<SUP><UP>fast</UP></SUP><SUB><UP>epp</UP></SUB></NU><DE>dt</DE></FR>=k′<SUB>i, <UP>app</UP></SUB>[<UP>F</UP><SUP>−</SUP>]<SUP>2</SUP>(100−r)−k′<SUB>r</SUB>r (Eq. 11)
The ratio of ki, app/feppfast, the true rate constant for F- binding to enzyme-PPi complex (ki, app refers to total enzyme), is decreased only in the D117E variant by comparison with wild type Y-PPase (Table IV).


                              
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Table IV
Enzyme-bound PPi formation and enzyme inactivation in the presence of 2 mM F- at pH 7.2

Inactivation by Fluoride in the Presence of Pi-- Upon the incubation with Pi and F- at pH 7.2, the variant PPases were diluted 900-4500-fold into assay medium, and the initial velocity of PPi hydrolysis was estimated from the slope of the Pi production curve 1 min after the dilution (the dead-time of the Pi analyzer increased by comparison with Fig. 3 because of the Pi present in the enzyme solution). The extensive dilution and the 1-min delay were expected to completely reverse the fast fluoride binding step seen in Fig. 4 for the E48D, Y93E, and D115E variants and to partially reverse the slower initial binding step observed with the D120E and D117E variants. In agreement with the data reported above, the preincubation with fluoride did not affect the activity of the D152E variant, slightly inactivated the D115E and D120E variants and more deeply inactivated the E48D, Y93E, and D117E variants (Figs. 5 and 6). Low degree of the inactivation seen with the D115E variant is consistent with its high kr value (Table IV). For all the variants, the degree of the inactivation observed after 80 min was smaller than for wild type Y-PPase (11), consistent with lower ki, app or higher kr value (Table IV). The values of these parameters estimated with Equation 1 for the two variants exhibiting >30% inactivation in Figs. 5 and 6 (Y93F and D117E) were similar to those derived from Fig. 4 (Table IV). For the D117E variant, only points with t > 5 min were used in this estimation.



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Fig. 5.   Inactivation of Y-PPase variants during incubation with 2 mM F- in the presence of 20 mM Pi and 5 mM Mg2+ at pH 7.2. The incubation was carried out in a total volume of 70 µl at 6-850 µM enzyme concentration. Aliquots of 2-12 µl were withdrawn at indicated time intervals and immediately added to the activity assay containing 1 mM PPi, 0.2 mM MgCl2, 10 µM EGTA, and 0.05 M Tris-HCl, pH 8.5. The line for the Y93F variant was obtained with Equation 1 using parameter values found in Table IV, the line for wild type Y-PPase is from Baykov et al. (11), and the other lines were drawn by eye.



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Fig. 6.   Inactivation of D117E-PPase during incubation with F- in the presence of 20 mM Pi and 5 mM Mg2+ at pH 7.2. F- concentrations in mmol/liter are shown at the curves. Other conditions were as for Fig. 5, except that the activity assay was started by adding PPi 1 min after enzyme was added; this 1-min delay was introduced to completely reverse the first, faster step of F- binding. The lines are the best fits of Equation 1.

Increasing the F- concentration decreased D117E-PPase inactivation (Fig. 6), a phenomenon previously observed with wild type Y-PPase and explained by binding of two F- ions to enzyme-Pi complex (11). Such binding decreases the concentration of the enzyme·PPi complex (the true binding species) proportionally to [F]2, and this is only partly compensated by an increase in the fluoride binding rate, which is proportional to [F].

Fluoride Inhibition of D117E-PPase during PPi Synthesis-- Studies of PPi synthesis provided further evidence for the ability of D117E-PPase to bind two F- ions in the presence of Pi (Fig. 7). The effect of [F] on the initial velocity of PPi synthesis was only poorly described by Equation 3 (with v'0 = 0), implying binding of only one F- ion but was well described by Equation 12, implying binding of two F- ions (Fig. 7, right panel).



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Fig. 7.   The effects of NaF on PPi synthesis catalyzed by D117E-PPase. Left panel, progress curves at indicated F- concentrations (in mmol/liter). Curves were normalized to 1.2 µM enzyme concentration; actual enzyme concentrations were 1.2-2.3 µM. One unit of luminescence corresponds to 1 µM PPi at zero time (luciferase fully active). Right panel, dependence of v0 on NaF concentration. The dotted and solid lines show the best fits of Equations 3 (with v'0 = 0) and 12, respectively.


v<SUB>0, <UP>app</UP></SUB>=<FR><NU>v<SUB>0</SUB></NU><DE>1+[<UP>F</UP>]<SUP>2</SUP>/K<SUB>F1</SUB></DE></FR> (Eq. 12)
The nonlinearity of the luminescence versus time curves (Fig. 7, left panel) was similar at different [F] and could be fully explained by fluoride-independent inactivation of luciferase (11). This means that the slow fluoride binding step is not evident over the 1-4.5-min time interval in the presence of Pi, consistent with the data in Fig. 4, showing no significant increase in fepp during this time interval.


