ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 196, No. 2, September, pp. 552-556, 1979
The Relation between Activity and Zinc and Chloride of Escherichia co/i Alkaline Phosphatase
Binding
JAN-ERIK NORNE, HELENA SZAJN,” HEDVIG CSOPAK,” PfiTUR REIMARSSON, AND BJORN LINDMAN Physical
Chemistry&
Chemical Center, P.O.B. ‘740, S-220 07Lund 7, Sweden, and *Institution ofBiochemistry, University of Giiteborg, Fack, S-.402 20 GBteborg, Sweden
Received August 3, 1978; revised December 21, 19’78 The relation between Zn*+ binding of E. coli alkaline phosphatase and enzymatic activity and anion binding (using Wl NMR) has been investigated. The results suggest the existence of two forms of the enzyme with different zinc binding properties. The anion binding associated with the enzyme’s function appears to be an amino acid residue and not the Zn2+ ions; furthermore, there is a rapid internal motion at the anion binding site. 35C1relaxation studies in the presence of Mg*+ ions point to a marked interdependence of Mg2+ and ZnZf binding.
The Eschwichia coli alkaline phosphatase (orthophosphoricmonoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is a dimeric metalloenzyme with a molecular weight of about 80,000. The requirement of zinc for both catalysis and induction of appropriate conformational changes has been established (l-5). However, as regards metal stoichiometry there has been a long dispute, the zinc content of purified phosphatase as reported by different authors varying between two and six per enzyme molecule (6-10). The data giving the number of zinc ions essential for activity show a corresponding wide variation. Among recent reports discussing the metal content of alkaline phosphatase may be mentioned those of Vallee and co-workers (11, 12) giving 4 Zn per enzyme molecule and that of Coleman and co-workers (13) giving 2 Zn necessary for full activity. Other recent contributions to the problem of metal ion binding to alkaline phosphatase may be found in Refs. (14-18). In our recent measurements to be reported here we have found that titration of the apoenzyme with Zn2+ leads to a linear increase in activity with the maximal activity being obtained at 4 Zr?+/dimer. This is also reflected in the 35C1 NMR relaxation where the first four zinc ions 0003~9861/79/100552-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
were found not to influence the chloride relaxation. These results are at variance with our previous findings where the stoichiometry of the zinc was found to be two per enzyme molecule (19-22). The most plausible explanation of these findings seems to be the existence of two forms of the enzyme interacting differently with zinc but displaying no pronouncedly different properties in other respects. Alkaline phosphatase was isolated and purified from E. coli bacteria strain CW 3747 as described previously and the conditions of bacteria growth and harvest were carefully controlled (8, 19). Enzyme activity assays and protein concentration measurements were performed as reported previously (8). The apoenzyme was prepared by treating the metalloprotein with Chelex 100 (23). The recent preparation was analyzed for Mg2+ by atomic absorption spectroscopy. Metal solutions were prepared from spectroscopically pure metal chlorides and all the other chemicals were of analytical grade. Metal-free solutions were made by extraction with dithizone (23) and glassware was treated according to Thiers (24). 35C1 NMR linewidth studies were performed at 9.80 MHz using a modified Varian XL-loo-15 spectrometer operating in the Fourier transform mode. The number
552
Zn AND Cl BINDING
TOE.
