Mg2+and Other Polyvalent Cations Catalyze Nucleotide Fluorolysis

Mg2+and Other Polyvalent Cations Catalyze Nucleotide Fluorolysis

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 334, No. 2, October 15, pp. 332–340, 1996 Article No. 0462 Mg2/ and Other Polyvalent Cations Catalyze N...

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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 334, No. 2, October 15, pp. 332–340, 1996 Article No. 0462

Mg2/ and Other Polyvalent Cations Catalyze Nucleotide Fluorolysis Robert E. London1 and Scott A. Gabel Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Box 12233, Research Triangle Park, North Carolina 27709

Received March 21, 1996, and in revised form August 6, 1996

The reaction of fluoride with adenosine triphosphate has been studied as a nonenzymatic analog of the pyruvate kinase-catalyzed fluorokinase reaction. The production of fluorophosphate, as well as adenosine 5*-O-fluorophosphate (FAMP) and adenosine 5*-O(2-fluorodiphosphate) (bFADP) was found to be dependent on the presence of polyvalent metal ions. All ions tested showed significant activity. Two catalytic regimes for the cations could be distinguished: a less specific enhancement of product formation at lower fluoride/cation ratios, and a considerably more active and specific (for fluorophosphate production) enhancement at high fluoride/cation ratios. A comparison of the results with studies of cation-catalyzed nucleotide hydrolysis indicates that the fluorolysis mechanisms are analogous to the hydrolysis by hydroxyl ions observed at high pH. In addition to these nonenzymatic studies, experiments performed using several commercially available kinases indicated significant fluorokinase activity for two: glycerokinase and acetate kinase, although the activities were much below that of pyruvate kinase. With the exception of the concentrations used in these studies, these reactions proceed under physiological conditions, yielding products at sufficient concentrations to be readily detected by 19F NMR spectroscopy. Key Words: Fluoride; fluorolysis; nucleotide fluorolysis; fluorophosphate; adenosine 5*-O-fluorophosphate; adenosine 5*-O-(2-fluorodiphosphate); 19F NMR.

Fluoride is a constituent of tooth enamel and bone (as fluorapatite) (1, 2) and has been demonstrated to play a significant role in the prevention of dental caries (3). At high levels, it can also produce dental defects 1

To whom correspondence and reprint requests should be addressed.

and other toxic symptoms (3). Epidemiological studies continue to suggest a possible link between high fluoride exposure and bladder cancer (4). As a consequence of the chronic exposure of the population to fluoride which results from fluoridation of the water supply as well as endogenous and exogenous catabolic processes which degrade fluorinated drugs, pesticides, and herbicides to inorganic fluoride, it is important to understand fluoride biochemistry. In addition to its extensively studied properties as an inhibitor of various metalloenzymes (5–10), and the more recently proposed role of metal fluorides as phosphate-mimetic effectors of enzyme function (11–15), biochemical incorporation of fluoride into other metabolites has received little attention. Nevertheless, the formation of the highly toxic compound monofluoroacetate from fluoride by various species of plants is well documented (16, 17). Additionally, it has been known for nearly 40 years that mammalian cells have the capability of converting fluoride into monofluorophosphate as a consequence of the ‘‘fluorokinase’’ activity of pyruvate kinase (18–21): Pyruvate kinase

ATP

40

/F

0

[1]

and this reaction has recently been demonstrated for PEPCK2 as well (22). Given the ubiquitous presence of fluoride in biological tissues and the many biochemical studies in the literature using fluoride as an effector of 2 Abbreviations used: ADP, adenosine diphosphate; CDTA, trans-1,2diaminocyclohexane-N,N,N*,N*-tetraacetic acid; bFADP, adenosine 5*O-(2-fluorodiphosphate); FAMP, adenosine 5*-O-fluorophosphate; FUMP, uridine 5*-O-fluorophosphate; FPPi , monofluoropyrophosphate; Hepes, N-[2-Hydroxyethyl]piperazine-N*-[2-ethanesulfonic acid]; NMR, nuclear magnetic resonance; PAPS, 3*-phosphoadenosine-5*-phosphosulfate; PADPbF, 3-phosphoadenosine-5*-bfluorodiphosphate; PEPCKphosphoenol pyruvate carboxykinase; Tris, Tris(hydroxymethyl)amino methane.

