ARCHIVES OF BIOCHEMISTRY ANII BIOPHYSICS Vol. 294, No. 1, April, pp. 238243,1992

A Two-Step Mechanism of Fluoride Inhibition of Rat Liver Inorganic Pyrophosphatase Alexander A. Baykov,’ Anatoliy P. Alexandrov, and Irina N. Smirnova A. N. Belozersky Laboratory of Molecular Biology and Bioorgank Chemistry, Moscow State University, Moscow 119899, USSR

Received October 18,199l

Product formation curves for inorganic pyrophosphatase-catalyzed hydrolysis of pyrophosphate in the presence of fluoride were analyzed in order to get insight into the mechanism of its inhibitory action on this enzyme. The enzymatic reaction was monitored with a phosphate analyzer operating on the time scale of seconds. Inhibition patterns were virtually identical for cytosolic and mitochondrial pyrophosphatases. The effect of fluoride was biphasic: it caused a rapid (tllB < 1 s) decrease in the initial velocity of the reaction followed by slow (tuz 3 4 s) inactivation of the enzyme during catalysis. The slow phase resulted in trapping intact substrate at the active site, and the resulting complex could be isolated by gel filtration. Pyrophosphatase remained active when incubated with fluoride in the absence of pyrophosphate or in the presence of its bisphosphonate analogs, which are bound to but not hydrolyzed by this enzyme. These features of the inhibition are consistent with the mechanism in which rapid binding of the inhibitor to the enzyme- substrate complex is followed by its slow isomerization. Kinetic parameters obtained in this work indicate that appreciable inactivation of pyrophosphatase can occur at fluoride concentrations found in human plasma. This e5ect may therefore be one of the major factors contributing to fluoride toxicity. @isea Academic hap, IDC.

Fluoride inhibits many enzymes but only a few of them, including inorganic pyrophosphatase (EC 3.6.1.1), are sensitive to micromolar concentrations of this substance (1,2). This inhibitor is therefore a useful probe of cellular pyrophosphate metabolism. Most mechanistic studies of fluoride effects were performed with baker’s yeast pyrophosphatase. Fluoride was found to inactivate this enzyme relatively slowly during Mg2+-supported hydrolysis of PPi (3). The inactivated enzyme, which is a tight EPPiMgzF complex (4,5), could ’ To whom correspondence 238

should be addressed.

be isolated by gel filtration since its reactivation and release of the bound ligands proceed on the time scale of hours. The reactivation could be accelerated manyfold by metal-chelating agents (6). No tight binding of fluoride was observed in the presence of other metal ions, such as Zn2+ and Mn2+ (7). These features of the inhibition reaction led to a hypothesis that fluoride substitutes for the metal-bound water molecule, which acts as the nucleophile during P-O bond cleavage (4). Not all pyrophosphatases, however, interact with fluoride in the same manner. Some bacterial pyrophosphatases are inhibited in a rapidly reversible manner (8) while some are not affected by fluoride at all (9-11). In this work, we studied the kinetic mechanism of fluoride action on animal cytosolic and mitochondrial pyrophosphatases. The results support the idea that pyrophosphatase inhibition may be related to the toxic effects exerted by fluoride. MATERIALS