coli ALKALINE
of transients was 1000 and the acquisition time 0.2 s. External lock was employed. The temperature was 28 & 1°C. The relaxation times were measured at 8.82 MHz on a Bruker BKr 322s spectrometer with homemade probes. T, was measured using a 180-T-90 pulse sequence and T, by means of the Meiboom-Gill-Carr-Purcell sequences. The signals were time averaged with a Varian V 71 computer using a homemade interface. The errors are in all cases less than 10%. Details of solution compositions, etc. are given in the figure legends. Figures 1 and 2 show previous and recent results of both enzyme activity and 35C1magnetic relaxation measurements as a function of zinc concentration. (It was established that the relaxation rate increases linearly with the enzyme concentration and that the relaxation rate is effectively independent of pH at low zinc contents.) The activity can be seen to be influenced by the first zinc ions added while beyond a certain amount of zinc no further effect is observed. The halide quadrupole relaxation method, which has become an established technique for monitoring protein-anion interactions (25), gives results which indicate that the first zinc ions added have no effect on chloride binding to the enzyme (very long-lived interactions, which are improbable, may escape detection) while further zinc ion additions give very large relaxation rate enhancements. (The effect of free Znz+ ions on 35C1- relaxation is much smaller than the relaxation enhancements encountered here (25); for the maximal Zn2+ concentrations used it would amount to less than 2 Hz.) A correlation of the two types of measurements shows that zinc ions are bound in addition to those influencing the enzymatic activity and that these additional zinc ions coordinate chloride ions. From Fig. 1, which shows our previous results, it is inferred that two zinc ions per enzyme molecule are required for full activity and this stoichiometry is also reflected in the chloride relaxation data (22). Electron paramagnetic resonance (EPR) studies of the binding of two CL?+ to alkaline phosphatase revealed that the two copper ions appear to occupy only one type of site. When more than two equivalents
PHOSPHATASE
553
of Cuz+ were added, the EPR spectrum showed at least two types of Cu2+ binding sites (20, 26). 31P NMR relaxation studies carried out with Zn2+-, Co2+-, and Mn2+alkaline phosphatases were consistent with the existence of two spectroscopically identical metal binding sites (21). In addition, the spectral titrations of the apoenzyme with Zn2+ Co2+ Mn2+, and Cd2+ indicated that the binding of two metal ions produced similar spectral changes and that the nature of the binding of the third and the fourth Zn2+ or Co2+ ions was different from that observed for the two first metal ions and different from each other (5). From Fig. 2, which shows our recent results, it is inferred that the stoichiometry of the zinc ion is four per enzyme molecule as reflected both in the activity and chloride relaxation measurements. The results are in other ways similar to those obtained before (27). In summary, we have by the two methods used obtained evidence for a variability of zinc binding to E. coli alkaline phosphatase. Careful examination of the isolation techniques, the apoenzyme preparation procedure, the solution preparation, etc. has not indicated any difference between the cases giving different zinc binding stoichiometries. In view of these findings it seems reasonable to believe that E. coli alkaline phosphatase may appear in two different forms having different zinc binding properties. The conversion from one form to the other may occur in some part of the preparation and be induced by small changes in preparation conditions. The occurrence of two different enzyme forms would explain why very careful investigations of different laboratories have given conflicting results as regards the number of zinc ions needed for full activity. It can be seen from these results that there is no contribution to the 35C1-relaxation from the minimum amount of the Zn2+ ions needed for full activity. This applies to both forms of the enzyme. There seemsthus to be no chloride or phosphate (cf. Ref. (22)) binding to the Zn2+ ions associated with the enzymatic activity. In order to further characterize the anion binding at the functional site, some 35C1relaxation time
NORNE ET AL.
554
(T, and T2) measurements were performed. For a 0.263 IIIM enzyme solution (with 4 ZrP per protein dimer) containing 0.5 M KC1 the reduction in the relaxation rates on addition of a stoichiometric amount of orthophosphate was
and
0 = 28 s-l
(at 28°C).
