- Email: [email protected]
Yeast inorganic pyrophosphatase v. binding of Eu3+
BIOINORGA_NIC
CHEMISTRY,
SHORT Yeast Inorganic JAMES W. SPEROW
Z&87-91
(19i2)
87
COMMUNICATION
Py-rophosphatase V.
Binding
of EUJ+
and LARR.Y G. BUTLER
Department of Biochemistry,
Purdue Unicerszly,
KEY
West Lafayefte, Indiana $“907
WORDS
Yeast Pyrophosphatase Europium Binding The interaction of yeast inorganic pyrophosphatase (EC 3.6 1.1) with metal ions has been shown both kinetically [I, 21 and statically [‘2, 31; the enzyme is maximally activated by hIg2+ [4] and strongly inhibited by Ca2* [4, 51. In conjunction with our studies on the role of the metal ion we investigated the binding of lanthanide ions, which are chemically similar to the alkaline earths [6] and because of their unique spectral properties have been suggested to be convenient probes of electrostatic binding sites in proteins [i, S]. Metal ions (>Ig=+, i\In=+ Zn”*, and Caz+) which are active in this system quench the intrinsic protei; fluorescence, with characteristic saturation behavior, whereas an ion which has no effect on the enzyme activity, SF, does not affect the protein Suorescence, indicating that quenching is specifically associated with metal ion binding [3]. Of the lanthanides tested using this technique, Kd3+, Sm3+, and Gd3+ strongly quenched the fluorescence, but the effects were time-dependent, diflicult to reproduce, and even at relatively high concentrations (approximately 1 mM) of lanthanide ion did not exhibit saturation. Under the conditions of the titration, pyrophosphatase was irreversibly inactivated by 5 m&Z Kd3+; we attribute the quenching of fluorescence observed with Xdsf: Sm3f and Gd3f to denaturation of the protein. This contributionis paper number 4838 of the AgriculturalELxperiment Station, Purdue UniversityCopyright @ 1972 by American Elsevier Publishing Company, Inc.
St3
JaMEs
w. SPEROW
AND LARRY G. BUTLER
Saturation of the quenching was observed with Eu3+. Moreover, the effects were time-independent and reproducible, and the enzyme remained active for weeks at 3” in the presence of saturating concentrations of EuJ+. The enzyme bmds Euj+ so strongly that KD could not be determined by conventional graphical procedures which assume the total &and concentration is always in excess over the protein concentration. As estimated from the concentration of free Eu J+ at the midpoint of the titration plot (Fig. lA), the apparent KD is 0.23 &I_ The corresponding KD’S for h9n2+, 2X+, &lg=*, and Ca+ were 2, 11, 16 and SO0 PM, respectively 131. Note that the concentration of fofal rather than free Eu3+ is plotted on the ab-
Figure 1. Titration of yest p>;rophosphatase with I&‘+. Crystallineenzyme, preparedaccordingto Ridlingtonetal. 191,was dialyzed against lo-’ M EDTA, then against 0.01 M Tris (Ci), pH 7-4, wbicb bad been specially extrscted [3] to remove auy contaminating metal ions_ Experimental wnditions were 0.24 M Tris (Cl), pH 7.4, and 6.0 X lo+ M enzyme in a volume of 1.0 ml, equilibrated to 30”. Fluorescence measure-
described[3]; wavelengthsfor excitationand emission were283 nm and 340 nm, respectively. Successive 5 111additions of 0.02 mM EuCL u-ere
ments were made as pevionsly
made until no further ffuorescencequenching was observed. The solid lime in A was drawn to fit the data, and the dashed line is a theoretical curve for two independentbinding sites with & = 0.23 &L The same data is plottedin A and EL
SHORT
COMMIJNICATION
89
100
75
s L
50
2 2s
0