AND

METHODS

Hat liver cytosolic (12) and mitochondrial(13) pyropbosphatases were isolated as described. Solutions of the enzymes (0.25-5 mg/ml) in 25 mM Tris-HCl buffer (pH 7.5 at 4’C) containing 1 mM MgSO,, 1 mM dithiothreitol, 10 pM EGTA,’ and 0.05% bovine serum albumin were kept frozen at -25“C. After being thawed, they were supplemented with fresh dithiothreitol to 5 mM and allowed to stand for at least 24 h at 4°C. On the day of use they were diluted to 25-50 pg/ml with 0.1 M Tris-HCl (pH 7.2) containing 10 pM EGTA, 1 mM dithiothreitol, 0.05% albumin, and MgC!lz at a concentration used in subsequent experiments. The final stock solutions were kept at room temperature with no loss of activity for at least 6 h. Most chemicals were obtained from Sigma Chemical Co. Sodium fluoride (ultrapure grade) was purchased from Heachim (USSR). Continuous recordings of Pi liberation during PPi hydrolysis by pyrophosphatase were obtained with an automatic analyzer (14) at a sensitivity of 50-200 gM Pi per recorder scale. The basic principle of the analyzer operation is to mix portions of the sample with acidic molybdate and Malachite green solutions and measure the resulting absorbance in a continuous way. The inlet system of the analyzer was modified to

2 Abbreviation used: EGTA, ethylene glycol bis(@-aminoethyl N, N’-tetraacetate.

ether)

ooo3-9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

FLUORIDE

INHIBITION

OF ANIMAL

decrease time between feeding the reaction medium into the analyzer and inactivating the enzyme on mixing with the acid/molybdate solution (Fig. 1). The solution to be analyzed was continuously withdrawn from the reaction vessel at a rate of 1.2 ml/min due to differences in the flow rates for the two tubes and terminated in 1 s in the T-joint. The compositions of the dye and acid/molybdate solutions were suitably changed to ensure that the concentrations of all reagents in the measuring cuvette be identical to those used in the original method (14). PPi hydrolysis was monitored for 5-20 min at 25“C and was initiated by adding lo-50 pl of enzyme solution to a lo-ml reaction mixture containing 0.1 M buffer, 10 pM EGTA, 0.2 mM PPi, and varying concentrations of MgClz and NaF. The buffers used were N-4-morpholineethanesulfonic acidNaOH (pH 6.0), Tris-HCl (pH 7.2, 7.4, and 8.5) or 2-amino-1,3-propanediol-HCl (pH 9.3). Incubations of pyrophosphatase with NaF in the presence of substrate analogs were performed at 25’C in a total volume of 0.1 ml. The incubation medium contained 2.5 fig/ml enzyme, 10 mM NaF, 0.1 M TrisHCl (pH 7.2), 1 mM MgClz, 0.2 mM PPi analog, 5 mM dithiothreitol, 10 pM EGTA, and 0.05% bovine serum albumin. Pi was added at a conwas increased to 5 centration of 10 pM, and the MgCl, concentration mM in this case. Aliquots of the incubation mixture (50 ~1) were withdrawn in 5 min and assayed for activity in the presence of 2 mM MgCl, as described above. For measurement of PPi content of the inactivated enzyme, the reaction with fluoride was performed in the following way. A solution of 0.8 mg/ml enzyme in 0.1 M 4-morpholineethanesulfonic acid-Tris buffer (pH 6.2) containing 2 mM MgClr ,2 mu dithiothreitol, and 20 NM EGTA was mixed with an equal volume of 0.2 M NaF containing 1.35 mM PPi and incubated for 3 min at 22°C. A 100~~1 portion of the mixture was subjected to centrifugal gel filtration (15) using 1 ml of Sephadex G-50 equilibrated with 0.1 Tris-HCl (pH 7.2) containing 2 mM MgClz, 2 mM dithiothreitol, and 20 pM EGTA. The amount of PPi in the eluate was assayed enzymatically with ATP-sulfurylase and luciferase as described previously (16). Appropriate controls indicated that the gel filtration procedure could separate all unbound PPi . To compensate for partial sequestration of Mg2+ due to formation of the MgF+ complex, which has a dissociation constant of 48 mM (17), the concentration of MgClz in the reaction mixture was increased by a factor of (1 + [F]/48) compared to that in the related experiment without any Auoride added. Here, [F] is inhibitor concentration in millimoles per liter. All concentrations of MgCIZ mentioned in the text do not include MgF+. Curve fitting was performed using a program for nonlinear regression analysis (18).