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various enzymatic processes, a more extensive study of this reaction seemed warranted. The biochemistry of ATP most typically involves nucleophilic substitution at one of the phosphorus atoms, with the displacement of pyrophosphate, AMP, or ADP as leaving groups. This biochemistry typically involves at least one divalent cation (23–25) such as Mg2/ or Mn2/. It is therefore not surprising that the fluoride ion could play the role of the nucleophile, and it appeared reasonable that analogous polyvalent cation-catalyzed, nonenzymatic reactions could exist. We have therefore analyzed the effects of various metal ions on the reaction of fluoride with purine nucleotides. In addition, the possibility that other kinases possess fluorokinase activity has been investigated. MATERIALS AND METHODS All chemicals used, as well as Type III rabbit muscle pyruvate kinase, Escherichia coli glycerokinase, E. coli acetate kinase, adenosine-5*-triphosphate sulfurylase (derived from Brewer’s yeast), and inorganic pyrophosphatase (derived from bakers yeast) were obtained from Sigma (St. Louis, MO). One unit of pyruvate kinase is defined as the enzyme activity which will convert 1 umol of phosphoenolpyruvate to pyruvate/min at pH 7.6, 377C; 1 unit of glycerokinase will convert 1 umol of glycerol and ATP to L-a-glycerophosphate and ADP per min at pH 9.8, 257C; 1 unit of acetate kinase will phosphorylate 1 umol of acetate to acetyl phosphate per min at pH 7.6, 257C. Sodium fluorophosphate was obtained from Strem Chemicals, Inc. (Newburyport, MA). NMR studies were performed on solutions immersed in boiling water for 2 h or in solutions maintained at 377C for longer periods of time, unless otherwise noted. Depending on the composition of the sample, the pH typically dropped by several tenths to 1 pH unit over the course of the experiment. Since there is minimal titration of the products occurring near neutral pH, no pH adjustments were made, unless otherwise stated. However, further lowering of the pH improves the resolution of the fluorophosphate and b-fluoronucleoside diphosphate resonances due to the 0.84 ppm 19 F titration shift of the former. o-fluorobenzamide was used as an internal shift and intensity standard, but 19F shift data are referenced to external trifluoroacetate (d(o-fluorobenzamide) Å 038.8 ppm). 31P shifts are referenced to external 85% H3PO4 . Adenosine 5*-O-(2-fluorodiphosphate) (bFADP) was prepared from adenosine5*-triphosphate and monofluorophosphate using sulfurylase and inorganic pyrophosphatase, following the procedure of Satischandran et al. (26). Kinetic rate constants at 377C were obtained by measuring resonance areas as a function of time after making up the solutions. The sum of the areas of the two fluorophosphate doublet resonances was compared with the area of 1 mM o-fluorobenzamide which was present in the solution. Both 19F– 31P dipolar and chemical shift anisotropy relaxation mechanisms contribute significantly to the relaxation rates of fluorophosphate resonances. As a result of the cross correlation between the two mechanisms, the relaxation times of the two fluorophosphate resonances differ significantly (27). Inversion recovery determinations of the T1 values at 377C on a solution containing fluorophosphate and o-fluorobenzamide gave T1 values of 4.77 s, 2.54 s, and 3.24 s for the two fluorophosphate resonances and o-fluorobenzamide peaks, respectively. The fact that the T1 for o-fluorobenzamide is intermediate between the value for the two fluorophosphate resonances suggests that even under overpulsed conditions, the errors involved in comparing the intensities of the fluorophosphate resonances and the o-fluorobenzamide resonance will largely cancel. This conclusion is readily verified by considering the expression for

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the signal intensity of a resonance as a function of the flip angle, u, and the ratio of interpulse delay to T1 , r Å Dt/T1 :

