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Multiple roles of metal ions in the reaction catalyzed by yeast inorganic pyrophosphatase
BIOINORGANICCHEAUSTRY
7,141-150
(1977)
141
Multiple Roles of Metal Ions in the Reaction Catalyzed by Yeast Inorganic Pyrophosphatase”
LARRY
G_ BUTLER
and JAMES W. SPEROWi
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
ABSTRAClYeast inorganic pyrophosphatase has three roles for metal ions in its reaction: activator, substrate and structural. Out of a wide variety of metal ions tested > only Mg2’, Zn”+, Mn2+ and Co2+ can fulfill both the activator and substrate roles. Several other metal ions inhibit the Mg2+-stirnulated activity; the strong inhibition by Ca2’ (and probably Cd29 is due to interference with both activator and substrate roles, while the weaker inhibition by Sr2+ (and possiily cu2+ and Ni2+) is due to interference with only the substrate role. Rare earth ions strongly stimulate nonenzymic PPi hydrolysis but do not activate the enzyme. Despite its ability to fulfill both the activator and substrate roles, Zn2+ causes inactivation of the enzyme, probably by interference with the “structural” Mg21-- The results suggest that the three roles for metal ions are independent (an individual metal ion can satisfy only one at a time) and that the metal ion specificity for the three roles declines in the order: structural > substrate > activator.
INTRODUCTION The hydrolysis phosphatase occurs are obtained in the (and independent) the enzyme 12, 3]
of inorganic pyrophosphate @‘Pi) by yeast inorganic pyroonly in the presence of multivalent metal ions; maximal rates presence of Mg2* [l ] , which appears to fulfill three different roles in the enzymic reaction_ Free Mg2+ is an activator of ; MgPPi’ is the substrate [2-41, which is bound via the Mg2+
* This is Journal Paper 5581 from the Purdue University Agricultural Experiment Station, W. Lafayette, IN 47907. f Present address: William H. Rorer, Inc., Fort Washington, PA 19034_ r Nonstandard abbreviations include: EDTA, ethylenediamine tetraacetic acid; EGTA, ethyleneglycol-bis @-aminoethyl ether)-N,N’-tetraacetic acid; Tris, tris( hydroxymethyl) aminomethane; PIPES, piperazinc-N,N’-bis (Z-ethane sulfonic acid); and CaPPi, MgPPi, EuPPf and BaPPf, the 1:l complex of PPi with Ca2+, Mg2*, Eu3’ and Ba2+, respectively. 0 Elsevier North-Holland, Inc., 1977
L. G_ BUTLER AND J. W. SPEROW
142
rather than the phosphates [S]; in addition, the enzyme has been reported to contain tightly bound “structural” Mg2* [6]. If the three roles for metal ions are independent (in the sense that an individual ion can satisfy only one of them at a time) the specificity and relative effectiveness of different metal ions need not be the same in each role_ Because of the complex equilibria involved, it is generally not possible to examine the effects of various metal ions separately in each role. Experimentally accessible observations with Ca2*, which is chemically similar to Mg2 but strongly inhibitory, susest that the metal ion complexed with PPr (substrate role) has a very large and critical effect on the rate of the hydrolytic reaction and a smaller but measurable effect on the strength of binding of the complex to the enzyme 17, 8]_ Both CaPPi and MgPPi interact with the Mg2+-activated enzyme; MgPPi is hydro!yzed at least lo5 times faster than CaPPi, although CaPPi binds much more strongly (60-fold difference in dissociation constants) [7-93 _ Asmeasured by the strength of binding CaPPi to either Mg2+- or Ca2+-activated enzyme, it appears to make little difference which metal ion satisfies the activation requirement_ Thus, the metal ion requirement for the substrate role is apparently much more specific for Mg’+ than is the metal ion requirement for the activator role. Preliminary studi s indicate the structural Mg2* can not be replaced by Zn2+ cr Mn2+ with retention of activity [6], suggesting considerable metal ion specificity for this role. On removal of this tightly bound Mg2+, the enzyme is susceptible to proteolysis and to dissociation to subunits [6] _ We have examined the effects of a variety of metal ions on the rate of yeast pyrophosphatase-catalyzed PPi hydrolysis and on the stability of the enzyme. The results are consistent with independence of the three roles for metal ions in this reaction_ MATERIALS
AND METHODS
Rare earth chloride hexahydrates (99.9%) were obtained from Alfa Inorganics. Cr(NOa)a-9HaO was purchased from the General Chemical Division, Allied Chemical Company. EGTA and PIPES were obtained from Sigma Chemical Company. Tris (ultra pure grade) was purchased from Mann Research Laboratories_ [32P] Pyrophosphate (minimum specific activity 1 Ci/mole) was obtained from New England Nuc!ear Corporation_ Heavy metal contaminants were removed from buffer solutions for pyrophosphatase assays by extraction with 0.003% dithizone in CC14 (IO). All other chemicals were the best grade available from standard sources, and were used without further purification_ Solutions were prepared from doubly deionized glass-distilled water, with precautions to avoid contamination by metal ions [lo] _ Crystalline yeast inorganic pyrophosphatase was prepared in this laboratory [6] and stored as the (NH&S04 suspension. The specific activity of the
METAL
IONS AND INORGANIC
PYROPHOSPHATASE TABLE
Activation
Metal ion Mgaf Zn2+ Mn** co2*
1
of Pyrophosphatase pmoles mine1 mg-l 402 32.4 23.3, 12.3
143
+15 5 4.0 f 1.7 + 0.83
by Metal Ione Relative velocity 1.000 0.081 0.058 0.030
o Conditions were 0.45 M Tris HCI, pH 7.4, 0.2 M KzS04, ionic strength 1.0 M, 0.3 mM PPi, and 2.0 mM metal ion: the radioisotope assay [ 21 was utilized. Temperature was 30°C. Samples were run in triplicate; velocities are reported + the average deviation from the mean. Controls lacking enzyme or metal ion gave a maximum relative velocity of less than 0.001.