RESULTS

Kinetics of Cytosolic Pyrophosphatase Fluoride

239

PYROPHOSPHATASE

inactivation and reactivation, respectively, v. is initial rate of Pi formation and t is time.

This equation was computer-fitted to each separate product formation curve represented with 20-25 pairs of [Pi] and t values, of which at least half belonged to the initial nonlinear part. This procedure yielded the values of vo, #, and k, and their standard errors. Differences between measured values of [Pi] and those predicted by Eq. [l] did not exceed 2% of the total “burst” values. The dependencies of v. and kf on inhibitor concentration in the presence of 5 mM MgC& at pH 7.2 are shown in Fig. 3. Both dependencies were clearly hyperbolic. The value of lz,, on the other hand, did not change with fluoride concentration. Qualitatively similar results were obtained at all sets of pH and MgClz concentrations used in this work. Such behavior is characteristic of a two-step mechanism of slow-binding inhibition (19) which is shown in Scheme I. According to this mechanism, fluoride rapidly binds to the enzyme. PPi complex and the resulting fluoride complex undergoes slow isomerization. Both complexes of the enzyme with fluoride are catalytically inactive. Formation of EPPiF is characterized by the dissociation constant KF and accounts for the decrease in v. in the presence of fluoride (Fig. 2). Thus, the inhibitor is proposed to bind to the enzyme-substrate complex, and data supporting this feature of the inhibition mechanism are presented below. E-EPPi-E+P-

r

KP I J EPP,F )

Inactivation

1

by SCHEME

ki

k

EPPiF*

1

In the absence of fluoride, hydrolysis of PPi proceeded linearily until most of the substrate was converted. If, The equations relating v. and kf to fluoride concentrahowever, fluoride was added, the reaction rate decreased tion in terms of the mechanism in Scheme I are gradually until a constant level was reached (Fig. 2). The initial rate of Pi formation was also lower compared to 4 that measured in the absence of the inhibitor. Further ” = 1 + [F]/K, ’ addition of PPi after onset of the enzyme-substrate equilibrium did not affect the rate of Pi formation while ad- and dition of fresh enzyme resulted in a new “burst” of Pi formation (data not shown). kf = ki [31 At a constant concentration of the inhibitor, the in1 + KF/[FI . activation could be treated as a reversible first-order reaction as described earlier for the yeast pyrophosphatase Figure 3 shows that these equations fitted the experi(3). Product formation curves could be analyzed using mental data quite satisfactorily. The fitting procedure Eq. [l], where kf and kr refer to the rate constants of yielded the values of KF and $. All parameters obtained

240

BAYKOV, ALEXANDROV,

AND SMIRNOVA

TO PHOTOMETER

I I

100

PERISTALTIC

60 8

4.4 I

DYE I

INLET TUBING

60

9 40 20

VESSEL FIG. 1. The modified part of the automatic ures at the pump refer to flow rates (ml/min). inlet tubing was 0.02 ml.

phosphate analyzer. FigThe inner volume of the

in this way at different sets of pH and Mg2+ concentration are summarized in Table I. The values of KF obtained from the dependencies of u. and kt agreed reasonably well in all cases. Kinetics of Mitochondrial Pyrophosphatase Inactivation by Fluoride The effect of 1.75-20 mM fluoride on mitochondrial pyrophosphatase in the presence of 1 mM MgC12 at pH 7.4 was analyzed in the same way. The results were found to agree with Scheme I, and the following values of the parameters were obtained: KF = 3.0 f 0.6 mM, h = 4.8 + 0.4 min-‘, lz, = 0.04 ? 0.09 min-‘. The last parameter was estimated with a large error because of the limited amount of the enzyme available. It is seen that the values of all parameters are quite close to those obtained for the cytosolic enzyme. Substrate Requirement for Cytosolic Pyrophosphatase Inactivation The experiments reported above indicate that fluoride effects inactivation of pyrophosphatase during catalysis.