scissa in Fig. IA; estimation of Ko from these data. assumes an equivalent change in fluorescence for each Eu3f bound. The tight Eu3+ binding facilitated determination of the binding stoichiometry. Figure 1B indicates the presence of approximately 2.5 Euj* binding sites per mole of enzyme. This correlates reasonably well with the dimeric nature of the enzyme [9] which contains 3 sites per mole for l\Ig’li and Xgz+ [lo] and for CaPPi 131. As can be seen from the fluorescence titration curves in Fig. l-4 the plot is steeper than the theoretical curve for independent binding sites; sin&~ but less pronounced results are observed for >Igz+ binding 131. The value for R, [II] for Eu3f binding calcuIated from these data is approsimately 10; the corresponding vaIue for Xg’f binding [3] is approximately 30. Values of R, less than Sl indicate positive cooperativity between the sites Ill]. These results imply that Eu3+and >Igz+ bind in a like manner. The degree of fluorescence quenching at saturation by Errs+, approximateIy 4070, is considerabIy greater than the 6% observed with ;\lgnt, &I&+, Zns+ and Caz+ 131. The additional quenching observed with Eu3+
90
JAMES W_ 3PEZOW
AND LARRY
G. BUTLER
may be due to energy transfer from the protein to Eu3+ [S]; however, the fluorescenceintensity of Eu3f was not enhanced. This strong quenching as -fveUas the tight binding observed iGrthis system suggests that lanthanide ions may indeedprcvide a sensitivetool for studying metal ion interactions with enzymes. Attempts to replace31gz+with Eu3* in the enzymatic reaction were complicated by the formation of insolublecomplexesof Eu3+ and PPi. The rate of enzymatic hydrolysis of PPi in the presenceof Eu3+ was Iess than 10% of the enzymatic rate observed with Jig” under otherwisesimilar conditions, and was completely accounted for by a nonenzymatic reaction catalyzed by Eu 3f.We have observedwith several other lanthauidesa nonenzymatic-catalysis of PPi hydrolysis at a similar rate, which is not accelerated by addition of pyrophosphatase-Bamann and Trapmann have shown that hmthauidehydroxide gels form under physiological conditions and cause hydrolysis of phosphate esters and pyrophosphates [12]. Although Eu3+ apparently binds to the enzyme in a manner similar to XIg=+, the most effective activating ion [4], it does not appear to be functional in the enzymatic reaction. In terms of the roles previously postulated for Mg* in this reaction [I, 21, either binding of free Eu~* does not activate the enzyme as ilIg*+ does, or the structure of the Eu3+-PPi complexes are not similar enough to the structure of the big’+-PPi complexesto interact with the activated enzyme. The simihuities in binding of free Eu3+ and 3fgz+ to the enzyme, and the crucial nature of the metal in the formation of an active substrate complex [5], suggest that the latter factor may be most important_ The authors ac?zm.c.?edge support by NIH Training Grant No. GM 1195 from National Institute of General Medical Sciences_ L.G.B. is recipient of a Research Career Development Award (GM 46404) f~cnn the U_ S_ Public Health Serufce.
RETEXENCES 1. 0. A. Moe and L. G. Butler, J- BioL Chem. (1972), inpress. 2. T. _A_Rapoport, W. E. Hohne, J. G. Reich, P. Heitmann and S. M. Rapoport, Eur. J. Bikhem.
26,9Z37 (1972).
3. J. W. Ridiington and L. G. Butler, J. BioZ. Chem. (1972), in press. 4- M. Ku&z, J. Gen. Physiol. 35,423 (x952)_ 5. 0. ..k Moe and L. G. Butler, J. BtiZ. Chem. (1972). in press. 6%D- G- Karracl;er, J. Chem. Educ., 45,424 (1970)_ 7. E. R. Birnbaum, J. E- Gomez and D_ W_ Darnal& J. Amer. Chem. Sot, 92,5287 (19iO).
SHORT
COMMUNICATION
91
8. C. K Luk, Biochemistry, IO,2838 (197i). 9. J- W. Ridlington, Y. Yang and L. G. Butler, (unpublshed). 10. L. G. Butler, in The Enzymes, (P_ D. Bayer, Ed.), Academic, New York (1971), 3rd Ed-, Vol. 4, p_ 529. 11. D. E. &x&land, Jr., in Current Topics in CeUu?.ut Regulation, (33. L. Horecker and E. R. §titman, Eids.),Academic, New York (1969), Vol. 1, p- 1. 12. E. Bamann and H. Trapmann, Admn. EnzymoZ. 21, 169 (1959).