enzyme at 30°C in 0.1 M Tris HCI, pH 7.4, using 2 mM PPi and 3 mM MgClz, was 600-650 moles PPi hydrolyzed min-l mg-l . For testing with metal ions, the enzyme was dialyzed for several hours against 0.05 M Tris HCI, pH 7.4, containing lo-* M EDTA, and then for 3 days against several changes of the same buffer without EDTA. Because dialysis against EDTA was brief and was carrried out in the cold room, no loss of activity was observed [6] _ In order to obtain maximum sensitivity, data presented in Table I and Fig_ 1 were obtained with the radioisotope assay previously described [2] ; the remaining data were obtained with a spectrophotometric assay [6] from which EGTA was omitted. RESULTS Metal Ion Specificity of the Activator and Substrate Roles The ability of Mg2+, Mn2+, and Co2+ to activate PPi hydrolysis catalyzed by yeast pyrophosphatase was demonstrated by Kunitz [1] ; later Zn2+ was also shown to be an activator [l l] _ The relative efficacy of these ions as activators depends somewhat on the conditions under which they are tested. The rates observed under the conditions we have previously employed for kinetic analysis of Mg2+ activation [2] are presented in Table I _ Eighteen other di- and trivalent metal ions (Ba*+, Be*+, Ca**, Cd2+, Cr3+, Cu*+, Fe*+, Fea+ 7 Ni*+ , Sr** 7 Dy3+, Er3*, Eu3+, Gda+, Ho3+, Nda+, Pra+ and yba+) were examined under the same conditions and found to be ineffective (no more than 0.1% of the activity observed with Mg2’). Thus, of the metal ions tested, only Mg*+, Zn*+, Mn*“. and Co*+. are able to fulfill both the activator and substrate roles.
L. G_ BUTLER
144
AND 3. W. SPEROW
-L
0
200
400
600
800
looo
l200
8
Moo
MINUTES FIG_ I_ Stimulation
of nonenzymic PPi hydrolysis by rare earth ions. (a) Conditions were O-45 M Tris HCl. pH 7.4.0.54 M KCl, 0-l mM 32PPi (8 X 10’ cpm), tCJm&f Gd(N03)3 and 1.2 yg/ml of yeast pyrophosphatase. Values shown are averages of duplicate sampIes_ Complete mixture (0); omit enzyme (0); omit Gd(NO& (A). B. Conditions were 0.1 M Tris HCI, pH 9.4, 1 mM 32PPi (7 X IO5 cpm), and OS mM NdCis (0) or MgCI, (0). No enzyme was present.
METAL
IONS AND INORGANIC
PYROPHOSPIIATASE TABLE
Inhibition
2
of Mg2+-Stimulated
Addition
145
Activity=
5%of Control
None Ba2+ Be2+ Ca2+
100 105_9b
71.1 32.5 22.1 97.2b 85.7 78.7b 75.4 64.1
$Z (32+
Eu3+ Ni2+ .%a+
a Conditions were 0.45 M Tris HCl, pH 7.4; 0.2 M K2SOB, 5 mM MgCl, 2 mM PPi and OS mM inhibitory metal ion; the spectrophotometric assay [6] was employed. Values reported are averages of duplicate samples. b incipient precipitation in the assay mix.
The rare earth ions exhibited anomalous behavior; all of those tested catalyzed the nonenzymic hydrolysis ofPP,_ Under a variety of conditions, including those utilized in Table 1, rare earth ions caused a rapid burst of PPi hydrolysis followed by a slower reaction; typical examples obtained under widely differing conditions are shown in Fig. 1. The rate of hydrolysis was dependent on pH, nature of the buffer, and concentrations of rare earth ions and PPi- In some cases visible precipitates formed within a few hours of mixing. In no case was the rate of hydrolysis increased by addition of pyrophosphatase. Of the ions tested, only the rare earths cairsed significant nonenzymic hydrolysis. Inhibition
of the Mg2+-StimuIated
Activity
Several metal ions which do not generate enzymic activity nevertheless strongly interact with the system as shown by their inhibition of the Mg2’stimulated activity (Table 2)_ For these inhibition experiments we chose conditions (5 mM Mg2*, 2 mM PPi, OS mM inhibitor) in which the concentration of free Mg2+ is we11 above its K, of I.3 mM [2], and the PPi concentraticn is much greater than the concentration of inhibitory metal ion, so that the concentration of MgPPr will remain well above its K, of 1.7 X 1O-5 M 121. Thus any observed inhibition cannot be due to deficiency of substrate or activator but must be due to interaction of inhibitor with the enzyme. Even under these stringent conditions Ca2* and Cd2+ are strongly inhibitory, and
L. G. BUTLER AND J. W. SPEROW
,
, I
I
1
50
loo
MINUTES (a) FIG. 2. Effect of Conditions were 2 6.5 (b). Zero time with cold solution
k
t
4
HRS
j‘k. 1 MINUTES
HRS
(b)
metal ions on yeast pyrophosphatase in the absence of PPi_ m&l metal ion in 0.1 M Tris HCl, pH 7.4 (a) or 0-l M PIPES, pH assays were carried out immediately after mixing cold enzyme of metal ion in buffer; solutions were then incubated at 30 OC
and aliquots removed for assay at various times. Activity was determined by the spectrophotometric assay; data points are averages of duplicate values_ Controi sample (no added metal ion) (@)I Mg2* (0); Eu3+ (0); Zn2+ (a) and Cr3* (A). SoIutions containing Eu3+ and Cr3+ developed a light precipitate during the experiment. several other ions show significant inhibition. In some cases thsapparent tion is at least partially due to inactivation of the enzyme (see below).