These can be analyzed to give an apparent correlation time of 8 ns and an apparent quadrupole coupling constant of 1.4 MHz (cf. Refs. (28) and (29)). Comparison ofthese values with the overall reorientation correlation time of the enzyme and known values of the quadrupole coupling constant for Cl- binding to positively charged amino acid side chains and for Cl- coordination to metal ions (25, 28, 30) gives the following important conclusions about the functional anion binding site in alkaline phosphatase. (1) The quadrupole coupling constant is much smaller than that characteristic of metal-coordinative binding but corresponds
Ratio CZd+l /[Enzyme1 FIG. 1. Titration of apoalkalme phosphatase with Zn*+, as obtained by previous enzyme preparation. (0) Percentage enzyme activity as a function of added Zn*+. The solution contained 15 pM enzyme in 0.01 M Tris/Cl- buffer at pH 8.0; 100% enzyme activity corresponds to a specific activity of 2,200 U/mg protein. (V) Excess 35C1linewidth as a function of added ZnZ+. The solution contained 0.22 mM enzyme in 0.5 M KCl, 0.05 M Tris/Cl- at pH 7.7. The right-hand scale pertains to enzyme activity and the left-hand scale to the excess linewidth. The lines are drawn for visual aid.
’
6 a Ratio CZn “I / [Enzyme1 2
4
on
FIG. 2. Titration of apoalkaline phosphatase with Znzf, as obtained by recent enzyme preparation. (0) Percentage enzyme activity as a function of added Zn2+. The solution contained 15 pM enzyme in 0.01 M Tris/Cl- buffer at pH 8.0; 100% enzyme activity corresponds to a specific activity of 1950 U/mg protein. (V) Excess 35C1linewidth as a function of added Zn2+. The solution contained 0.11 mM enzyme in 0.5 M KCl, 0.05 M Tris/Cl- at pH 8.3. The right-hand scale pertains to enzyme activity and the left-hand scale to the excess linewidth. The lines are drawn for visual aid.
closely to that for interaction with a positive amino acid residue. (A second sphere enzyme Zn(H,O)Cl- complex may not be excluded on the basis of these data but is not consistent with the Zn titrations.) (2) There is a very rapid (characteristic time < 10Pgs) internal motion of the anion binding group (28). In order to shed further light on the anion binding characteristics of alkaline phosphatase, variable temperature (in the range 4-30°C) 35C1linewidth studies were performed at different zinc contents. Below 4 Zn per enzyme molecule the relaxation rate decreases (Arrhenius’ activation energy 23 kJ/mol) with increasing temperature at all Zn contents pointing to rapid exchange conditions for Cl- (Cf. Ref. (25)). On the other hand, the Zn-induced relaxation enhancement obtained at higher Zn contents corresponds to slow-exchange conditions. Thus this relaxation contribution increases strongly with increasing temperature (activation energy about 39 kJ/mol). One would expect that the lifetime of a bound Cl- ion is much longer for metal-coordinative binding than for binding to an amino acid residue and indeed most proteins with
Zn AND Cl BINDING TOE. coli ALKALINE
32 -
22.
dtio
C3n2+l&nzy4ne] . (dwithout
5 i!n*+
(42.0$$g&m (43.6
--^-
PHOSPHATASE
555
Zn contents. From Fig. 3 it can be inferred that the presence of Mg2+ does not alter the main features of the titration curves, i.e., Zn stoichiometry and the magnitude of the relaxation rate. However, it can be seen that addition of Mgz+ to the metal-free enzyme causes a small increase (ea. 40%) in the 35C1 excess relaxation. Furthermore, in the presence of Mg2+ (1.5 per enzyme molecule) the increase in relaxation rate in the range of O-2 Zn*+ is suppressed and the relaxation rate is nearly constant in the whole range O-4 Zn*+. Vallee and co-workers (12) found a significant coupling between Zn*+ and Mg*+ binding and the present data support this view. Particularly significant is the decrease in relaxation rate on addition of Mg*+ to the 2 Zn-enzyme (Fig. 3). REFERENCES 1. PLOCKE, D. J., LEVINTHAL, C., AND VALLEE, B. L. (1962) Biochemistry 1, 373-378.