NaF, mM FIG. 3. Dependencies of IA, and # for cytosolic pyrophosphatase on fluoride concentration at pH 7.2 in the presence of 5 mM MgClr. The curves show the best fit for Eqs. [2] and [3].

When incubated with fluoride in the absence of any substrate, the enzyme retained its full activity (Table II). Moreover, no inactivation was observed when pyrophosphatase was incubated with fluoride in the presence of a number of bisphosphonate analogs of PPi which are tightly bound to but not hydrolyzed by the rat liver enzyme (12). Substantial inactivation occurred, on the contrary, in the presence of imidodiphosphate, which is slowly hydrolyzed by this enzyme (12), or Pi, which is the substrate for the reaction in the direction of PPi synthesis. Pyrophosphute Content of the Inactivated Cytosolic Pyrophosphutase Previous studies have indicated that intact PPi molecule is trapped at the active site of yeast pyrophosphatase during its inactivation by fluoride (4). Experiments of similar kind were performed with the rat liver pyrophosphatase using, however, a more specific, enzymatic assay for PPi. Pyrophosphatase was incubated with 0.1 M fluoride in the presence of PPi and separated from the reTABLE I of the Kinetic Parameters for Cytosolic Pyrophosphatase Inhibition by Fluoride

Summary

Time FIG. 2. Actual recordings of Pi formation during PPi hydrolysis by pyrophosphatase (0.2 pg/ml) in the presence of 10 mu NaF. (a) Complete system, (b) no fluoride added, (c) no enzyme added The arrow shows the moment of enzyme addition. Conditions: pH 7.2,0.2 mM MgCl,.

PH

lM&l,I bM)

6.0 7.2 7.2 1.2 8.5 8.5 9.3

1 0.2 1 5 0.2 1 1

ki

k,

(min-‘)

(min-‘)

KF'

bM) 0.98 11.7 4.3 5.6 10.6 4.2 11.8

+ 0.12 _+ 0.8 + 0.4 + 0.2 + 6.7 k 0.2 + 1.5

(0.99 (9.3 (3.0 (7.7 (8.8 (5.7 (7.3

f 0.17) -t 2.9) f. 0.6) + 0.6) z!c1.4) k 0.8) + 0.9)

8.0 7.6 6.8 6.9 10.6 5.2 7.4

f k f + f + +

0.4 0.3 0.3 0.3 2.7 0.9 0.6

0.044 0.023 0.014 0.013 0.037 0.042 0.041

+ 0.012 -+ 0.002 f 0.001 + 0.001 2 0.002 + 0.005 + 0.002

’ Values without parentheses and in parentheses were obtained from the dependencies of kj and v,, respectively.

FLUORIDE TABLE

INHIBITION

OF ANIMAL

II

The Effect of Substratesand Their Analogs on the Inactivation of Cytosolic Pyrophosphataseupon Incubation with 10 mM Fluoride

Ligand

No. 1 2 3 4 5 6 7

None Pyrophosphate (0.2 mM) Imidodiphosphate (0.2 mM) Phosphate (10 mM) Hydroxymethylenebisphosphonate (0.2 mM) Aminomethylenebisphosphonate (0.2 mM) 1-Methyl-l-hydroxymethylenebisphosphonate (0.2 mM)

