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Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum
Biochimica et Biophysica Acta, 995 (1989) 291-294
291
Elsevier BBA 33351
Studies on the mechanism of action of the alkaline phosphatase from Dictyostefium discoideum Pradeep Bhanot 1 and Gerald Weeks 1,2 Departments of i Microbiology and 2 Medical Genetics, University of British Columbia, Vancouver(Canada)
(Received 27 December 1988)
Key words: Alkalinephosphatase; Enzymekinetics; (D. discoideum) The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activRy in an uncompetifive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced a¢ a|ka|ine pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a confolmational change that renders the enzyme susceptible to irreversible inhibition by phosphate.
Introduction
Membrane bound alkaline phosphatase activity is regulated both temporally and spatially during the differentiation of Dictyostelium discoideum [1-4]. In previous reports from this laboratory, evidence was presented to suggest that the increase in activity during differentiation was not due to an increase in enzyme synthesis but to the unmasking of preexisting enzyme The enzyme is unusual among alkaline phosphatases in that it has a relatively restricted substrate specificity [8]. It is most active on AMP and p-nitrophenyl phosphate (pNPP), although the binding characteristics of the two substrates are somewhat different [7-9]. Despite its restricted substrate specificity, the enzyme is capable of transferring phosphate from either AMP or pNPP to an organic acceptor molecule [9], a property that is more characteristic of the broad specificity alkaline phosphatases. It is apparently the only major AMPase in the cell [8] and is responsible for the production of adenosine, which has been implicated recently as a morphogen responsible for cell type determination [10,11]. In view of the potential importance of the
Abbreviations: pNPP, p-nitrophenyl phosphate: Tris, 2-amino-2-hydroxymethylpropane-l,3-diol. Correspondence: G. Weeks, Department of Microbiology,University of British Columbia, Vancouver, BC V6T 1W5, Canada.
activation of alkaline phosphatase during development, we have attempted to further define its reaction mechanism. Materials and Methods Organism and culture conditions D. discoideum, strain Ax-2 was grown axenically on
HL-5 media to a density of 5 × 106 cells/ml [12]. Cells were harvested by centrifugation at 700 × g, washed twice with cold water and then washed with 5 mM Tris-HCl (pH 7.4) containing 8.6% sucrose (Trissucrose). Materials
pNPP and Tris were from Sigma, Triton X-100 was from Amersham Corporation and enzyme grade sucrose was from Schwarz-Mann. All other chemicals were the best available grade from Fisher Chemical Co. Membrane preparation and enzyme purification
Membranes were prepared by slight modifications of a previously published procedure [6]. Washed cells were resuspended at a density of 10 8 cells/ml in Tris-sucrose containing freshly dissolved 0.1 mM phenylmethylsulfonyl fluoride. 1.65 g of glass beads (0.45-0.50 mm diameter) were added per l0 s cells and mechaaical grinding was accomplished by stirring the suspension with a magnetic stirring bar for 10-15 rain. Almost 100% cell breakage was obtained as assessed by microscopy. The unbroken cells and glass beads were removed by centrifugation at 700 × g for 5 n~n. The
0167-4838/89/$03.50 © 1989 ElsevierScience Publishers B.V.(Biomedical Division)
292 supernatant was centrifuged at 105 000 × g to obtain a membrane pellet which was resuspended in Tris-sucrose and recentrifuged as before. The final pellet was then resuspended in 5 mM Tris-HCl (pH 7.4) to give a protein concentration of approx. 25 m g / m l and stored at - 7 0 °C. Membranes were extracted with Triton X100 and alkaline phosphatase was purified as described previously [7]. For some experiments the Triton X-100 extracts were dialyzed against 5 mM Tris-HCl (pH 7.4) for 24 h. Protein was determined by the method of Sandermann and Strominger [13].