S(u, r) Å

KN1/2(sin u)(er 0 1) , er 0 cos u

[2]

where N is the number of free induction decays collected and K is the appropriate constant of proportionality which includes M0 and the various spectrometer response coefficients (28). For an interpulse delay of 1.6 s (the shortest q value used in these studies, and a flip angle of 707, and setting K N Å 1, the values of S(u,r) Å 0.361, 0.541, and 0.469 for the fluorophosphate doublet and o-fluorobenzamide, respectively. The ratio of the mean fluorophosphate saturation factor to that for o-fluorobenzamide is 0.963. The ratio for an interpulse delay of 5.1 s increases to 0.969. These factors were considered sufficiently close to 1 so that no further corrections were introduced in comparing the total fluorophosphate resonance intensity to that of the o-fluorobenzamide standard. It was assumed that all changes in concentration are linearly time dependent, so that the intensities observed for spectra obtained between t and t / Dt were plotted vs t / Dt/2. In a few cases where the appearance of fluorophosphate required days to develop, samples were maintained in a constant temperature bath and then placed in the spectrometer for the determination. Kinetic measurements on pyruvate kinase were also performed in solutions in sealed NMR tubes under an argon atmosphere after a 10-min period of bubbling with argon gas in order to remove dissolved CO2/HCO0 3 . In the studies with acetate kinase and glycerokinase, CDTA or EGTA (4 mM) was added in order to suppress activity due to contamination with other divalent cations such as Ca2/. NMR experiments were performed on a General Electric GN-500 NMR spectrometer using a 5-mm 19F probe or, for 19F {31P} 2D NMR studies, a 10-mm 31P probe in which the decoupler coil was tuned to the 19F NMR frequency of 470.528 MHz. The 2D HMQC experiment (29, 30) was performed in the phase-sensitive, absorption mode (31). A delay of 2 s between scans was employed.

RESULTS AND DISCUSSION

Product Identification The 19F NMR spectra obtained from several studies in which ATP or other nucleotides, MgCl2 , and NaF were immersed in boiling water at an initial pH of 7.0 for a period of 2 h are summarized in Fig. 1. The reaction of ATP with fluoride in the presence of magnesium yields three doublets: d1 Å 2.25 ppm, 1JFP Å 934 Hz; d2 Å 1.94 ppm, 1JFP Å 868 Hz; d3 Å 04.34 ppm, 1JFP Å 934 Hz, with 1JFP coupling constants typical of the fluorophosphates (32–36). Identification of the 1.94 ppm doublet as monofluorophosphate based on shift and coupling constant data, and ultimately on the addition of authentic material, is unequivocal. The reaction of fluoride with ADP (Fig. 1B) results in the production of both monofluorophosphate and a second, upfield doublet at 04.34 ppm, also seen in the ATP-derived spectrum. This doublet is assigned to the adenosine 5*-Ofluorophosphate (FAMP) based on coupling constant data (33) and on expectations based on the analogy to ADP hydrolysis chemistry (24). Since the a-phosphate group is sufficiently close to the nucleoside base to exhibit a significant shift sensitivity, the reaction corresponding to Fig. 1A was repeated substituting a 50:50

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FIG. 1. 19F NMR spectra of the products formed by heating mixtures of 0.1 M NaF, 0.09 M MgCl2 , and (A) 0.1 M ATP; (B) 0.1 M ADP; (C) 0.05 M ATP / 0.05 M UTP, at 1007C for a period of 2 h. Chemical shifts are relative to (external) trifluoroacetate. The inorganic fluoride resonance is typically at Ç044 ppm on this shift scale and generally exhibits significant broadening due to exchange due to formation of cation complexes.