Residual activity 6) 100 3 I!I 0.5 19 f 2 70 + 6 104 + 6 95 2 4 97 + 2

action mixture by gel filtration. The incubation was performed at pH 6.2 in order to ensure that substantial inactivation occurred before all substrate could be hydrolyzed by the relatively high amounts of enzyme used in this experiment. The enzyme, whose residual activity was 27% after the gel filtration, was treated with perchloric acid to release any bound PI’,, and its amount was measured with a coupled-enzyme assay utilizing ATP-sulfurylase and luciferase (16,20). The stoichiometry of PP, incorporation could be roughly estimated to be 0.7 f. 0.1 mol/mol, assuming M, = 70,000 (12) and a tentative value of 10 for && No bound PPi was found if NaF was omitted from the incubation medium, in accord with the finding that native pyrophosphatase of rat liver does not contain any PPi (16). DISCUSSION Three critical observations were made in this work regarding the fluoride inhibition of rat liver pyrophosphatase. First, the inhibition occurs in two steps: an instant decrease in PPi hydrolysis rate followed by a more slow inactivation step. Second, the occurrence of, at least, the slow step requires that the enzyme be active as a catalyst. And finally, intact substrate becomes trapped at the active site in the presence of fluoride. The simplest rationale for these observations is provided by the model presented in Scheme I. According to this model, the rapid inactivation step refers to inhibitor binding while the slow step represents isomerization of the enzyme * inhibitor complex. The first step could not be resolved in time using the current assay procedure and is therefore characterized by the dissociation constant KF. Rapidly reversible binding of fluoride to the free enzyme, although quite possible, was not considered here because it represents less than 1% of total enzyme in the presence of 200 I.LM PPi used in this work (21). For the second step, which occurs only in the enzyme * substrate complex, both

PYROPHOSPHATASE

241

forward and backward rate constants could be estimated from continuous recordings of PPi hydrolysis in the presence of fluoride. Interestingly, the value of Iziwas virtually independent of pH and Mg2+ ion concentration (Table I) although two other constants and enzyme activity (21) varied considerably. The value of the backward rate constant lz, was quite low at pH 7.2 in the presence of 1-5 mM Mg2+ and increased severalfold if the metal concentration was decreased or the pH was varied in either direction, in accord with the previous data on yeast pyrophosphatase (5). Among the parameters which characterize the interaction between pyrophosphatase and fluoride, KF was the one most affected by changes in reaction conditions. At a constant concentration of the metal ion activator (1 mM), KF decreased by one order of magnitude when the pH was decreased from 9.3 to 6.5 (Table I). This effect may be, at least, partly explained by assuming that the active fluoride species is HF, in view of the kinetic and X-ray data favoring HF as the binding species for cytochrome c peroxidase (22, 23). Dependence of KF on Mg2+ concentration at a constant pH (Table I) can be explained by the fact that the enzyme * PPi complex, which is a predominant form of the cytosolic enzyme at the high substrate concentration used in the present work, can bind from two to four Mg2+ ions (21). At least three metal ions are required for catalysis to occur, and the fourth one is partially inhibitory. The dissociation constants characterizing the binding of the third and fourth metal ions have been determined to be 0.15 and 0.43 mM, respectively, at pH 7.2 (the value of the latter constant is not mentioned in the cited paper but can be calculated as K&“/K,,, (21)). Using these constants one can estimate relative amounts of the three forms of the enzyme *PPi complex in the presence of 0.2, 1, and 5 mM MgC& and further correlate them with the affinity to fluoride. Such analysis, which is not given in detail here, showed that the enzyme * PPi complex containing two Mg2+ ions does not bind fluoride at all while the two other complexes containing 3 and 4 Mg2+ bind fluoride with nearly equal dissociation constants of 3.7 and 6.5 mM. This means that fluoride binding requires the presence of at least three Mg2+ ions, in addition to the substrate, in the active site. Qualitatively, the inhibition pattern for rat liver pyrophosphatase is similar to that reported previously for yeast pyrophosphatase (3,4, 24) since inhibition is timedependent on the usual time scale of enzyme assays and results in trapping PPi at the active site. For both enzymes, inactive complex with PPi is very stable and can be isolated by gel filtration. The strength of the inhibition is, however, higher for the rat liver enzyme. Thus the apparent inhibition constant, which is defined as the concentration of the inhibitor decreasing the activity by onehalf after onset of the equilibrium of the enzyme-fluoride