CHEMISTRY,
SHORT Yeast Inorganic JAMES W. SPEROW
Z&87-91
(19i2)
87
COMMUNICATION
Py-rophosphatase V.
Binding
of EUJ+
and LARR.Y G. BUTLER
Department of Biochemistry,
Purdue Unicerszly,
KEY
West Lafayefte, Indiana $“907
WORDS
Yeast Pyrophosphatase Europium Binding The interaction of yeast inorganic pyrophosphatase (EC 3.6 1.1) with metal ions has been shown both kinetically [I, 21 and statically [‘2, 31; the enzyme is maximally activated by hIg2+ [4] and strongly inhibited by Ca2* [4, 51. In conjunction with our studies on the role of the metal ion we investigated the binding of lanthanide ions, which are chemically similar to the alkaline earths [6] and because of their unique spectral properties have been suggested to be convenient probes of electrostatic binding sites in proteins [i, S]. Metal ions (>Ig=+, i\In=+ Zn”*, and Caz+) which are active in this system quench the intrinsic protei; fluorescence, with characteristic saturation behavior, whereas an ion which has no effect on the enzyme activity, SF, does not affect the protein Suorescence, indicating that quenching is specifically associated with metal ion binding [3]. Of the lanthanides tested using this technique, Kd3+, Sm3+, and Gd3+ strongly quenched the fluorescence, but the effects were time-dependent, diflicult to reproduce, and even at relatively high concentrations (approximately 1 mM) of lanthanide ion did not exhibit saturation. Under the conditions of the titration, pyrophosphatase was irreversibly inactivated by 5 m&Z Kd3+; we attribute the quenching of fluorescence observed with Xdsf: Sm3f and Gd3f to denaturation of the protein. This contributionis paper number 4838 of the AgriculturalELxperiment Station, Purdue UniversityCopyright @ 1972 by American Elsevier Publishing Company, Inc.
St3
JaMEs
w. SPEROW
AND LARRY G. BUTLER
Saturation of the quenching was observed with Eu3+. Moreover, the effects were time-independent and reproducible, and the enzyme remained active for weeks at 3” in the presence of saturating concentrations of EuJ+. The enzyme bmds Euj+ so strongly that KD could not be determined by conventional graphical procedures which assume the total &and concentration is always in excess over the protein concentration. As estimated from the concentration of free Eu J+ at the midpoint of the titration plot (Fig. lA), the apparent KD is 0.23 &I_ The corresponding KD’S for h9n2+, 2X+, &lg=*, and Ca+ were 2, 11, 16 and SO0 PM, respectively 131. Note that the concentration of fofal rather than free Eu3+ is plotted on the ab-
Figure 1. Titration of yest p>;rophosphatase with I&‘+. Crystallineenzyme, preparedaccordingto Ridlingtonetal. 191,was dialyzed against lo-’ M EDTA, then against 0.01 M Tris (Ci), pH 7-4, wbicb bad been specially extrscted [3] to remove auy contaminating metal ions_ Experimental wnditions were 0.24 M Tris (Cl), pH 7.4, and 6.0 X lo+ M enzyme in a volume of 1.0 ml, equilibrated to 30”. Fluorescence measure-
described[3]; wavelengthsfor excitationand emission were283 nm and 340 nm, respectively. Successive 5 111additions of 0.02 mM EuCL u-ere
ments were made as pevionsly
made until no further ffuorescencequenching was observed. The solid lime in A was drawn to fit the data, and the dashed line is a theoretical curve for two independentbinding sites with & = 0.23 &L The same data is plottedin A and EL
SHORT
COMMIJNICATION
89
100
75
s L
50
2 2s
0