10 8
"! >
6
Alkaline phosphatase assay The ability of the enzyme to hydrolyze pNPP was assayed by a slight modification of a previously published procedure [2]. Incubations contained 20 mM MgCIz; 20 mM Tris-HCI (pH 8.5); 30 mM sodium fluoride and, unless otherwise indicated, 1 mM pNPP in a final wlume of 1.0 ml. Incubations were initiated by the addition of protein at the levels indicated in the text and were terminated after 10 rain at 3 0 ° C by the addition of 1.0 ml 1 M Na2CO 3. Any resulting precipitate was removed by centrifugation at 2500 rpm for 10 rain. Absorbance at 410 nm was measured and a molar absorption coefficient of 1.62.104 was used to calculate the amount of p-nitrophenol formed. The ability of the enzyme to hydrolyze AMP was assessed by a previously detailed procedure [2]. Both enzyme assays were linear over the entire period of the incubation. Results and Discussion
o
0
1
2
•
[]
3
4
5
11S
Fig, 1. Double-reciprocal plots showing the effect of Tris-HC! on enzyme activity. Purified enzyme (4 ttg) was incubated under standard assay conditions at the indicated reciprocal concentrations of pNPP (raM) with: o, no addition; ra, 500 mM NaCI; ®, 50 mM Tris-HCI (pH 8.5); and B, 500 mM Tris-HCl (pH 8.5). Activity is expressed as the reciprocal of A41o produced per min during the standard assay. Data points are the meansof triplicate determinations and standard deviations have been omitted in the interests of clarity. Lines of best-fit weredrawn using a linear regression analysis.
Biphasic kinetics of p-nitroDhenyl phosphate hydrolysis It should be possible to directly confirm a double displacement mechanism for the enzyme, because the nucleophilic substitution of enzyme for the alcoholic portion of the substrate should occur at a greater rate than the subsequent turnover of the enzyme [16]. Fig. 2 shows that there was a significant burst of pNPP hydrolysis prior to the steady-state rate, when the reaction
The effects of Tris on the substrate kinetics of alkaline phosphatase In order to further investigate the mechanism of action of the alkaline phosphatase of D. discoideum, a kinetic analysis was undertaken with the purified enzyme, in the presence and absence of Tris. With increasing concentrations of Tris, p-nitrophenol formation was stimulated and Lineweaver-Burk plots of the data were parallel (Fig. 1), suggesting that Tris is either a non-essential uncompetitive activator [14] or a substrate in the reaction [15]. The indirect evidence obtained earlier that indicated that Tris was phosphorylated during the course of the reaction [9] suggests that the latter possibility is the more likely. The transfer of phosphate to Tris during the hydrolysis of pNPP is characteristic of a double displacement mechanism [16]. The activity observed in the presence of a low concentration of Tris-HCl buffer was unaffected by the addition of 500 mM NaCI, indicating that the stimulatory effect of Tris-HCl was not merely due to increased ionic strength. Similar results v : ~ obtained when the detergent-solubilized membranes were used or when AMP was substituted for pNPP (data not shown).
0.48 ttl O
~0.36 ¢t:
o
¢0
~ o.24 I,u Q 0.12
0
I 0.04
.
I 0.08
I . 0.12
I 0.16
0.2
SECONDS Fig. 2. Stopped-flowrapid spectrophotometric assay of enzyme activity. Standard conditions were used, except that assays contained 8 mg/ml dialyzed Triton X-100 extract and 8 mM pNPP. Delta absorbance was measured a 410 nm and the plot shown ~s the computed average of five ,~:~periments. A dead time of 0.01 s was used before initiating data collection. The pronounced lag observed was an instrumental artifact, since it was also obtained in a experiment with E. coli alkaline phosphatase.
293 was rapidly assayed using a stopped-flow spectrophotometer. The steady-state rate obtained was consistent with the anticipated overall rate of reaction. Previous studies have demonstrated that AMP and pNPP are hydrolyzed more rapidly than other phosphorylated substrates and that the enzyme displays restricted substrate specificity [8]. The data presented above, however, are consistent with earlier findings [9], suggesting a double displacement mechanism characteristic of a broad-spectrum alkaline phosphatase [16]. Thus, despite the fact that the enzyme is the only major AMPase activity in Dictyostelium [8] and despite the fact that it has restricted substrate specificity, the enzyme should continue to be defined as an alkaline phosphatase.