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Roles for the Six Residues in Metal Binding and Catalysis-- There is a substantial increase in KM2 with five of the six variants under study (Table I), indicating the M2 site to be highly sensitive to changes in the delicate network of interactions in the active site of Y-PPase (Fig. 1). The most drastic effects on KM2 are seen with the E48D and D120E variants, which is consistent with the x-ray crystallographic data showing Glu48 and Asp120 to be most closely associated with M2 (Fig. 1). The M1 site is clearly much more resistant to amino acid substitutions than the M2 site; a significant effect is only seen with the D120E(N), D152E, and D117E variants. This is compatible with changes seen between the EM2 and EM2(MPi)2 complexes (6). In these structures, the M2 site showed itself to be more rigidly fixed than the M1 site, so that the loops carrying Asp115 and Asp152 swing in toward a rigid M2 binding site in the product complex structure. Consequently, variation at the M1 ligands Asp115 and Asp152 is more readily tolerated than variation at the M2 ligands Asp120 and Glu48. However, it is unclear why D115E and D152E substitutions have much larger effect on M2 than on M1 binding (Table I). One possible explanation is that the substitutions effectively misposition M1 and Asp120 and so prevent the rigid M2 binding site from forming properly. Consistent with this notion is the fact that the changes seen at M2 for the D115E and D152E substitutions, although large, are smaller than that seen for directly mutating Asp120, as if the effects of the D115E and D152E substitutions were to misalign Asp120. As expected, mutating Tyr93, which is not involved in metal binding (Fig. 1), had no effect on KM1 and only a minor effect on KM2 (Table I).

In general, the effects of the mutations on PPi hydrolysis (as characterized by kh and k3') are larger than the effects on PPi synthesis (as characterized by kpp, off) (Table II). All the mutations, with the only exception of E48D at pH 8.5, decrease kh, whereas kpp, off is increased in three variants (E48D, Y93F, and D115E) at pH 8.5. The latter effect appears to result from increased kB (see Equation 7), because the kB step is rate-limiting in PPi synthesis by wild type PPase (11). A similar stimulating effect of an E20D substitution on PPi synthesis was earlier observed with E. coli PPase (Glu20 in E. coli PPase corresponds to Glu48 in Y-PPase) (22).

Combining Equation 6 with the expression for kh (11), one obtains
<FR><NU>1</NU><DE>k<SUB>h</SUB></DE></FR>=<FR><NU>1</NU><DE>k<SUB><UP>A</UP></SUB></DE></FR>+<FR><NU>1</NU><DE>k′<SUB>3</SUB>(1−P<SUB><UP>c</UP></SUB>)</DE></FR>+<FR><NU>1</NU><DE>k<SUB>5</SUB></DE></FR>+<FR><NU>1</NU><DE>k<SUB>7</SUB></DE></FR> (Eq. 13)
At pH 7.2, kA (840-2200 s-1), k3' (1200 s-1), k5 (800 s-1), and k7 (960-3300 s-1) are nearly equal for wild type Y-PPase (11), and (1 - Pc) does not differ much from unity (Table II). Therefore, a mutation decreasing only k3' by a factor of n would decrease kh by a factor approaching (n + 3)/4. This criterion is met for most mutations, except for D120E and D152E, at pH 7.2 (Table II). For the latter mutations, the effect on kh is nearly equal to (D120E) or exceeds (D152E) the effect on k3', which can only result if, at least, one of kA, k5, and k7 is also decreased. The anomalous behavior of the D152E variant is preserved at pH 8.5. Moreover, despite a decrease in k3', kh is slightly increased in the E48D variant at pH 8.5, suggesting an increase in kA, k5, and/or k7. Thus, both PPi hydrolysis (k3') and Pi release are decelerated in the D152E variant, PPi hydrolysis is decelerated, and Pi release is accelerated in the E48D variant, and only PPi hydrolysis is affected in the other four variants. According to Equation 6, the effects on k3' may result from a decrease in k3 or KAB. At present, we cannot distinguish between these alternatives. The invariance of k5 in the Y93F, D115E, and D117E variants would mean that the decrease in their Pc values (Table II) results from a drop in k4. This effect is the largest with the D117E variant (6-13-fold) but, nevertheless, is much less than the effect on k3'.