mixture of ATP:UTP for the ATP. The resulting spectrum shown in Fig. 1C contains two sets of upfield doublets with shifts d1 Å 04.32 ppm and d2 Å 04.39 ppm, which are assigned to the two fluoro mononucleotides: FAMP and FUMP. Finally, a low intensity doublet appears slightly downfield of the fluorophosphate resonances at d Å 2.25 ppm in both the ATP and ATP/UTPderived products (Figs. 1A and C). This shift and the 934 Hz coupling constant are typical of fluorophosphate groups located at the end of polyphosphate chains (32– 36). Based on mechanistic considerations and the analogous ATP hydrolysis chemistry, this resonance would presumably correspond to either bFADP or to fluoropyrophosphate, FPPi . The latter assignment was considered less likely since: (1) the 19F resonances did not shift with titration between pH 3 and 7, although FPPi would have a titrable phosphate group, and (2) Pronkin et al. (36) give a 19F shift for FPPi which is upfield of fluorophosphate and a coupling constant which differs significantly from the observed value. As expected, 31P NMR analysis of the above samples reveals that there is substantial but incomplete hydrolysis of the ATP under the conditions used. In order to correlate 31P and 19F shift data, a 2D 19 F{31P} HMQC experiment was performed (Fig. 2). The fluorine-observe, phosphorus-decouple approach optimizes sensitivity and represents an ideal application of this approach since the isotopic abundance of both

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species is 100%. The 31P shifts obtained for the three components of the mixture in Fig. 1A are consistent with the shift data given by Vogel and Bridger (33). As a final assignment tool, authentic bFADP was prepared as described by Satischandran et al. (26) using the sulfurylase-catalyzed reaction of monofluorophosphate with ATP. The resulting spectral parameters were found to be consistent with the proposed assignment. Catalytic Effects of Polyvalent Cations In the absence of added polyvalent cations and the presence of chelators such as CDTA, formation of fluorophosphate can be observed by 19F NMR after a 2-h period of boiling. The fluorophosphate concentration shows a linear dependence on [NaF]. FAMP can also be observed if the fluoride concentration is sufficiently high (Fig. 3). Although these products presumably arise from non-metal-catalyzed reactions, it is important to note that since nucleoside triphosphates are strong metal chelators, low levels of metal–nucleoside complexes may be present even in the presence of a strong competing chelator such as CDTA. Addition of MgCl2 and deletion of the CDTA results in the formation of observable levels of FAMP at much lower [NaF] concentrations (Fig. 3). However, the production of fluorophosphate is not much greater than

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FIG. 2. Two-dimensional 19F {31P} HMQC experiment with 31P decoupling on a sample similar to that shown in A. Pulsing parameters: 19 F sweep width, 5 kHz; 31P sweep width, 6 kHz; 19F 907 pulse (decouple coil), 67 usec; interpulse delay, 2 s, 512 data points in both dimensions. The pH was set at 5.4 to improve the resolution of the fluorophosphate and bFADP resonances. The 2D spectra were recorded at T Å 227C.

observed in the absence of metal ions, if the [NaF] concentration is held below 200 mM. We note, however, that data obtained at these high concentrations of fluoride must be evaluated semiquantitatively due to some precipitation of fluoride salts. Qualitatively similar data are obtained at lower Mg2/ concentrations with significantly less precipitation. Nevertheless, at higher NaF concentrations, a dramatic increase in fluoro-

phosphate production is observed. Although considerably more FAMP was produced in the presence of Mg2/ , FAMP production falls off at the highest [NaF] levels. The nonlinear dependence of FAMP on [NaF] concentration presumably arises from the fact that at high fluoride concentrations, the fluorophosphate reaction successfully competes for the available ATP substrate, so that less ATP is available for the FAMP reaction. In summary, these results show that: (1) fluorophosphate and FAMP are produced in the absence of added metal cations and even in the presence of added chelators; (2) Mg2/ is catalytic for the formation of FAMP; (3) at high fluoride concentrations, a second, distinct Mg2/dependent catalytic process specific for the formation of fluorophosphate becomes significant. The rate constants for reaction with the nucleotide phosphate groups thus follow the pattern apparent from Fig. 1, i.e., kg ú ka ú kb . At high [NaF] the relative rate constants follow the pattern: kg @ ka ú kb . For the sake of the discussion, these qualitatively distinct regimes are referred to as type I and type II catalysis, respectively. With the exception of some specific cases such as the high [NaF] study in Fig. 3, 31P NMR spectra tended to be fairly similar, consistent with the conclusion that under most of the protocols used hydrolysis was the predominant reaction. At the end of the 2-h boiling period, 31P resonances corresponding to ATP, ADP, AMP, inorganic phosphate, and, in some cases, fluorophosphate are readily observed. The 31P resonances corresponding to FAMP and bFADP were in most cases too small to be detected without very lengthy signal averaging times. In general, final [ATP] levels Ç10 mM,