2O
==
10
m
•
0
I
1
"
I
"
2
I
il
3
4
"
5
115
Inhibition of enzyme activity by phosphate In view of the evidence in favour of a double displacement mechanism for alkaline phosphatase, phosphate should be a competitive inhibitor of the reaction. However, earlier reports have indicated that the interaction of the enzyme with phosphate is more complex. Gezelius and Wright [17] had originally reported that p~osphate inhibition was reversible, but Armant and Rutherford [18] showed that phosphate inhibition was irreversible. A more detailed investigation of the mechanism of phosphate inhibition was therefore undertaken. Phosphate was found to be a competitive inhibitor of pNPP hydrolysis by the purified enzyme when the ionic strength was low (Fig. 3), consistent with the existence of an enzyme-phosphate intermediate. Similar r~sults were obtained either with AMP as substrate or with detergent-solubilized membranes (data not shown). However, the interaction of pl-osphate with the purified enzyme was dramatically different at high ionic strength. Phosphate at a concentration of only 1 mM irreversibly inhibited the enzyme activity in the presence of 200 mM KC1 (Table I). Similar results were obtained with AMP as substrate (data not shown). This irreversible inhibition of alkaline phosphatase by phosphate is presumably due to a conformational change in the protein induced by high ionic strength. Even in the absence of phosphate, prolonged incubation at high ionic strength produces some inhibition cff enzyme activity (Table I), although short incubations clearly did not have a noticeably deleterous effect on enzyme activity (Fig. 1). Elevated ionic strength has been shown to elicit a conformational change in the E. coli alkaline phosphatase [19]. Other alkaline phosphatases are also affected by ionic strength, but there is no consistent pattern in that some enzymes are stimulated, while other are inhibited [16]. The inhibition by phosphate at high ionic strength was enhanced by elevated pH (Fig. 4), which may also involve a pH-dependent conformational change in the protein. Pre':i,~us studies had revealed a pronounced
Fig. 3. Double-reciprocal plots showing the effect of phosphate on enzyme activity. Purified enzyme (4 /~g) was incubated under standard assay conditions at the indicated reciprocal concentrations of pNPP (raM) with: o, no addition; A, 2 mM K2HPO4; and El, 10 mM K 2 HPO 4. Activity is expressed as the reciprocal of ,,141o produced per rain during the standard assay. Data points are the means of triplicate determinations, and standard deviations have been omitted in the interests of clarity. Lines of best-fit were drawn using linear regress ~)n.
~:hange in substrate binding to the enzyme with increased pH [9], consistent with a conformational change. Furthermore, inhibition by high ionic strength alone was enhanced at elevated pH (Fig. 4). The irreversible inhibition by phosphate presumably involves phosphate binding to the active site and it can be partially prevented by the substrate, AMP (Table II). Attempts to demonstrate covalent attachment of phosphate to the enzyme under these conditions were unsuccessful (data not shown).
TABLE I Irreversible inhibition of the enzyme by phosphate Treatment a
No addition + 200 mM KCI + 200 mM KCI, 1 mM K 2HPO4 +200 mM KCI, 1 mM KeHPO4 followed by dialysi~
Enzyme activity b (/~mol/min per mg protein) 1.32+ 0.04 1.08+ 0.06 0.23 ~_0.04 0.18+_0.04
d Purified enzyme (160/tg protein/ml) was incabated for 60 rain at 30 o C in the pr¢~enceof 5 mM Tris-HCI (pH 9.0) and the indicated additions. At the end of this incubation aliquots (0.05 ml) were assayed for enzyme activity using the standard assay conditions. Where indicated, samples were dialyzed against 5 mM Tris.HCi (pH 9.0) for 3 days at 0 °C prior to assay. b The results are the means±standard deviation for three experiments. The activitybefore the incubationcommencedwas 1.39+ 0.05 Fmol/min per mg protein.
294
lar weight inhibitor [7]. It is not likely that this putative inhibitor is phosphate, since neither the reversible or the irreversible inhibition reported here resembles the characteristics of the endogenous modulation [6,7]. The 13. discoideum alkaline phosphatase is clearly an unusual membrane-bound enzyme and a more detailed elucidation of its structure will be important for an understanding of its regulation during development.
D
E m
E m o o
m
E
3
Acknowledgments m m 41a
01-6.5
7.5
8.5
9.5
pH Fig. 4. pH-dependence of phosphate inhibition of enzyme activity. Purified enzyme (4 ~tg) was incubated at 30°C for 60 rain in the pre~nce of 200 mM Tris.HCI at the indicated pH values with either no addition (11) or ! m M K2HPO4 (13) in a final volume of 0,1 ml. Enzyme activity was then as~yed under standard conditions, except that 500 mM Tris-HCI (pH 8.5) replaced the usual buffer concentration and 4.0 mM pNPP was used as substrate.