FIG. 3. Titration of apoalkaline phosphatase with Zn*+ and Mg2+ followed by the excess 35C1linewidth. The solutions contained 0.2 M KC1 and 0.05 M Tris/Clat pH 8.1. The enzyme concentration was always within 20% of 0.31 mM and has been normalized to this value. Upper: The W- linewidth as a function of added Zn2+ without Mg2+ (0) and with 1.5 mol M$+/mol enzyme (W). Lower: The 35C1-linewidth as a function of added Mgl+ without Zn2+ (V), and in a solution containing 2.0 mol Zn2+/mol enzyme (0) and 3.6 mol Zn2+/mol enzyme (W). The curves are drawn as a visual aid.
metal-coordinative Cl- binding seem to be characterized by slow Cl- exchange while in general rapid exchange seems to apply in the other case (25, 31, 32). Thus the variable temperature studies indicate that Cl- coordinates to Zn only above the minimum amount of Zn needed for full activity. In view of the interesting findings (11, 12) recently that there is a considerable intrinsic content of magnesium in alkaline phosphatase and that magnesium affects the binding of other metal ions as well as enzyme activity it was important to investigate in detail the effect of Mg2+ concentration on the 35C1relaxation enhancement due to alkaline phosphatase at different
2. DAEMEN, F. J. M., AND RIORDAN, J. M. (1974) Biochemistry 13, 2865-2871. 3. LAZDUNSKI, C., PETITCLERC, C., AND LAZDUNSKI, M. (1969) Eur. J. Biochem. 8, 510-517. 4. APPLEBURY, M. L., JOHNSON, B. P., AND COLEMAN, J. E. (197O)J.Biol. Ch.em. 245,4968-4976. 5. SZAJN, H., AND CSOPAK, H. (1977) Biochem. Biophys. Acta 480, 143-153. 6. HARRIS, M. J., AND COLEMAN, J. E. (1968) J. Biol. Chem. 243, 5063-5073. 7. SIMPSON, R. T., VALLEE, B. L., AND TAIT, G. H. (1968) Biochemistry ‘7, 4336-4342. 8. CSOPAK, H., GARELLICK, G., AND HALLBERG, B. (1972) Acta Chem. Stand. 26, 2401-2411. 9. REID, T. W., AND WILSON, J. B. (1971) in The Enzymes (Boyer, P., ed.), Vol. 4, pp. 373-415,
Academic Press, New York. 10. REYNOLDS, J. A., AND SCHLESINGER, M. J. (1968) Biochemistry
11. ANDERSON,
12.
13.
14.
15.
R. B.
‘7, 2080-2085. A., KENNEDY, F. S., L. (1976) Biochemistry
AND VALLEE, 15, 3710-3716. BOSRON, W. F., ANDERSON, R. A., FALK, M. C., KENNEDY, F. S., AND VALLEE, B. L. (1977) Biochemistry 16, 610-614. CHLEBOWSKI, J. F., ARMITAGE, I. M., AND COLEMAN, J. E. (1977) J. Biol. Chem. 252, 7053-7061. ARMITAGE, I. M., UITERKAMP, A. J. M. S., CHLEBOWSKI, J. F., AND COLEMAN, J. E. (1978) J. Map. Reson. 29, 375-392. CHLEBOWSKI,J. F., AND COLEMAN,J. E.(1976) Metal Ions Biol. Syst. 6, l-140.
556
NORNE
16. LEVINE, H., TSONG, T. Y., AND HOLLIS, D. P. (1976) I$e Sci. 19, 859-865. 17. HULL, W. E., AND SYKES, B. D. (1976) Biochemistry 15, 1535- 1546. 18. HULL, W. E., HALFORD, S. E., GUTFREUND, H., AND SYKES, B. D. (1976) Biochemistry 15, 1547-1561. 19. CSOPAK, H., AND SZAJN, H. (1973) Arch. Biochem. Biophys. 157, 374-379. 20. CSOPAK, H., AND FALK, K. E. (1974) Biochim. Biophys. Acta 359,22-32. 21. CSOPAK, H., AND DRAKENBERG, T. (1973) FEBS Lett. 30, 296-300. 22. NORNE, J.-E., CSOPAK, H., AND LINDMAN, B. (1974) Arch. Biochem. Biophys. 162, 552-559. 23. CSOPAK, H. (1969) Eur. J. Biochem. 7, 186-192. 24. THIERS, R. E. (1957) Methods Biochxm. Anal. 5,273-335. 25. LINDMAN, B., AND FORSI?N, S. (1976) Chlorine,
ET AL.