inhibi-
Effects of Me&I Ions on the Enzyme in the Absence of PPi Exposure of the enzyme to metal ions in the absence of PPi can resutt in protection, inactivation or stimulation of the catalytic activity. The effects are dependent not oniy on the nature of the metalion,but also upon pH, temperature and other factors such as previous dialysis of the enzyme against an EDTA
METAL IONS AND IN0 , GANIC PYROPHOSPHATASE
I47
E solution in order to rem ve traces of contaminating metal ions, as described in Methods. Rest&s of typ caI experiments documenting these effects are shown in Fig. 2. In order to min- ize the effect of free Mg 2+ which can reactivate inactive -P the standard assay was modified to include a twofold enzyme in the assay [6&, excess of total PPi ove$ total Mg2’ so that most of the Mg2+ was complexed, ,a+ at a low level. At pH 7.4 (Fig. 2a), keeping the concentratton of free MO exposure to 2 mM Mg2’ and Eu3* had little effect, but Zn2+ and Cra+ inactivate the enzyme under these conditions. Addition of EDTA, which strongly complexes most metals, but binds Mg2+ relatively weakly (121, arrests but does not reverse the Zn2+dependent inactivation (data not shown). In other experiments at pH 7.4, Ca2+, Cd2 *,Cu2+, Mn2*, Nd2+ and Ni 2+ showed small effects similar to Mg2+ and Eu3*_ At pH 6.5 (Fig . 2b), the enzyme is unstable, Zn2’ has little effect and Cra+ causes an even more rapid loss of activity_ Mg2+ and Eu3+ (and in other experiments, Co2+) protect the enzyme against this inactivation. DISCUSSION Specificity of Metal ion Activation
In order to make comparisons with previous results on the Mg2+-dependent reaction, the conditions employed for testing for activation by various metal ions were similar to those previously utilized in our kinetic studies of this enzyme [2, 71; these conditions are near optimal with Mg2+ but may not be optimal with other metal ions. Because each metal ion was tested separately it must be able to satisfy the requirements for both activator and substrate in order to generate enzyme activity. The ability to fulfill one but not both of these roles would not be detected in this experiment. In interpreting the effects of various metal ions in the role of substrate, it should be kept in mind that the substrate (metal-ion-PPi complex) probably binds to the enzyme through the metal ion rather than through the phosphate groups [S] _ The only four metal ions which generate pyrophosphatase activity (Mg2+, Zn2+, Mn2+, and Co2+) also activate the Pi * HOH exchange catalyzed by this enzymee2 Our assays of PPr hydrolysis under several conditio-ns (not shown) are in agreement with those of Kunitz who showed that the relative rates obtained with these ions depend on factors such as pH, PPi concentration and ratio of metal ion concentration to PPi concentration [l] _ Mn2+ can form slowIy dissociating agregates with PPi which are not substrates for yeast pyrophosphatase [13] ; the catalytic efficiency of any other metal ions which act similarly might thus be underestimated. The test for aggregation is nonlinearity of measured velocity as a function of enzyme * P. D. Boyer, personal communication.
L_ G_ BUTLER
148
AND J. W. SPEROW
concentration; by this criteria, aggregation did not occur with Mg2+ or Mn2+ in our assays. We did not mix the metal ion with PPi until immediately before starting the assay, so aggregation, which is a relatively slow process, does not account for the lack of activity of other metal ions in our assays. We have occasionally observed small amounts of activity with some of these other metal ions, but careful exclusion from the assay of extraneous Mg2+, Zn2+, Mn2+ and Co2+ eliminated this apparent activity_ We attribute reports of low amounts of activity with other metal ions to contamination by one of these “active” ions, although under somewhat different conditions, such as utilization of ATP as substrate, other ions could also be effective [ 1 l] _ a Rare Earth Ions Although rare earths do not activate the enzyme, they nevertheless stimulate the reaction nonenzymically. Catalysis of hydrolysis of esters of phosphates, phosphonates and pyrophosphates by gels of rare earth hydroxides has previously been reported [14- 171 _The biphasic nature of the plots shown in Fig_ 1 suggests that rare earth ions, in the soluble form in which they are added, are even more efficient at promoting rapid PPr hydrolysis than the aggregated gels [13, 151 which form during the reaction_ It is possible that the enzyme may function by altering the bound MgPPi to a form somewhat resembling a rare-earth-PPi complex but the inability of rare earth ions to stimulate enzymic PPi hydrolysis provides no support for this model. The inefficiency of rare earth ions in the enzymic reaction is not due to their failure to bind to the enzyme; Eu3+ and other rare earth ions have previously been shown to strongly bind to the enzyme in the absence of PPi, presumably at the activator site [ 18, 191, in accord with the similar effects of Mg2+ and Eu3+ observed in Fig. 2. Moreover, the inhibition of Mg2*-stimulated activity by Eua+ under the conditions employed in Table 2 is probably due to EuPPi binding. These data do not indicate whether it is the activator or substrate role which rare earth ions are incapable of fulfilling in the enzymic reaction_ Inhibition of Mg2*Stimnlated
Activity