The increase in alkaline phosphatase activity during the development of Dictyostelium (1-4), which may be important for localized production of adenosine, a potential morphogen [10,11] is due to the gradual unmasking of the enzyme [6,7]. This unmasking was accomplished in vitro by elevated temperatures or by prolonged dialysis [5,6] and reconstitution experiments indicated that it involved the removal of a low molecuTABLE !1
AMP protection of activity against phosphate inactivation Treatment *
Enzyme activity b (It mo!/min per m8 protein)
None + I mM KaHPO,, + ! mM K2HPO4, 1 mM AMP + i mM K2HPO4, 2 mM AMP + ! m M K2HPO4, 4 mM AMP
1.29+-0.06 0.31 +-0.05 0.54+-0.03 0.81 + 0.06 1.02+0.06
" Purified enzyme (160 ~tg protein/ml) was incubated in the presence of 200 raM Tris-HC! (pH 7.5) and the indicated additions for 10 rain at 30 o C. After incubation, aliquots (0.05 ml) were assayed for enzyme activity using the standard assay conditions. b Th• ~s,dtg ate. the means of triplicate experiments + standard deviation.
This work was supported by a grant from the Natural Science and Engineering Research Council of Canada. One of us (P.B.) was in receipt of NSERC and UBC Frank Wesbrook graduate fellowships during the course of these studies. We wish to thank Dr. Grant Mauk of the Biochemistry Department at U.B.C. for making the stopped-flow spectrophotometer available to us and for help in its use. References 1 Loomis, W.J., Jr. (1969) J. Bacteriol. 100, 417-422. 2 Lee, A., Chance, K., Weeks, C. and Weeks, G. (1975) Arch. Biochem. Biophys. 141, 407-417. 3 Quiviger, B., Bemicho, J.C. and Ryter, A. (1980) Biol. Ceil. 37, 241-250. 4 Arman~, D.R., Stettler, D.A. and Rutherford, C.L. (1980) J. Cell Sci. 45, 119-129. 5 Das, D.V.M. and Weeks, G. (1980) Nature 288, 166-167. 6 Das, D.V.M. and Weeks, G. (1981) FEBS Lett. 130, 249-252. 7 Das, D.V.M. and Weeks, G. (1984) Can. J. Biochem. Cell Biol. 62, 970-974. 8 Armant, D.R. and Rutherford, C.L. (1981) J. Biol. Chem. 256, 12710-12718. 9 Bhanot, P. and Weeks, G. (1985) Arch. Biochem. Biophys. 236, 497-505. 10 Weijer, C.J. and Durston, A.J. (1985) J. Embryoi. Exp. Morph. 86, 19-37. 11 Schaap, P. and Wang, M. (1986) Cell 45, 137-144. 12 Weeks, C. and Weeks, G. (1975) Exp. Cell Res. 92, 372-382. 13 Sandermann, H., Jr. and Strominger, J.L. (1972) J. Biol. Chem. 247, 5123-5131. 14 Frieden, C. (1964) J. Biol. Chem. 239, 3522-3531. 15 Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd Edn., pp. 114-117, W.H. Freeman and Co., New York. 16 MacComb, R.B., Bowers, G.N., Jr. and Posen, S. (1979) Alkaline phosphatase, pp. 229-287, Plenum Press, New York. 17 Gezelius, K. and Wright, B.E. (1965) J. Gun. Microbiol. 38, 309-327. 18 Armant, D.R. and Rutherford, C.L. (1979) Mech. Aging Dev. 10, 199-217. 19 Halford, S.E. (1982) Biochem. J. 126, 727.
291
Elsevier BBA 33351
Studies on the mechanism of action of the alkaline phosphatase from Dictyostefium discoideum Pradeep Bhanot 1 and Gerald Weeks 1,2 Departments of i Microbiology and 2 Medical Genetics, University of British Columbia, Vancouver(Canada)
(Received 27 December 1988)
Key words: Alkalinephosphatase; Enzymekinetics; (D. discoideum) The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activRy in an uncompetifive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced a¢ a|ka|ine pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a confolmational change that renders the enzyme susceptible to irreversible inhibition by phosphate.