242

BAYKOV,

ALEXANDROV,

interaction (K&/kJ, is 7 pM for the rat liver enzyme and 21 PM for the yeast enzyme (3) at pH 7.2,l mM MgCl,. The effects of fluoride on several other enzymes are similar in some respects to those observed for pyrophosphatase. Thus temperature-jump studies of fluoride binding to peroxidase has revealed three steps, of which the last two have been identified as isomerization reactions (25). Low rates of fluoride inhibition have been observed for lactase (26), esterase (27, 28), enolase (29), and ATPase (30). Positive cooperativity of substrate (Pi) and fluoride binding has been reported for enolase (31) and succinate dehydrogenase (32). Pyrophosphatase inhibition is unique in that its mechanism combines all features observed separately with other enzymes. The molecular mechanism of the fluoride effect on pyrophosphatase still remains to be elucidated. The observations that inhibition depends on the presence of enzyme-bound metal ion and is more strong in the presence of Mg2+ than in the presence of Zn2+ (7) suggest that the fluoride-binding site is closely associated with the metal ion. It is clear that fluoride blocks the P-O breakage step since the inactivated enzyme contains intact PPi. This effect can be caused by displacement of the active water molecule from the coordination sphere of Mg2+ or by affecting its nucleophilicity if the binding of the two ligands is not mutually exclusive. It is noteworthy that NMR studies of enolase have revealed that fluoride displaces one of the two water molecules from the inner coordination sphere of the enzyme-bound Mn2+ (33, 34). The nature of the slow isomerization step provides another challenging problem. Its rate is virtually independent of pH and Mg2+ concentration, as well as the source of pyrophosphatase (3). Isomerization may represent a process specifically induced by fluoride or a true catalytic step whose rate is suppressed by the inhibitor. Kuranova and Sokolov have recently suggested that PPi hydrolysis involves formation of a neutral bicyclic intermediate in which two Mg2+ ions are linked to two pairs of oxygen atoms belonging to different phosphate residues (35). One can hypothesize that fluoride stabilizes this intermediate by preventing the attack of the water nucleophile on it. The absence of the isomerization step with the bisphosphonate analogs of PPi may then mean that the formation of this intermediate critically depends on the interaction of the third Mg2+ ion, which contains the water molecule in its coordination sphere, with the bridge oxygen atom of PPi. Evidence for the importance of such an interaction in catalysis has been obtained previously in the studies of imidodiphosphate hydrolysis by pyrophosphatase (36). Clearly, further work is required to elucidate the molecular mechanism of fluoride effects and the results of such work may prove very helpful in defining the mechanism of pyrophosphatase catalysis. Although fluoride has long been known to be a toxic agent, the mechanism of its toxicity has not yet been fully characterized. The present data show that pyrophospha-

AND SMIRNOVA

tase is most sensitive to fluoride among the enzymes whose activity is affected by this substance. Thus the equilibrium inhibition constant for rat liver cytosolic pyrophosphatase is an order of magnitude lower compared to that for enolase, whose inactivation has been implicated in the effect of fluoride on glycolysis (31). Significant inhibition of pyrophosphatase therefore can occur even at the low fluoride concentration found in human saliva, about 5.5 PM (37). This may lead to an increase in cellular PPi, which is a potent inhibitor of a number of vital processes, such as fatty acid oxidation (38), protein and nucleic acid synthesis (39, 40), and bone formation and growth (41, 42). Pyrophosphatase inhibition may thus be one of the major reasons of fluoride toxicity. ACKNOWLEDGMENT We thank Dr. A. Unguryte

for her help in this work.

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FLUORIDE 24. Smirnova,

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INHIBITION

A. A. (1983) Biokhimiya

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A two-step mechanism of fluoride inhibition of rat liver inorganic pyrophosphatase.

Product formation curves for inorganic pyrophosphatase-catalyzed hydrolysis of pyrophosphate in the presence of fluoride were analyzed in order to get...
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