Under the conditions utilized in Table 2 inhibition will be observed only if the inhibitory ion strongZy interferes with either the activator or substrate role of Mg2+_ Although Ba2+ complexes almost as strongly with PPi as does Mg2+ [12], no inhibition is observed (Table 2): indicating not only that adequate MgPPi is present to maintain full activity but that the enzyme must bind BaPPi weakly compared to MgPPr- The strong inhibition by Ca2* has been shown to be due to interference with both activator and substrate roles [7] ; presumably the same is true for Cd2+. Inhibition by other ions such as Cu2+, Ni2’ and Sr2+ is less marked but nevertheless quite significant under these conditions_ According
METAL
IONS AND INORGANIC
PYROPHOSPHATASE
149
to fluorescence titration data, Sr z+ does not bind to the enzyme at the activator site [8]. It is thus possible that the effect of this metal ion (and perhaps others) is due solely to its interference with the substrate role of Mg2+ by formation of a metal-ion-PPi complex which is bound but not hydrolyzed. Effect of Metal Ions on the Enzyme in the Absence of PI’,
It is of interest that Zn2+, which generates pyrophosphatase activity and thus can fulfill both the substrate and activator roles for metal ion, can inactivate the enzyme. It has previously been reported that Zn2+ catalyzes the thermal inactivation of the enzyme under somewhat different conditions [20] _ Inactivation by Zn2+ under our conditions at 30°C does not appear to be associated with binding at the tight binding site for Zn 2+; binding at this site is rapid, does not result in appreciable loss of activity, and is competitive with binding of other metal ions which do not inactivate the enzyme [8, 19]_ Thus inactivation by Zn2+ must be due to interaction at some other site such as the second, weaker Zn’*-binding site [21] _ Inactivation by Zn*+ may be related to formation of hydroxides, since it occurred at pH 7.4, but not at pH 6.5. It is possible that inactivation by Zn2+ and by Cra+ as previously noted [S] is due to displacement of the “structural” Mg2+ [3] _ It has been reported that Mg2+, but not Zn2+, partially restores catalytic activity to the enzyme which has been inactivated by exposure to EDTA [6] _ As shown in Fig. 2, enzyme dialyzed briefly against EDTA shows a time-dependent increase in activity on exposure to Mg2+ but not to other ions. In order to demonstrate this effect it is necessary to employ assay conditions in which the concentration of free Mg2+ is kept low to prevent rapid reactivation by Mg2* m _ the assay [6] _ These results lend further credence to suggestions of a structural role for tightly bound Mg2+, which no other ion can replace with retention of activity_ The observations reported here are consistent not only with three different functional roles for metal ions in this reaction, but also with the suggestion that a single metal ion can satisfy only one role at a time. Mg2+ is the only metal ion known to be capable of fulfilling all three roles, and the metal ion specificity of the roles apparently decreases in the order: structural > substrate > activator_ In the presence of M,e2+, the enzyme is extremely specific for PP, as substrate [22]. Full understanding of the structural and functional relationships of metal ions in these roles and a satisfying correlation of the properties of various metal ions with their relative effectiveness in each of these roles must await detailed structural analysis_
This investigation was supported by NIH Training Grant Number GM1 195 from National Institute of General Medical Sciences LGB is a recipient of a Research Career Development Award (GM 46404) from the US_ Public Health Service. We are grateful to Dr. S. M. Avaeva and Dr. Paul Boyer for making manuscripts available to us pnbr to publicatiorz
150
L. G_ BUTLER AND J. W. SPEROW
REFERENCES 1. M. Kunitz,f_ Gen Physiol. 35,423 2. 3. 4. 5. 6. 7. 8_ 9_ IO. 11. 12. 13. 141.5. 1617. 1s. 19. 20_ 21. 22.
(1952)0. A. Moe and L. G_ Butler,/. Biol. Chem. 247,7308 (1972). E. A. Braga and S_ Avaeva, Biochem Biophyr Rer. Commwr 49,528 (1972). I-. A_ Rapoport. W. E. HGhne. J. G. Reich, P. Heitmann and S. M. Rapoport, Eur. J. Biochem. 26,237 (1972). J. W. Sperow and L. G. Butler, J. BioL chern Xl, 2611 (1976). J. W. RJdJJngton, Y. Yang andL. G. Butler,ArcJr_ Biochem Biophrs 152,714 (1972). 0. A. Moe and L. G. Butler, J. Biol. Chem 247,731s (1972). J. W. Ridlington and L. G. Butler, J. Biol. Chem. 247,7303 (1972). T. A_ Rapoport. W. E. HShne, P. Heitmann and S- Rapoport, Eur. J. Biochem. 33,341 (1973). J. W. Sperow, M.S. thesis, Purdue University (1972). M. J. Schlesinger and M. J. Coon, B&chin Biophys. Actn 41,30 (1960). L. G. Siilen and A_ E_ Martell, in Stability Constants of Metal-Ion Complexes, Special PubIication No. 17, The Chemical Society, London (1964). B.S. Cooperman and D. H. Mark,Biochim Biophys. At% 252,221 (1971). E- Bamann and H. Trapmann,Advan. Enzymol. 21,169 (1959). P-Watts aad F- M. B1ewett.J. them SOC_B. 881 (1971). D-J- Howelks and D. B. CouIt, Biochim- Biophys. Acfa 244,427 (1971). G. Rutherford and A. R. Morgan, CQIL J. Biochem. 50,287 (1972). 1. W. Sperow and L. G. Butler, Bioinorg. Chem. 2 87 (1972) B. S. Cooperman and N. Y. Chiu, Biochem. 12,167O (1973). Y. A. Shafmnskii and S. M. Avaeva, Biokhimiya 38-1248 (1974). A. A. Baykov and S. M. Avaeva. Eur. J. Biochem 47,57 (1974). J- W. Sperow. 0. A. Moe, J. W. Ridlington and L. G. Butler,J_ BioL Chem 248,2062 (1973).