Introduction
Membrane bound alkaline phosphatase activity is regulated both temporally and spatially during the differentiation of Dictyostelium discoideum [1-4]. In previous reports from this laboratory, evidence was presented to suggest that the increase in activity during differentiation was not due to an increase in enzyme synthesis but to the unmasking of preexisting enzyme The enzyme is unusual among alkaline phosphatases in that it has a relatively restricted substrate specificity [8]. It is most active on AMP and p-nitrophenyl phosphate (pNPP), although the binding characteristics of the two substrates are somewhat different [7-9]. Despite its restricted substrate specificity, the enzyme is capable of transferring phosphate from either AMP or pNPP to an organic acceptor molecule [9], a property that is more characteristic of the broad specificity alkaline phosphatases. It is apparently the only major AMPase in the cell [8] and is responsible for the production of adenosine, which has been implicated recently as a morphogen responsible for cell type determination [10,11]. In view of the potential importance of the
Abbreviations: pNPP, p-nitrophenyl phosphate: Tris, 2-amino-2-hydroxymethylpropane-l,3-diol. Correspondence: G. Weeks, Department of Microbiology,University of British Columbia, Vancouver, BC V6T 1W5, Canada.
activation of alkaline phosphatase during development, we have attempted to further define its reaction mechanism. Materials and Methods Organism and culture conditions D. discoideum, strain Ax-2 was grown axenically on
HL-5 media to a density of 5 × 106 cells/ml [12]. Cells were harvested by centrifugation at 700 × g, washed twice with cold water and then washed with 5 mM Tris-HCl (pH 7.4) containing 8.6% sucrose (Trissucrose). Materials
pNPP and Tris were from Sigma, Triton X-100 was from Amersham Corporation and enzyme grade sucrose was from Schwarz-Mann. All other chemicals were the best available grade from Fisher Chemical Co. Membrane preparation and enzyme purification
Membranes were prepared by slight modifications of a previously published procedure [6]. Washed cells were resuspended at a density of 10 8 cells/ml in Tris-sucrose containing freshly dissolved 0.1 mM phenylmethylsulfonyl fluoride. 1.65 g of glass beads (0.45-0.50 mm diameter) were added per l0 s cells and mechaaical grinding was accomplished by stirring the suspension with a magnetic stirring bar for 10-15 rain. Almost 100% cell breakage was obtained as assessed by microscopy. The unbroken cells and glass beads were removed by centrifugation at 700 × g for 5 n~n. The
0167-4838/89/$03.50 © 1989 ElsevierScience Publishers B.V.(Biomedical Division)
292 supernatant was centrifuged at 105 000 × g to obtain a membrane pellet which was resuspended in Tris-sucrose and recentrifuged as before. The final pellet was then resuspended in 5 mM Tris-HCl (pH 7.4) to give a protein concentration of approx. 25 m g / m l and stored at - 7 0 °C. Membranes were extracted with Triton X100 and alkaline phosphatase was purified as described previously [7]. For some experiments the Triton X-100 extracts were dialyzed against 5 mM Tris-HCl (pH 7.4) for 24 h. Protein was determined by the method of Sandermann and Strominger [13].
10 8
"! >
6
Alkaline phosphatase assay The ability of the enzyme to hydrolyze pNPP was assayed by a slight modification of a previously published procedure [2]. Incubations contained 20 mM MgCIz; 20 mM Tris-HCI (pH 8.5); 30 mM sodium fluoride and, unless otherwise indicated, 1 mM pNPP in a final wlume of 1.0 ml. Incubations were initiated by the addition of protein at the levels indicated in the text and were terminated after 10 rain at 3 0 ° C by the addition of 1.0 ml 1 M Na2CO 3. Any resulting precipitate was removed by centrifugation at 2500 rpm for 10 rain. Absorbance at 410 nm was measured and a molar absorption coefficient of 1.62.104 was used to calculate the amount of p-nitrophenol formed. The ability of the enzyme to hydrolyze AMP was assessed by a previously detailed procedure [2]. Both enzyme assays were linear over the entire period of the incubation. Results and Discussion
o
0
1
2
•
[]
3
4
5
11S
Fig, 1. Double-reciprocal plots showing the effect of Tris-HC! on enzyme activity. Purified enzyme (4 ttg) was incubated under standard assay conditions at the indicated reciprocal concentrations of pNPP (raM) with: o, no addition; ra, 500 mM NaCI; ®, 50 mM Tris-HCI (pH 8.5); and B, 500 mM Tris-HCl (pH 8.5). Activity is expressed as the reciprocal of A41o produced per min during the standard assay. Data points are the meansof triplicate determinations and standard deviations have been omitted in the interests of clarity. Lines of best-fit weredrawn using a linear regression analysis.