FIG. 3. Fluorophosphate (A) or FAMP (B) concentrations produced as a function of initial [NaF] concentration in the presence of 100 mM CDTA (h) or 90 mM MgCl2 (s). Other sample constituents were: 100 mM ATP, 25 mM Tris–HCl, pH 6.5. Samples were immersed in boiling water for a 2-h period prior to NMR analysis. Final concentrations were determined based on the addition of a 1 mM o-fluorobenzamide standard. Concentrations of reactants in the MgCl2 studies represent quantities added rather than true solution concentrations due to precipitation of MgF2 .

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[ADP] Ç60 mM, and [AMP] Ç30 mM. ATP consumption was somewhat greater in the high NaF concentration studies, consistent with the high level of fluorophosphate production. However, even in this case, there was some unreacted ATP present at the end of the study. In general, final phosphate concentrations exceed fluorophosphate concentrations, except at the highest [NaF] concentrations used. From these spectra, it is apparent that nucleotide hydrolysis predominates over fluorolysis until the NaF concentration becomes very high (ú0.5 M). One further question which can be readily addressed concerns the production of FAMP, which can be formed either directly from ATP with loss of pyrophosphate, or derived from the reaction of fluoride with ADP which builds up during these studies as a result of ATP hydrolysis. The latter might be supposed to represent a less significant pathway, for the same reasons that bFADP production is very low. This issue is readily addressed by evaluating the time-dependent production of ADP. In order to facilitate data accumulation over a reasonable time period, a study of the NaF-Mg2/-ATP system was performed at 707C (Fig. 4). In this case, 0.1 M sodium citrate was also added to chelate excess Mg2/ in order to avoid a large build-up of the free magnesium concentration as the ATP was hydrolyzed. The timedependent formation of both fluorophosphate and FAMP is observed to be linear over the course of several hours (Fig. 4). This result is consistent with the first proposed mechanism, i.e., direct formation of FAMP from ATP, since if ADP were required for FAMP formation, the accumulation would be expected to exhibit a more significant curvature as the ADP concentration increases over the course of the study. Studies of the effects of different reaction conditions and metal ions indicate that all of the ions tested: Mg2/,

FIG. 4. Time-dependent production of fluorophosphate (small circles) and FAMP (large circles) at 707C monitored by 19F NMR. Sample contained, 100 mM ATP, 100 mM MgCl2 , 100 mM sodium citrate, pH 7, and 200 mM NaF. Calibration of signal intensities was based on comparison of resonance areas with o-fluorobenzamide, added subsequent to the study. Rates of fluorophosphate and FAMP production in this study are 0.41 mM/h and 0.12 mM/h, respectively.

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FIG. 5. Time-dependent formation of fluorophosphate from a solution initially containing 10 mM CaCl2 , 150 mM NaF, 100 mM ATP, 25 mM Tris–Hepes buffer (pH 7.0), 1 mM o-fluorobenzamide (as an intensity standard), at T Å 377C. The straight line represents the initial slope.

Ca2/, Sr2/, Mn2/, Zn2/, Co2/, Cd2/, Pb2/, Sn2/, Al3/, La3/, and Lu3/ exhibit at least some catalytic potency, and that, depending on conditions, the ions can play at least two distinct catalytic roles. Product ratios are variable, with Mg2/, Zn2/, Co2/, Al3/, and Sn2/ giving higher FAMP/fluorophosphate ratios. At some Sn2/ (added as SnCl2) concentrations studied, production of FAMP exceeds production of fluorophosphate. Alternatively, Ca2/ and La2/ are much more effective catalysts than Mg2/ for the formation of fluorophosphate (type II catalysis). Thus, the product distribution observed in the presence of Ca2/ resembles that observed in the presence of Mg2/ at high [NaF], i.e., with [fluorophosphate] @ [FAMP]. The potency of Ca2/ ions as a fluorokinase catalyst is illustrated by the time-dependent production of fluorophosphate at 377C (Fig. 5). Catalytic Mechanisms Based on proposals for the role of metal ions in nucleotide hydrolysis, a number of catalytic roles for multivalent cations can be postulated. Several of these, outlined in Fig. 6, are closely analogous to proposals for cation-catalyzed nucleotide hydrolysis (23–25). The less specific catalytic effects observed at lower fluoride/ cation ratios may be due to the effects of the divalent ion in neutralizing the negative charges of the ionized phosphate groups, facilitating the approach of the nucleophilic fluoride ion and decreasing the electron density on the phosphorus (Fig. 6). Additionally, the chelated species may form a better leaving group (Figs. 6-II and 6-IV). The well-established preference for coordination of divalent cations to the b and g phosphate groups (37, 38), combined with the data above indicating that FAMP is formed directly by an interaction of fluoride with ATP, provide strong support for mechanism 6-II as an explanation for the cation catalysis of FAMP formation.