Received
20 July 2 9 76
7,141-150
(1977)
141
Multiple Roles of Metal Ions in the Reaction Catalyzed by Yeast Inorganic Pyrophosphatase”
LARRY
G_ BUTLER
and JAMES W. SPEROWi
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
ABSTRAClYeast inorganic pyrophosphatase has three roles for metal ions in its reaction: activator, substrate and structural. Out of a wide variety of metal ions tested > only Mg2’, Zn”+, Mn2+ and Co2+ can fulfill both the activator and substrate roles. Several other metal ions inhibit the Mg2+-stirnulated activity; the strong inhibition by Ca2’ (and probably Cd29 is due to interference with both activator and substrate roles, while the weaker inhibition by Sr2+ (and possiily cu2+ and Ni2+) is due to interference with only the substrate role. Rare earth ions strongly stimulate nonenzymic PPi hydrolysis but do not activate the enzyme. Despite its ability to fulfill both the activator and substrate roles, Zn2+ causes inactivation of the enzyme, probably by interference with the “structural” Mg21-- The results suggest that the three roles for metal ions are independent (an individual metal ion can satisfy only one at a time) and that the metal ion specificity for the three roles declines in the order: structural > substrate > activator.
INTRODUCTION The hydrolysis phosphatase occurs are obtained in the (and independent) the enzyme 12, 3]
of inorganic pyrophosphate @‘Pi) by yeast inorganic pyroonly in the presence of multivalent metal ions; maximal rates presence of Mg2* [l ] , which appears to fulfill three different roles in the enzymic reaction_ Free Mg2+ is an activator of ; MgPPi’ is the substrate [2-41, which is bound via the Mg2+
* This is Journal Paper 5581 from the Purdue University Agricultural Experiment Station, W. Lafayette, IN 47907. f Present address: William H. Rorer, Inc., Fort Washington, PA 19034_ r Nonstandard abbreviations include: EDTA, ethylenediamine tetraacetic acid; EGTA, ethyleneglycol-bis @-aminoethyl ether)-N,N’-tetraacetic acid; Tris, tris( hydroxymethyl) aminomethane; PIPES, piperazinc-N,N’-bis (Z-ethane sulfonic acid); and CaPPi, MgPPi, EuPPf and BaPPf, the 1:l complex of PPi with Ca2+, Mg2*, Eu3’ and Ba2+, respectively. 0 Elsevier North-Holland, Inc., 1977
L. G_ BUTLER AND J. W. SPEROW
142
rather than the phosphates [S]; in addition, the enzyme has been reported to contain tightly bound “structural” Mg2* [6]. If the three roles for metal ions are independent (in the sense that an individual ion can satisfy only one of them at a time) the specificity and relative effectiveness of different metal ions need not be the same in each role_ Because of the complex equilibria involved, it is generally not possible to examine the effects of various metal ions separately in each role. Experimentally accessible observations with Ca2*, which is chemically similar to Mg2 but strongly inhibitory, susest that the metal ion complexed with PPr (substrate role) has a very large and critical effect on the rate of the hydrolytic reaction and a smaller but measurable effect on the strength of binding of the complex to the enzyme 17, 8]_ Both CaPPi and MgPPi interact with the Mg2+-activated enzyme; MgPPi is hydro!yzed at least lo5 times faster than CaPPi, although CaPPi binds much more strongly (60-fold difference in dissociation constants) [7-93 _ Asmeasured by the strength of binding CaPPi to either Mg2+- or Ca2+-activated enzyme, it appears to make little difference which metal ion satisfies the activation requirement_ Thus, the metal ion requirement for the substrate role is apparently much more specific for Mg’+ than is the metal ion requirement for the activator role. Preliminary studi s indicate the structural Mg2* can not be replaced by Zn2+ cr Mn2+ with retention of activity [6], suggesting considerable metal ion specificity for this role. On removal of this tightly bound Mg2+, the enzyme is susceptible to proteolysis and to dissociation to subunits [6] _ We have examined the effects of a variety of metal ions on the rate of yeast pyrophosphatase-catalyzed PPi hydrolysis and on the stability of the enzyme. The results are consistent with independence of the three roles for metal ions in this reaction_ MATERIALS
AND METHODS
Rare earth chloride hexahydrates (99.9%) were obtained from Alfa Inorganics. Cr(NOa)a-9HaO was purchased from the General Chemical Division, Allied Chemical Company. EGTA and PIPES were obtained from Sigma Chemical Company. Tris (ultra pure grade) was purchased from Mann Research Laboratories_ [32P] Pyrophosphate (minimum specific activity 1 Ci/mole) was obtained from New England Nuc!ear Corporation_ Heavy metal contaminants were removed from buffer solutions for pyrophosphatase assays by extraction with 0.003% dithizone in CC14 (IO). All other chemicals were the best grade available from standard sources, and were used without further purification_ Solutions were prepared from doubly deionized glass-distilled water, with precautions to avoid contamination by metal ions [lo] _ Crystalline yeast inorganic pyrophosphatase was prepared in this laboratory [6] and stored as the (NH&S04 suspension. The specific activity of the
METAL
IONS AND INORGANIC
PYROPHOSPHATASE TABLE
Activation
Metal ion Mgaf Zn2+ Mn** co2*
1
of Pyrophosphatase pmoles mine1 mg-l 402 32.4 23.3, 12.3
143
+15 5 4.0 f 1.7 + 0.83
by Metal Ione Relative velocity 1.000 0.081 0.058 0.030
o Conditions were 0.45 M Tris HCI, pH 7.4, 0.2 M KzS04, ionic strength 1.0 M, 0.3 mM PPi, and 2.0 mM metal ion: the radioisotope assay [ 21 was utilized. Temperature was 30°C. Samples were run in triplicate; velocities are reported + the average deviation from the mean. Controls lacking enzyme or metal ion gave a maximum relative velocity of less than 0.001.