Biphasic kinetics of p-nitroDhenyl phosphate hydrolysis It should be possible to directly confirm a double displacement mechanism for the enzyme, because the nucleophilic substitution of enzyme for the alcoholic portion of the substrate should occur at a greater rate than the subsequent turnover of the enzyme [16]. Fig. 2 shows that there was a significant burst of pNPP hydrolysis prior to the steady-state rate, when the reaction
The effects of Tris on the substrate kinetics of alkaline phosphatase In order to further investigate the mechanism of action of the alkaline phosphatase of D. discoideum, a kinetic analysis was undertaken with the purified enzyme, in the presence and absence of Tris. With increasing concentrations of Tris, p-nitrophenol formation was stimulated and Lineweaver-Burk plots of the data were parallel (Fig. 1), suggesting that Tris is either a non-essential uncompetitive activator [14] or a substrate in the reaction [15]. The indirect evidence obtained earlier that indicated that Tris was phosphorylated during the course of the reaction [9] suggests that the latter possibility is the more likely. The transfer of phosphate to Tris during the hydrolysis of pNPP is characteristic of a double displacement mechanism [16]. The activity observed in the presence of a low concentration of Tris-HCl buffer was unaffected by the addition of 500 mM NaCI, indicating that the stimulatory effect of Tris-HCl was not merely due to increased ionic strength. Similar results v : ~ obtained when the detergent-solubilized membranes were used or when AMP was substituted for pNPP (data not shown).
0.48 ttl O
~0.36 ¢t:
o
¢0
~ o.24 I,u Q 0.12
0
I 0.04
.
I 0.08
I . 0.12
I 0.16
0.2
SECONDS Fig. 2. Stopped-flowrapid spectrophotometric assay of enzyme activity. Standard conditions were used, except that assays contained 8 mg/ml dialyzed Triton X-100 extract and 8 mM pNPP. Delta absorbance was measured a 410 nm and the plot shown ~s the computed average of five ,~:~periments. A dead time of 0.01 s was used before initiating data collection. The pronounced lag observed was an instrumental artifact, since it was also obtained in a experiment with E. coli alkaline phosphatase.
293 was rapidly assayed using a stopped-flow spectrophotometer. The steady-state rate obtained was consistent with the anticipated overall rate of reaction. Previous studies have demonstrated that AMP and pNPP are hydrolyzed more rapidly than other phosphorylated substrates and that the enzyme displays restricted substrate specificity [8]. The data presented above, however, are consistent with earlier findings [9], suggesting a double displacement mechanism characteristic of a broad-spectrum alkaline phosphatase [16]. Thus, despite the fact that the enzyme is the only major AMPase activity in Dictyostelium [8] and despite the fact that it has restricted substrate specificity, the enzyme should continue to be defined as an alkaline phosphatase.
2O
==
10
m
•
0
I
1
"
I
"
2
I
il
3
4
"
5
115
Inhibition of enzyme activity by phosphate In view of the evidence in favour of a double displacement mechanism for alkaline phosphatase, phosphate should be a competitive inhibitor of the reaction. However, earlier reports have indicated that the interaction of the enzyme with phosphate is more complex. Gezelius and Wright [17] had originally reported that p~osphate inhibition was reversible, but Armant and Rutherford [18] showed that phosphate inhibition was irreversible. A more detailed investigation of the mechanism of phosphate inhibition was therefore undertaken. Phosphate was found to be a competitive inhibitor of pNPP hydrolysis by the purified enzyme when the ionic strength was low (Fig. 3), consistent with the existence of an enzyme-phosphate intermediate. Similar r~sults were obtained either with AMP as substrate or with detergent-solubilized membranes (data not shown). However, the interaction of pl-osphate with the purified enzyme was dramatically different at high ionic strength. Phosphate at a concentration of only 1 mM irreversibly inhibited the enzyme activity in the presence of 200 mM KC1 (Table I). Similar results were obtained with AMP as substrate (data not shown). This irreversible inhibition of alkaline phosphatase by phosphate is presumably due to a conformational change in the protein induced by high ionic strength. Even in the absence of phosphate, prolonged incubation at high ionic strength produces some inhibition cff enzyme activity (Table I), although short incubations clearly did not have a noticeably deleterous effect on enzyme activity (Fig. 1). Elevated ionic strength has been shown to elicit a conformational change in the E. coli alkaline phosphatase [19]. Other alkaline phosphatases are also affected by ionic strength, but there is no consistent pattern in that some enzymes are stimulated, while other are inhibited [16]. The inhibition by phosphate at high ionic strength was enhanced by elevated pH (Fig. 4), which may also involve a pH-dependent conformational change in the protein. Pre':i,~us studies had revealed a pronounced
Fig. 3. Double-reciprocal plots showing the effect of phosphate on enzyme activity. Purified enzyme (4 /~g) was incubated under standard assay conditions at the indicated reciprocal concentrations of pNPP (raM) with: o, no addition; A, 2 mM K2HPO4; and El, 10 mM K 2 HPO 4. Activity is expressed as the reciprocal of ,,141o produced per rain during the standard assay. Data points are the means of triplicate determinations, and standard deviations have been omitted in the interests of clarity. Lines of best-fit were drawn using linear regress ~)n.