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has been shown to bind to the enzyme via the formation of an oxygen bond to Mn2/ (44, 45). Room Temperature Kinetics The Mg2/-catalyzed reaction of fluoride with ATP proceeds very slowly at physiological temperature. In a sample containing 0.1 M ATP, 0.09 M MgCl2 , 25 mM Tris–Hepes, pH 7.0, and 50 mM NaF maintained at 377C, fluorophosphate was formed at a rate of 1.9 1 1003 mM/h (Table I), while the formation rate of FAMP was 7.2 1 1004 mM/h. These rates compare with a value of 1.58 mM/h for pyruvate kinase measured under the same conditions. Hence, the nonenzymatic reaction would appear to make negligible contributions to the in vivo formation of fluorophosphate relative to the enzyme. In contrast, the Ca2/-catalyzed production of fluorophosphate is much more rapid (Fig. 5, Table I). FIG. 6. Proposed catalytic mechanisms for cation catalysis of nucleotide fluorolysis. In I and III, catalysis results primarily from the increase in electron density on phosphorus, increasing the susceptibility to nucleophilic attack. In II and IV, the chelated pyrophosphate moiety forms a better leaving group. In V, reaction involves a second, Metal-fluoride species, in addition to the chelated ATP structures shown in I–IV. The possible involvement of a ternary complex is shown in VI. Analogous mechanisms involving ADP will yield fluorophosphate and FAMP.

The qualitative change in the product ratios which occurs in the presence of Mg2/ at high [F0]/Mg2/] ratios requires some additional explanation. Two possible explanations for this change are: (1) the involvement of a ternary metal-fluoride-nucleotide complex, or (2) a reaction involving a chelated nucleotide plus a metal fluoride (Figs. 6-V and 6-VI). The inhibition of enolase by fluoride has been suggested to result from the formation of a ternary fluoride-Mg2/-phosphate complex (5– 7). NMR measurements indicate binding of F0 to enzyme-bound Mn2/ (7). ternary complexes have also been demonstrated to form in mixtures of aluminum, fluoride, and nucleoside diphosphates (39, 40). Alternatively, involvement of two metal ions has sometimes been proposed to explain the effects on the hydrolytic rate (41–43). It is of particular interest to note in this context that the fluoride inhibition of inorganic pyrophosphatase arises due to the fluoride inhibition of inorganic pyrophosphatase arises due to the formation of a tight Enzyme-PPi-Mg2F complex (8, 9). Kinetic analysis of the phosphorylation of fluoride by rabbit muscle pyruvate kinase as a function of pH suggests that fluoride is directly bound to the Mg2/ ion (21). However, 19 F NMR studies of fluoride in the presence of Mn2/ / pyruvate kinase have been interpreted as providing no indication of the formation of a ternary enzyme-Mn2/F-bridge structure (44). In contrast, fluorophosphate

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Enzyme Fluorokinase Activity Although the active site of pyruvate kinase is unique in utilizing two divalent metal cations (46), it seemed likely that the adventitious association of fluoride with other kinases could result in significant fluorokinase activity. This hypothesis was confirmed when two of six randomly selected kinases: acetate kinase and glycerokinase, were found to possess significant fluorokinase activity as well (Table I). Fluorokinase reactions