enzyme at 30°C in 0.1 M Tris HCI, pH 7.4, using 2 mM PPi and 3 mM MgClz, was 600-650 moles PPi hydrolyzed min-l mg-l . For testing with metal ions, the enzyme was dialyzed for several hours against 0.05 M Tris HCI, pH 7.4, containing lo-* M EDTA, and then for 3 days against several changes of the same buffer without EDTA. Because dialysis against EDTA was brief and was carrried out in the cold room, no loss of activity was observed [6] _ In order to obtain maximum sensitivity, data presented in Table I and Fig_ 1 were obtained with the radioisotope assay previously described [2] ; the remaining data were obtained with a spectrophotometric assay [6] from which EGTA was omitted. RESULTS Metal Ion Specificity of the Activator and Substrate Roles The ability of Mg2+, Mn2+, and Co2+ to activate PPi hydrolysis catalyzed by yeast pyrophosphatase was demonstrated by Kunitz [1] ; later Zn2+ was also shown to be an activator [l l] _ The relative efficacy of these ions as activators depends somewhat on the conditions under which they are tested. The rates observed under the conditions we have previously employed for kinetic analysis of Mg2+ activation [2] are presented in Table I _ Eighteen other di- and trivalent metal ions (Ba*+, Be*+, Ca**, Cd2+, Cr3+, Cu*+, Fe*+, Fea+ 7 Ni*+ , Sr** 7 Dy3+, Er3*, Eu3+, Gda+, Ho3+, Nda+, Pra+ and yba+) were examined under the same conditions and found to be ineffective (no more than 0.1% of the activity observed with Mg2’). Thus, of the metal ions tested, only Mg*+, Zn*+, Mn*“. and Co*+. are able to fulfill both the activator and substrate roles.
L. G_ BUTLER
144
AND 3. W. SPEROW
-L
0
200
400
600
800
looo
l200
8
Moo
MINUTES FIG_ I_ Stimulation
of nonenzymic PPi hydrolysis by rare earth ions. (a) Conditions were O-45 M Tris HCl. pH 7.4.0.54 M KCl, 0-l mM 32PPi (8 X 10’ cpm), tCJm&f Gd(N03)3 and 1.2 yg/ml of yeast pyrophosphatase. Values shown are averages of duplicate sampIes_ Complete mixture (0); omit enzyme (0); omit Gd(NO& (A). B. Conditions were 0.1 M Tris HCI, pH 9.4, 1 mM 32PPi (7 X IO5 cpm), and OS mM NdCis (0) or MgCI, (0). No enzyme was present.
METAL
IONS AND INORGANIC
PYROPHOSPIIATASE TABLE
Inhibition
2
of Mg2+-Stimulated
Addition
145
Activity=
5%of Control
None Ba2+ Be2+ Ca2+
100 105_9b
71.1 32.5 22.1 97.2b 85.7 78.7b 75.4 64.1
$Z (32+
Eu3+ Ni2+ .%a+
a Conditions were 0.45 M Tris HCl, pH 7.4; 0.2 M K2SOB, 5 mM MgCl, 2 mM PPi and OS mM inhibitory metal ion; the spectrophotometric assay [6] was employed. Values reported are averages of duplicate samples. b incipient precipitation in the assay mix.
The rare earth ions exhibited anomalous behavior; all of those tested catalyzed the nonenzymic hydrolysis ofPP,_ Under a variety of conditions, including those utilized in Table 1, rare earth ions caused a rapid burst of PPi hydrolysis followed by a slower reaction; typical examples obtained under widely differing conditions are shown in Fig. 1. The rate of hydrolysis was dependent on pH, nature of the buffer, and concentrations of rare earth ions and PPi- In some cases visible precipitates formed within a few hours of mixing. In no case was the rate of hydrolysis increased by addition of pyrophosphatase. Of the ions tested, only the rare earths cairsed significant nonenzymic hydrolysis. Inhibition
of the Mg2+-StimuIated
Activity
Several metal ions which do not generate enzymic activity nevertheless strongly interact with the system as shown by their inhibition of the Mg2’stimulated activity (Table 2)_ For these inhibition experiments we chose conditions (5 mM Mg2*, 2 mM PPi, OS mM inhibitor) in which the concentration of free Mg2+ is we11 above its K, of I.3 mM [2], and the PPi concentraticn is much greater than the concentration of inhibitory metal ion, so that the concentration of MgPPr will remain well above its K, of 1.7 X 1O-5 M 121. Thus any observed inhibition cannot be due to deficiency of substrate or activator but must be due to interaction of inhibitor with the enzyme. Even under these stringent conditions Ca2* and Cd2+ are strongly inhibitory, and
L. G. BUTLER AND J. W. SPEROW
,
, I
I
1
50
loo
MINUTES (a) FIG. 2. Effect of Conditions were 2 6.5 (b). Zero time with cold solution
k
t
4
HRS
j‘k. 1 MINUTES
HRS
(b)
metal ions on yeast pyrophosphatase in the absence of PPi_ m&l metal ion in 0.1 M Tris HCl, pH 7.4 (a) or 0-l M PIPES, pH assays were carried out immediately after mixing cold enzyme of metal ion in buffer; solutions were then incubated at 30 OC
and aliquots removed for assay at various times. Activity was determined by the spectrophotometric assay; data points are averages of duplicate values_ Controi sample (no added metal ion) (@)I Mg2* (0); Eu3+ (0); Zn2+ (a) and Cr3* (A). SoIutions containing Eu3+ and Cr3+ developed a light precipitate during the experiment. several other ions show significant inhibition. In some cases thsapparent tion is at least partially due to inactivation of the enzyme (see below).