scissa in Fig. IA; estimation of Ko from these data. assumes an equivalent change in fluorescence for each Eu3f bound. The tight Eu3+ binding facilitated determination of the binding stoichiometry. Figure 1B indicates the presence of approximately 2.5 Euj* binding sites per mole of enzyme. This correlates reasonably well with the dimeric nature of the enzyme [9] which contains 3 sites per mole for l\Ig’li and Xgz+ [lo] and for CaPPi 131. As can be seen from the fluorescence titration curves in Fig. l-4 the plot is steeper than the theoretical curve for independent binding sites; sin&~ but less pronounced results are observed for >Igz+ binding 131. The value for R, [II] for Eu3f binding calcuIated from these data is approsimately 10; the corresponding vaIue for Xg’f binding [3] is approximately 30. Values of R, less than Sl indicate positive cooperativity between the sites Ill]. These results imply that Eu3+and >Igz+ bind in a like manner. The degree of fluorescence quenching at saturation by Errs+, approximateIy 4070, is considerabIy greater than the 6% observed with ;\lgnt, &I&+, Zns+ and Caz+ 131. The additional quenching observed with Eu3+
90
JAMES W_ 3PEZOW
AND LARRY
G. BUTLER
may be due to energy transfer from the protein to Eu3+ [S]; however, the fluorescenceintensity of Eu3f was not enhanced. This strong quenching as -fveUas the tight binding observed iGrthis system suggests that lanthanide ions may indeedprcvide a sensitivetool for studying metal ion interactions with enzymes. Attempts to replace31gz+with Eu3* in the enzymatic reaction were complicated by the formation of insolublecomplexesof Eu3+ and PPi. The rate of enzymatic hydrolysis of PPi in the presenceof Eu3+ was Iess than 10% of the enzymatic rate observed with Jig” under otherwisesimilar conditions, and was completely accounted for by a nonenzymatic reaction catalyzed by Eu 3f.We have observedwith several other lanthauidesa nonenzymatic-catalysis of PPi hydrolysis at a similar rate, which is not accelerated by addition of pyrophosphatase-Bamann and Trapmann have shown that hmthauidehydroxide gels form under physiological conditions and cause hydrolysis of phosphate esters and pyrophosphates [12]. Although Eu3+ apparently binds to the enzyme in a manner similar to XIg=+, the most effective activating ion [4], it does not appear to be functional in the enzymatic reaction. In terms of the roles previously postulated for Mg* in this reaction [I, 21, either binding of free Eu~* does not activate the enzyme as ilIg*+ does, or the structure of the Eu3+-PPi complexes are not similar enough to the structure of the big’+-PPi complexesto interact with the activated enzyme. The simihuities in binding of free Eu3+ and 3fgz+ to the enzyme, and the crucial nature of the metal in the formation of an active substrate complex [5], suggest that the latter factor may be most important_ The authors ac?zm.c.?edge support by NIH Training Grant No. GM 1195 from National Institute of General Medical Sciences_ L.G.B. is recipient of a Research Career Development Award (GM 46404) f~cnn the U_ S_ Public Health Serufce.
RETEXENCES 1. 0. A. Moe and L. G. Butler, J- BioL Chem. (1972), inpress. 2. T. _A_Rapoport, W. E. Hohne, J. G. Reich, P. Heitmann and S. M. Rapoport, Eur. J. Bikhem.
26,9Z37 (1972).
3. J. W. Ridiington and L. G. Butler, J. BioZ. Chem. (1972), in press. 4- M. Ku&z, J. Gen. Physiol. 35,423 (x952)_ 5. 0. ..k Moe and L. G. Butler, J. BtiZ. Chem. (1972). in press. 6%D- G- Karracl;er, J. Chem. Educ., 45,424 (1970)_ 7. E. R. Birnbaum, J. E- Gomez and D_ W_ Darnal& J. Amer. Chem. Sot, 92,5287 (19iO).
SHORT
COMMUNICATION
91
8. C. K Luk, Biochemistry, IO,2838 (197i). 9. J- W. Ridlington, Y. Yang and L. G. Butler, (unpublshed). 10. L. G. Butler, in The Enzymes, (P_ D. Bayer, Ed.), Academic, New York (1971), 3rd Ed-, Vol. 4, p_ 529. 11. D. E. &x&land, Jr., in Current Topics in CeUu?.ut Regulation, (33. L. Horecker and E. R. §titman, Eids.),Academic, New York (1969), Vol. 1, p- 1. 12. E. Bamann and H. Trapmann, Admn. EnzymoZ. 21, 169 (1959).