TABLE I

Reaction Rates for Formation of Fluorophosphate Mg2/ (mM)

Ca2/ (mM)

NaF (mM)

Rateb mM/h

— —

90 90 10 10 — —





— — — — 10 10 10 10 10

50 10 400 800 25 50 100 150 250

1.9 1 1003 5.5 1 1004 4.6 1 1003 1.14 1 1001 1.5 1 1004 5.6 1 1003 .085 1.07 0.42c

Pyruvate kinase Pyruvate kinase Pyruvate kinase Acetate kinase Acetate kinase Glycerokinase Glycerokinase Glycerokinase

90 45 10 90 10 90 45 10

— — — — — — — —

50 50 50 50 50 50 50 50

1.58 0.22 1.2 1 1002 5.8 1 1002 3.6 1 1002 6.2 1 1002 2.2 1 1002 —

Enzymea — —

a

Enzyme levels were: pyruvate kinase, 1000 units; acetate kinase, 250 units; glycerol kinase, 200 units, in 0.5 ml. b Initial rates measured at 100 mM [ATP], 25 mM Tris–HCl, pH 7.0, T Å 377C. c Lower rate probably due to precipitation.

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with both acetate kinase and glycerokinase produce only fluorophosphate; no fluoronucleotides are formed, subject to the detection limits of the NMR experiment (Ç10 mM). Given the natural anionic substrate, the observation of fluorokinase activity for acetate kinase is perhaps not too surprising. These activities, summarized in Table I, while significantly greater than the nonenzymatic reaction, are also much lower than that of pyruvate kinase (Table I). The rate of fluorophosphate formation by pyruvate kinase determined at [Mg2/] Å 90 mM is fairly similar to the rate observed by LeBlond and Robinson for yeast pyruvate kinase under somewhat different conditions (20). This similarity suggests that higher substrate and magnesium concentrations used in the present study may compensate for the absence of added bicarbonate and fructose-1,6diphosphate used in the yeast pyruvate kinase study. Although initially proposed to be required for fluorokinase activity (18, 19), LeBlond and Robinson found that omission of the bicarbonate reduced the rate by 76%, but did not abolish it. Studies in which attempts were made to purge the system of dissolved CO2 exhibit reduced but significant activity, further supporting the conclusion that no absolute requirement for bicarbonate exists. The fluorokinase activity of both pyruvate kinase and glycerokinase was found to decrease sharply as the Mg2/ concentration was lowered (Table I), while acetate kinase showed a much more modest reduction in activity. CONCLUSIONS

Polyvalent cations catalyze the fluorolysis of nucleotides. The catalytic role of the ion is complex and appears to result from multiple effects. It is useful to consider both a general, type I catalytic effect, in which the formation of fluorophosphate as well as fluoronucleotides is accelerated, and a type II catalytic effect, which specifically involves the formation of fluorophosphate. Data presented here suggest that type II catalysis by Mg2/ involves either the formation of a ternary metal-fluoride-nucleotide complex, or a reaction involving both cation-fluoride and cation-nucleotide complexes. An illuminating comparison can be made between the present results and previous studies of nucleotide hydrolysis. Tetas and Lowenstein (23) found that the catalytic effects of divalent metal ions are strongly pH dependent, indicating that different mechanisms or intermediate chelated species are involved. Their data indicate that reactions involving water may be more significant at lower pH, while nucleophilic attack by hydroxyl ions becomes more significant at high pH. A comparison with the metal ion specificities observed in the hydrolysis reaction strongly suggests that the fluorolysis reaction studied here is closely analogous to