inhibi-
Effects of Me&I Ions on the Enzyme in the Absence of PPi Exposure of the enzyme to metal ions in the absence of PPi can resutt in protection, inactivation or stimulation of the catalytic activity. The effects are dependent not oniy on the nature of the metalion,but also upon pH, temperature and other factors such as previous dialysis of the enzyme against an EDTA
METAL IONS AND IN0 , GANIC PYROPHOSPHATASE
I47
E solution in order to rem ve traces of contaminating metal ions, as described in Methods. Rest&s of typ caI experiments documenting these effects are shown in Fig. 2. In order to min- ize the effect of free Mg 2+ which can reactivate inactive -P the standard assay was modified to include a twofold enzyme in the assay [6&, excess of total PPi ove$ total Mg2’ so that most of the Mg2+ was complexed, ,a+ at a low level. At pH 7.4 (Fig. 2a), keeping the concentratton of free MO exposure to 2 mM Mg2’ and Eu3* had little effect, but Zn2+ and Cra+ inactivate the enzyme under these conditions. Addition of EDTA, which strongly complexes most metals, but binds Mg2+ relatively weakly (121, arrests but does not reverse the Zn2+dependent inactivation (data not shown). In other experiments at pH 7.4, Ca2+, Cd2 *,Cu2+, Mn2*, Nd2+ and Ni 2+ showed small effects similar to Mg2+ and Eu3*_ At pH 6.5 (Fig . 2b), the enzyme is unstable, Zn2’ has little effect and Cra+ causes an even more rapid loss of activity_ Mg2+ and Eu3+ (and in other experiments, Co2+) protect the enzyme against this inactivation. DISCUSSION Specificity of Metal ion Activation
In order to make comparisons with previous results on the Mg2+-dependent reaction, the conditions employed for testing for activation by various metal ions were similar to those previously utilized in our kinetic studies of this enzyme [2, 71; these conditions are near optimal with Mg2+ but may not be optimal with other metal ions. Because each metal ion was tested separately it must be able to satisfy the requirements for both activator and substrate in order to generate enzyme activity. The ability to fulfill one but not both of these roles would not be detected in this experiment. In interpreting the effects of various metal ions in the role of substrate, it should be kept in mind that the substrate (metal-ion-PPi complex) probably binds to the enzyme through the metal ion rather than through the phosphate groups [S] _ The only four metal ions which generate pyrophosphatase activity (Mg2+, Zn2+, Mn2+, and Co2+) also activate the Pi * HOH exchange catalyzed by this enzymee2 Our assays of PPr hydrolysis under several conditio-ns (not shown) are in agreement with those of Kunitz who showed that the relative rates obtained with these ions depend on factors such as pH, PPi concentration and ratio of metal ion concentration to PPi concentration [l] _ Mn2+ can form slowIy dissociating agregates with PPi which are not substrates for yeast pyrophosphatase [13] ; the catalytic efficiency of any other metal ions which act similarly might thus be underestimated. The test for aggregation is nonlinearity of measured velocity as a function of enzyme * P. D. Boyer, personal communication.
L_ G_ BUTLER
148
AND J. W. SPEROW
concentration; by this criteria, aggregation did not occur with Mg2+ or Mn2+ in our assays. We did not mix the metal ion with PPi until immediately before starting the assay, so aggregation, which is a relatively slow process, does not account for the lack of activity of other metal ions in our assays. We have occasionally observed small amounts of activity with some of these other metal ions, but careful exclusion from the assay of extraneous Mg2+, Zn2+, Mn2+ and Co2+ eliminated this apparent activity_ We attribute reports of low amounts of activity with other metal ions to contamination by one of these “active” ions, although under somewhat different conditions, such as utilization of ATP as substrate, other ions could also be effective [ 1 l] _ a Rare Earth Ions Although rare earths do not activate the enzyme, they nevertheless stimulate the reaction nonenzymically. Catalysis of hydrolysis of esters of phosphates, phosphonates and pyrophosphates by gels of rare earth hydroxides has previously been reported [14- 171 _The biphasic nature of the plots shown in Fig_ 1 suggests that rare earth ions, in the soluble form in which they are added, are even more efficient at promoting rapid PPr hydrolysis than the aggregated gels [13, 151 which form during the reaction_ It is possible that the enzyme may function by altering the bound MgPPi to a form somewhat resembling a rare-earth-PPi complex but the inability of rare earth ions to stimulate enzymic PPi hydrolysis provides no support for this model. The inefficiency of rare earth ions in the enzymic reaction is not due to their failure to bind to the enzyme; Eu3+ and other rare earth ions have previously been shown to strongly bind to the enzyme in the absence of PPi, presumably at the activator site [ 18, 191, in accord with the similar effects of Mg2+ and Eu3+ observed in Fig. 2. Moreover, the inhibition of Mg2*-stimulated activity by Eua+ under the conditions employed in Table 2 is probably due to EuPPi binding. These data do not indicate whether it is the activator or substrate role which rare earth ions are incapable of fulfilling in the enzymic reaction_ Inhibition of Mg2*Stimnlated
Activity