The fluoride inhibition data support the notion that PPi conversion to Pi becomes rate-limiting in some variants. For D117E-PPase, values of ki in hydrolysis (Table III) and ki, app/feppfast (Table IV) are the same. This is possible if EM4PP* and EM4PP predominate during hydrolysis and are at equilibrium with each other, which is equivalent to k3 being the rate-determining step in hydrolysis. This is consistent with high KF1 value for this variant (Table III), indicating that the fraction of EM3P is quite low during hydrolysis. That EM3P, when present, binds fluoride well is indicated by its large effect on PPi synthesis by D117E-PPase (Fig. 7) by comparison with wild type PPase (11). The effect of the Y93F substitution on KF1 is also larger at pH 7.2 (Table III), where k3' is nearly rate-determining (Table II), than at pH 8.5, where k3' is not such.

Interestingly, changes in pH had an opposite effect on kh for all the variants, except for D115E, by comparison with wild type Y-PPase (Table II). This appears to result from a substantial alkaline shift in the bell-shaped pH profile for kh (28). As a result, the pH 7.2 and 8.5 points are on the descending limb of the pH rate profile for wild type Y-PPase and on its ascending limb for most variants. By contrast, the D115E substitution leaves the pH profile for kh nearly unchanged (28). The shift may be explained by a change in the pKa of the nucleophilic water (28, 29) or by inhibition of the three-metal pathway in the variant PPases (21).

Fluoride Binding Site-- The slow inhibition step results in the incorporation of one fluoride ion per substrate molecule, which remains intact and tightly bound to PPase (30), suggesting that fluoride replaces the nucleophilic water. Substitutions mispositioning its ligands are thus expected to affect fluoride binding. Computer modelling of the enzyme-PPi complex from the x-ray structure of Y-PPase and E-PPase suggested that the nucleophile is either Wat1, located between metal ions M1 and M2 and further liganded by Asp117 (Fig. 1 and Ref. 6) or the water molecule associated with M2 and Tyr93 (7). The results presented in Tables III and IV clearly favor Wat1 as the nucleophile, because they show that the D117E substitution does have the most drastic effect on F- binding, as characterized by ki, KF1, KF2, and ki, app/feppfast. Furthermore, a strong effect of the D120E substitution on F- binding is consistent with Asp120 being important for exact placing M1 and M2 and, hence, Wat1 (Fig. 1). The substitutions of Asp115 and Asp152, which are in the first coordination sphere of M1, and of Glu48, which is in the second coordination sphere of M2, apparently have smaller effects on Wat1 position, which correlates with much greater activity of the D115E, D152E and D48E variants by comparison with the D120E or D120N variants (Table II; see also Ref. 28). The Y93F substitution has only a moderate effect on fluoride inhibition, despite a more drastic nature of this substitution (the hydroxy group interacting with water is eliminated) by comparison with the D117E substitution (the side chain is elongated). For the same reason, similar effects of the two substitutions on different catalytic properties in Y-PPase (Table II; see also Ref. 28) and of the corresponding substitutions in E-PPase (29, 31) also point to a greater importance of Asp117 by comparison with Tyr93.

Several other lines of evidence support the identification of Wat1 as the water molecule substituted by fluoride. First, Wat1 is replaced with a Glu117 carboxylate oxygen in D117E-PPase (13), showing the largest decrease in fluoride binding. Second, as measured by differential UV spectroscopy, fluoride binding to Y-PPase in the absence of substrates requires metal ions M1 and M2 (10), the ligands to Wat1. Third, F- ion becomes entrapped together with two Mg2+ ions in the stable enzyme-F--PPi complex (12). Fourth, F- inhibition of E-PPase, in which the carboxylate of Asp67 (equivalent of Asp117 in Y-PPase) is moved away from M1 and M2 by about 1 Å by comparison with Y-PPase (7), exhibits only a fast step. Moreover, a D67N substitution results in appearance of the slow inhibition step in E-PPase (31), which is similar in this respect to Y-PPase. In a series of D/N substitutions in the active site of E-PPase, the D67N substitution had the largest effect on fluoride binding (31). Fifth, other dimetal hydrolases, including enolase (32, 33), aminopeptidase (34), purple acid phosphatase (35), and urease (36), presumably generating a hydroxide ion in the dimetal active site, are similarly inhibited by fluoride.

Mechanism of Fluoride Inhibition and Catalysis-- According to Scheme I, derived from studies of wild type Y-PPase (11), fluoride binds with different rates and stoichiometry to two enzyme intermediates. Effects of fluoride on Y-PPase variants provide further support to Scheme I. Thus, five of the six variants exhibit a biphasic transition in the amount of enzyme-bound PPi upon addition of fluoride (Fig. 4; compare values for fepp and feppfast in Tables II and IV), the rapid drop and slow rise corresponding to fluoride binding to EM3P and EM4PP*, respectively. These data are inconsistent with a consecutive binding model, in which rapid preassociation of a single enzyme form (EM4PP* in our case) with the inhibitor is followed by a slow conformational change, because such a model predicts an increase in the amount of enzyme-bound PPi during both rapid and slow phases. Besides, the involvement of two different intermediates is supported by the observation that the D120E (at pH 8.5) and D152E substitutions cancel the slow inhibition phase without significantly affecting the fast phase. These substitutions appear to greatly reduce the steady-state concentration of EM3P, which binds fluoride rapidly. The ability of EM3P to bind two F- ions, another important feature of Scheme I, is confirmed by the inactivation patterns seen in Figs. 6 and 7 for the D117E variant.