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nucleotide hydrolysis observed at high pH. In particular, Tetas and Lowenstein find that Ca2/ is fairly ineffective as a hydrolysis catalyst at low pH, but was the most effective ion studied as a hydrolysis catalyst at high pH. In contrast, Mg2/ gave only small hydrolysis rate enhancements over the metal-free case at pH 9. This pattern is similar to that obtained for fluorolysis catalysis. These results support the conclusion that the cation catalyzed fluorolysis reactions are closely analogous to nucleotide hydrolysis observed at high pH involving nucleophilic attack by hydroxyl ions. Given the complexity of the nucleotide hydrolysis which in general involves both the solvent water as well as hydroxyl ions, studies of fluorolysis may provide a useful model system for separating the effects of these two processes. Although consistent with the hydrolysis data, the greater effectiveness of Ca2/ than Mg2/ as a fluorokinase catalyst is somewhat surprising in light of the higher dissociation constants of Ca2/ for both ATP (47) and fluoride (48). Thus, Kd(Ca-ATP) Å 123 mM, Kd(CaF) Å 20 mM, while Kd(Mg-ATP) Å 51 mM, Kd(Mg-F) Å 48 mM. Therefore, smaller amounts of any of the intermediates illustrated in Fig. 6 will form with Ca2/ than with the same concentration of Mg2/. Presumably, the lower concentration of catalytic intermediates is more than compensated by a higher catalytic efficiency. One of the major differences in ligand binding of these two ions is the higher ligand exchange rate for Ca2/ vs Mg2/ complexes, typically a factor of 103 independent of the nature of the particular ligand (49). The greater lability of the intermediates may be related to the greater catalytic activity of Ca2/ ions. The cation-catalyzed fluorolysis of ATP, UTP, and presumably other nucleotides suggests the possibility of forming these molecules in vivo, and the consequent biochemical perturbations which they might exert require further consideration. Of course, whether these molecules could accumulate to any significant degree in cellular systems depends on the rate of formation as well as the rates of excretion and catabolism. Although fluorophosphate is widely considered to be unstable in physiological systems (50), NMR studies on erythrocytes indicate stability over a period of hours (34, 51; A. Xu, private communication). This stability may result from the fact that any fluoride produced will inhibit phosphatase enzymes which could be involved in the catabolic process, i.e., fluorophosphate degradation may be self-limiting. Uridine-5*-fluorophosphate (FUMP) is reported to be fully resistant toward alkaline phosphatase and acid phosphatase, but is hydrolyzed by 5*-nucleotidase (52). The biological activity of fluorophosphorylated nucleosides is generally quite variable. Although g-fluoronucleotides are generally poor analogs of ATP due to their reduced binding affinity for Mg2/ ions (33, 53– 55), fluorinated mono- and dinucleotides may be closer

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analogs of the nonfluorinated species. However, gFGTP is reported to be a nonhydrolyzable, potent inhibitor of ribosome-dependent EF-G GTPase (56). bFADP has been shown to be a purinoreceptor agonist (57, 58). It is an alternate substrate for adenosine phosphosulfate kinase, yielding the PADPbF analog of PAPS, and an alternate substrate for the reverse reaction of ATP sulfurylase, yielding ATP and fluorophosphate (26, 59). bFGDP was found to bind to protein synthesis initiation factor 2 from Xenopus laevis oocytes, with affinity intermediate between GDP and GTP (60). FAMP inhibits the AMP activation of glycogen phosphorylase b with inhibition constant Ki Å 3 mM (61). Thymidine-3*-fluorophosphate is reported to be an irreversible inhibitor of nuclear exoribonuclease derived from Ehrlich ascites tumor cell nuclei or HeLa cell nuclei (62). Perhaps of greatest significance, 3*fluoro-3*-deoxythymidine-5*-fluorophosphate has recently been shown to inhibit HIV reproduction in cell cultures (63). Since the g-substitution reaction of the kinases is found to utilize fluoride in some cases, it seems possible that enzyme catalyzed a and b substitutions may also occur, yielding the corresponding fluoronucleotides. Presumably, bFADP can be derived from fluoride in biological systems by the combined actions of pyruvate kinase and sulfurylase. While the rates of all reactions investigated to date will be extremely low in biological systems, it is important to note that exposure to fluoride is chronic. Fluorolysis reactions might also be more significant in organelles such as chromaffin granules which contain high concentrations of ATP (64). Based on the results summarized here, a wider investigation of the effects of fluoride on phosphorylation biochemistry is warranted. ACKNOWLEDGMENT The authors gratefully acknowledge the contributions of Dr. Donald Davis in setting up the 19F{31P} HMQC experiment.

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