Under the conditions utilized in Table 2 inhibition will be observed only if the inhibitory ion strongZy interferes with either the activator or substrate role of Mg2+_ Although Ba2+ complexes almost as strongly with PPi as does Mg2+ [12], no inhibition is observed (Table 2): indicating not only that adequate MgPPi is present to maintain full activity but that the enzyme must bind BaPPi weakly compared to MgPPr- The strong inhibition by Ca2* has been shown to be due to interference with both activator and substrate roles [7] ; presumably the same is true for Cd2+. Inhibition by other ions such as Cu2+, Ni2’ and Sr2+ is less marked but nevertheless quite significant under these conditions_ According
METAL
IONS AND INORGANIC
PYROPHOSPHATASE
149
to fluorescence titration data, Sr z+ does not bind to the enzyme at the activator site [8]. It is thus possible that the effect of this metal ion (and perhaps others) is due solely to its interference with the substrate role of Mg2+ by formation of a metal-ion-PPi complex which is bound but not hydrolyzed. Effect of Metal Ions on the Enzyme in the Absence of PI’,
It is of interest that Zn2+, which generates pyrophosphatase activity and thus can fulfill both the substrate and activator roles for metal ion, can inactivate the enzyme. It has previously been reported that Zn2+ catalyzes the thermal inactivation of the enzyme under somewhat different conditions [20] _ Inactivation by Zn2+ under our conditions at 30°C does not appear to be associated with binding at the tight binding site for Zn 2+; binding at this site is rapid, does not result in appreciable loss of activity, and is competitive with binding of other metal ions which do not inactivate the enzyme [8, 19]_ Thus inactivation by Zn2+ must be due to interaction at some other site such as the second, weaker Zn’*-binding site [21] _ Inactivation by Zn*+ may be related to formation of hydroxides, since it occurred at pH 7.4, but not at pH 6.5. It is possible that inactivation by Zn2+ and by Cra+ as previously noted [S] is due to displacement of the “structural” Mg2+ [3] _ It has been reported that Mg2+, but not Zn2+, partially restores catalytic activity to the enzyme which has been inactivated by exposure to EDTA [6] _ As shown in Fig. 2, enzyme dialyzed briefly against EDTA shows a time-dependent increase in activity on exposure to Mg2+ but not to other ions. In order to demonstrate this effect it is necessary to employ assay conditions in which the concentration of free Mg2+ is kept low to prevent rapid reactivation by Mg2* m _ the assay [6] _ These results lend further credence to suggestions of a structural role for tightly bound Mg2+, which no other ion can replace with retention of activity_ The observations reported here are consistent not only with three different functional roles for metal ions in this reaction, but also with the suggestion that a single metal ion can satisfy only one role at a time. Mg2+ is the only metal ion known to be capable of fulfilling all three roles, and the metal ion specificity of the roles apparently decreases in the order: structural > substrate > activator_ In the presence of M,e2+, the enzyme is extremely specific for PP, as substrate [22]. Full understanding of the structural and functional relationships of metal ions in these roles and a satisfying correlation of the properties of various metal ions with their relative effectiveness in each of these roles must await detailed structural analysis_
This investigation was supported by NIH Training Grant Number GM1 195 from National Institute of General Medical Sciences LGB is a recipient of a Research Career Development Award (GM 46404) from the US_ Public Health Service. We are grateful to Dr. S. M. Avaeva and Dr. Paul Boyer for making manuscripts available to us pnbr to publicatiorz
150
L. G_ BUTLER AND J. W. SPEROW
REFERENCES 1. M. Kunitz,f_ Gen Physiol. 35,423 2. 3. 4. 5. 6. 7. 8_ 9_ IO. 11. 12. 13. 141.5. 1617. 1s. 19. 20_ 21. 22.
(1952)0. A. Moe and L. G_ Butler,/. Biol. Chem. 247,7308 (1972). E. A. Braga and S_ Avaeva, Biochem Biophyr Rer. Commwr 49,528 (1972). I-. A_ Rapoport. W. E. HGhne. J. G. Reich, P. Heitmann and S. M. Rapoport, Eur. J. Biochem. 26,237 (1972). J. W. Sperow and L. G. Butler, J. BioL chern Xl, 2611 (1976). J. W. RJdJJngton, Y. Yang andL. G. Butler,ArcJr_ Biochem Biophrs 152,714 (1972). 0. A. Moe and L. G. Butler, J. Biol. Chem 247,731s (1972). J. W. Ridlington and L. G. Butler, J. Biol. Chem. 247,7303 (1972). T. A_ Rapoport. W. E. HShne, P. Heitmann and S- Rapoport, Eur. J. Biochem. 33,341 (1973). J. W. Sperow, M.S. thesis, Purdue University (1972). M. J. Schlesinger and M. J. Coon, B&chin Biophys. Actn 41,30 (1960). L. G. Siilen and A_ E_ Martell, in Stability Constants of Metal-Ion Complexes, Special PubIication No. 17, The Chemical Society, London (1964). B.S. Cooperman and D. H. Mark,Biochim Biophys. At% 252,221 (1971). E- Bamann and H. Trapmann,Advan. Enzymol. 21,169 (1959). P-Watts aad F- M. B1ewett.J. them SOC_B. 881 (1971). D-J- Howelks and D. B. CouIt, Biochim- Biophys. Acfa 244,427 (1971). G. Rutherford and A. R. Morgan, CQIL J. Biochem. 50,287 (1972). 1. W. Sperow and L. G. Butler, Bioinorg. Chem. 2 87 (1972) B. S. Cooperman and N. Y. Chiu, Biochem. 12,167O (1973). Y. A. Shafmnskii and S. M. Avaeva, Biokhimiya 38-1248 (1974). A. A. Baykov and S. M. Avaeva. Eur. J. Biochem 47,57 (1974). J- W. Sperow. 0. A. Moe, J. W. Ridlington and L. G. Butler,J_ BioL Chem 248,2062 (1973).
Received
20 July 2 9 76