The fluoride binding data obtained for the E48D and D115E variants confirm the occurrence of two PPi-containing intermediates (EM4PP* and EM4PP in Scheme I) in PPase catalysis. For both variants, ki values (Table III) are greater than ki, app/feppfast values (Table IV), despite the fact that the former parameter refers to the total enzyme, of which only a fraction contains bound PPi during steady-state hydrolysis, and therefore underestimates the rate constant for fluoride binding. Importantly, the ki, app/feppfast values are true rate constants for fluoride binding with the sum of EM4PP* and EM4PP. Like with wild type Y-PPase, this difference can be only explained by existence of two PPi-containing intermediates that react with fluoride at substantially different rates (11). Independent evidence for two PPi-containing intermediates was obtained in presteady-state measurements of PPi hydrolysis.2

The nature of the structural changes accompanying the EM4PP* to EM4PP transition still remains to be determined. An attractive hypothesis presented below is based on the observation that the metals M1 and M2 are bridged by two water molecules in EM2 (6, 9) and by only one water molecule in EM4P2 (6) and EM4PP.3 We suggest that the two-water bridge is preserved upon initial substrate binding (i.e. in EM4PP*), but one water molecule is expelled during the transition to EM4PP (Scheme III), consistent with a decrease in the pKa of the essential basic group, attributable to Wat1, from >7.0 to 5.8 (21). Recent model studies of Kaminskaia et al. (37) have indicated no substantial increase in nucleophilicity for a bridging water versus terminally bound water. Thus, the M1-Wat1-M2 structure apparently helps to favorably position Wat1 with respect to the attacked Pi residue. Scheme III differs from Scheme I in that Scheme I shows fluoride interaction with EM4PP* as one bimolecular reaction, whereas in Scheme III this interaction involves rapidly reversible binding of fluoride to EM4PP*, with a dissociation constant of K'F, and slow conversion of the resulting unstable intermediate, with a rate constant of k'A, to a form having a fluoride ion between M1 and M2. However, the two mechanisms are kinetically equivalent, provided that the bottom left species in Scheme III is stoichiometrically insignificant, i.e. K'F [F]. In terms of Scheme III, ki is equal to k'A/K'F. Given the chemistry of fluoride, the M1-F-M2 complex is likely to be very stable, making the reverse reaction, which requires insertion of a water molecule, very slow. For this reason, it is not shown in Scheme III. On the other hand, the bound PPi cannot hydrolyze and leave as Pi. This explains why fluoride stabilizes the enzyme-substrate intermediate to an extent allowing its isolation by gel filtration (30). At neutral pH, the bridging water molecule exists predominantly as OH- in EM4PP and, presumably, EM4P2 (21), which explains their very slow, if any, binding of the negatively charged fluoride ion. The proposed structure of EM4PP* is consistent with the results of recent x-ray crystallographic studies of the fluoride-inhibited PPi complex of Y-PPase, showing a single electron density between M1 and M2.3 By contrast, two water molecules are expected to be present between M1 and M2 in EM3P because fluoride binding to this species is relatively weak and proceeds in a rapidly reversible manner with a stoichiometry of two per active site. The unsurpassed stability of the F-H-F hydrogen bond (38) would favor the M1-F-H-F-M2 structure, despite the low pKa value (3.2) for hydrofluoric acid.



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Scheme III.   Proposed mechanism of fluoride binding to EM4PP*. W, water.



    FOOTNOTES

* This work was supported by Academy of Finland Grants 35736 and 47513 and Russian Foundation for Basic Research Grants 00-04-48310 and 00-15-97907.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.

Dagger Dagger To whom correspondence may be addressed. Tel.: 95-939-5541; Fax: 95-939-3181; E-mail: baykov@genebee.msu.su.

§§ To whom correspondence may be addressed. Tel.: 358-02-333-6845; Fax: 358-02-3336860; E-mail: reijo.lahti@utu.fi.

Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M007360200

2 P. Halonen et al., manuscript in preparation.

3 P. Heikinheimo, V. Tuominen, A.-K. Ahonen, A. Teplyakov, B. S. Cooperman, A. A. Baykov, R. Lahti, and A. Goldman, submitted for publication.


    ABBREVIATIONS

The abbreviations used are: PPase, inorganic pyrophosphatase; E-PPase, E. coli PPase; PPi, pyrophosphate; WT, wild type; Y-PPase, yeast (Saccharomyces cerevisiae) PPase; 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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