~:hange in substrate binding to the enzyme with increased pH [9], consistent with a conformational change. Furthermore, inhibition by high ionic strength alone was enhanced at elevated pH (Fig. 4). The irreversible inhibition by phosphate presumably involves phosphate binding to the active site and it can be partially prevented by the substrate, AMP (Table II). Attempts to demonstrate covalent attachment of phosphate to the enzyme under these conditions were unsuccessful (data not shown).
TABLE I Irreversible inhibition of the enzyme by phosphate Treatment a
No addition + 200 mM KCI + 200 mM KCI, 1 mM K 2HPO4 +200 mM KCI, 1 mM KeHPO4 followed by dialysi~
Enzyme activity b (/~mol/min per mg protein) 1.32+ 0.04 1.08+ 0.06 0.23 ~_0.04 0.18+_0.04
d Purified enzyme (160/tg protein/ml) was incabated for 60 rain at 30 o C in the pr¢~enceof 5 mM Tris-HCI (pH 9.0) and the indicated additions. At the end of this incubation aliquots (0.05 ml) were assayed for enzyme activity using the standard assay conditions. Where indicated, samples were dialyzed against 5 mM Tris.HCi (pH 9.0) for 3 days at 0 °C prior to assay. b The results are the means±standard deviation for three experiments. The activitybefore the incubationcommencedwas 1.39+ 0.05 Fmol/min per mg protein.
294
lar weight inhibitor [7]. It is not likely that this putative inhibitor is phosphate, since neither the reversible or the irreversible inhibition reported here resembles the characteristics of the endogenous modulation [6,7]. The 13. discoideum alkaline phosphatase is clearly an unusual membrane-bound enzyme and a more detailed elucidation of its structure will be important for an understanding of its regulation during development.
D
E m
E m o o
m
E
3
Acknowledgments m m 41a
01-6.5
7.5
8.5
9.5
pH Fig. 4. pH-dependence of phosphate inhibition of enzyme activity. Purified enzyme (4 ~tg) was incubated at 30°C for 60 rain in the pre~nce of 200 mM Tris.HCI at the indicated pH values with either no addition (11) or ! m M K2HPO4 (13) in a final volume of 0,1 ml. Enzyme activity was then as~yed under standard conditions, except that 500 mM Tris-HCI (pH 8.5) replaced the usual buffer concentration and 4.0 mM pNPP was used as substrate.
The increase in alkaline phosphatase activity during the development of Dictyostelium (1-4), which may be important for localized production of adenosine, a potential morphogen [10,11] is due to the gradual unmasking of the enzyme [6,7]. This unmasking was accomplished in vitro by elevated temperatures or by prolonged dialysis [5,6] and reconstitution experiments indicated that it involved the removal of a low molecuTABLE !1
AMP protection of activity against phosphate inactivation Treatment *
Enzyme activity b (It mo!/min per m8 protein)
None + I mM KaHPO,, + ! mM K2HPO4, 1 mM AMP + i mM K2HPO4, 2 mM AMP + ! m M K2HPO4, 4 mM AMP
1.29+-0.06 0.31 +-0.05 0.54+-0.03 0.81 + 0.06 1.02+0.06
" Purified enzyme (160 ~tg protein/ml) was incubated in the presence of 200 raM Tris-HC! (pH 7.5) and the indicated additions for 10 rain at 30 o C. After incubation, aliquots (0.05 ml) were assayed for enzyme activity using the standard assay conditions. b Th• ~s,dtg ate. the means of triplicate experiments + standard deviation.
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