26. 27. 28.
29. 30. 31.
32.
Bromine and Iodine NMR, Physico-Chemical and Biological Applications, Chap. 8, SpringerVerlag, Heidelberg. TAYLOR, J. S., AND COLEMAN, J. E. (1972) Proc. Nat. Acad. Sci. USA 69, 850-862. CSOPAK, H., FALK, K. E., AND SZAJN, H. (1972) Biochim. Biophys. Acta 258, 466-472. BULL, T., NORNE, J.-E., REIMARSSON, P., AND LINDMAN, B. (1978) J. Amer. Chem. Sot. 100, 4643-4647. BULL, T., LINDMAN, B., AND REIMARSSON, P. (1976) Arch. Biochem. Biophys. 176, 389-391. LINDMAN, B. (1978) J. Magn. Reson. 32, 39-47. NORNE, J.-E., BULL, T. E., EINARSSON, R., LINDMAN, B., AND ZEPPEZAUER, M. (1973) Chem. Ser. 3, 142-144. NORNE, J.-E., LILJA, H., LINDMAN, B., EINARSSON, R., AND ZEPPEZAUER, M. (1975) Eur. J. Biochem. 59, 463-473.
Vol. 196, No. 2, September, pp. 552-556, 1979
The Relation between Activity and Zinc and Chloride of Escherichia co/i Alkaline Phosphatase
Binding
JAN-ERIK NORNE, HELENA SZAJN,” HEDVIG CSOPAK,” PfiTUR REIMARSSON, AND BJORN LINDMAN Physical
Chemistry&
Chemical Center, P.O.B. ‘740, S-220 07Lund 7, Sweden, and *Institution ofBiochemistry, University of Giiteborg, Fack, S-.402 20 GBteborg, Sweden
Received August 3, 1978; revised December 21, 19’78 The relation between Zn*+ binding of E. coli alkaline phosphatase and enzymatic activity and anion binding (using Wl NMR) has been investigated. The results suggest the existence of two forms of the enzyme with different zinc binding properties. The anion binding associated with the enzyme’s function appears to be an amino acid residue and not the Zn2+ ions; furthermore, there is a rapid internal motion at the anion binding site. 35C1relaxation studies in the presence of Mg*+ ions point to a marked interdependence of Mg2+ and ZnZf binding.
The Eschwichia coli alkaline phosphatase (orthophosphoricmonoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is a dimeric metalloenzyme with a molecular weight of about 80,000. The requirement of zinc for both catalysis and induction of appropriate conformational changes has been established (l-5). However, as regards metal stoichiometry there has been a long dispute, the zinc content of purified phosphatase as reported by different authors varying between two and six per enzyme molecule (6-10). The data giving the number of zinc ions essential for activity show a corresponding wide variation. Among recent reports discussing the metal content of alkaline phosphatase may be mentioned those of Vallee and co-workers (11, 12) giving 4 Zn per enzyme molecule and that of Coleman and co-workers (13) giving 2 Zn necessary for full activity. Other recent contributions to the problem of metal ion binding to alkaline phosphatase may be found in Refs. (14-18). In our recent measurements to be reported here we have found that titration of the apoenzyme with Zn2+ leads to a linear increase in activity with the maximal activity being obtained at 4 Zr?+/dimer. This is also reflected in the 35C1 NMR relaxation where the first four zinc ions 0003~9861/79/100552-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
were found not to influence the chloride relaxation. These results are at variance with our previous findings where the stoichiometry of the zinc was found to be two per enzyme molecule (19-22). The most plausible explanation of these findings seems to be the existence of two forms of the enzyme interacting differently with zinc but displaying no pronouncedly different properties in other respects. Alkaline phosphatase was isolated and purified from E. coli bacteria strain CW 3747 as described previously and the conditions of bacteria growth and harvest were carefully controlled (8, 19). Enzyme activity assays and protein concentration measurements were performed as reported previously (8). The apoenzyme was prepared by treating the metalloprotein with Chelex 100 (23). The recent preparation was analyzed for Mg2+ by atomic absorption spectroscopy. Metal solutions were prepared from spectroscopically pure metal chlorides and all the other chemicals were of analytical grade. Metal-free solutions were made by extraction with dithizone (23) and glassware was treated according to Thiers (24). 35C1 NMR linewidth studies were performed at 9.80 MHz using a modified Varian XL-loo-15 spectrometer operating in the Fourier transform mode. The number
552
Zn AND Cl BINDING
TOE.
coli ALKALINE
of transients was 1000 and the acquisition time 0.2 s. External lock was employed. The temperature was 28 & 1°C. The relaxation times were measured at 8.82 MHz on a Bruker BKr 322s spectrometer with homemade probes. T, was measured using a 180-T-90 pulse sequence and T, by means of the Meiboom-Gill-Carr-Purcell sequences. The signals were time averaged with a Varian V 71 computer using a homemade interface. The errors are in all cases less than 10%. Details of solution compositions, etc. are given in the figure legends. Figures 1 and 2 show previous and recent results of both enzyme activity and 35C1magnetic relaxation measurements as a function of zinc concentration. (It was established that the relaxation rate increases linearly with the enzyme concentration and that the relaxation rate is effectively independent of pH at low zinc contents.) The activity can be seen to be influenced by the first zinc ions added while beyond a certain amount of zinc no further effect is observed. The halide quadrupole relaxation method, which has become an established technique for monitoring protein-anion interactions (25), gives results which indicate that the first zinc ions added have no effect on chloride binding to the enzyme (very long-lived interactions, which are improbable, may escape detection) while further zinc ion additions give very large relaxation rate enhancements. (The effect of free Znz+ ions on 35C1- relaxation is much smaller than the relaxation enhancements encountered here (25); for the maximal Zn2+ concentrations used it would amount to less than 2 Hz.) A correlation of the two types of measurements shows that zinc ions are bound in addition to those influencing the enzymatic activity and that these additional zinc ions coordinate chloride ions. From Fig. 1, which shows our previous results, it is inferred that two zinc ions per enzyme molecule are required for full activity and this stoichiometry is also reflected in the chloride relaxation data (22). Electron paramagnetic resonance (EPR) studies of the binding of two CL?+ to alkaline phosphatase revealed that the two copper ions appear to occupy only one type of site. When more than two equivalents
PHOSPHATASE
553
of Cuz+ were added, the EPR spectrum showed at least two types of Cu2+ binding sites (20, 26). 31P NMR relaxation studies carried out with Zn2+-, Co2+-, and Mn2+alkaline phosphatases were consistent with the existence of two spectroscopically identical metal binding sites (21). In addition, the spectral titrations of the apoenzyme with Zn2+ Co2+ Mn2+, and Cd2+ indicated that the binding of two metal ions produced similar spectral changes and that the nature of the binding of the third and the fourth Zn2+ or Co2+ ions was different from that observed for the two first metal ions and different from each other (5). From Fig. 2, which shows our recent results, it is inferred that the stoichiometry of the zinc ion is four per enzyme molecule as reflected both in the activity and chloride relaxation measurements. The results are in other ways similar to those obtained before (27). In summary, we have by the two methods used obtained evidence for a variability of zinc binding to E. coli alkaline phosphatase. Careful examination of the isolation techniques, the apoenzyme preparation procedure, the solution preparation, etc. has not indicated any difference between the cases giving different zinc binding stoichiometries. In view of these findings it seems reasonable to believe that E. coli alkaline phosphatase may appear in two different forms having different zinc binding properties. The conversion from one form to the other may occur in some part of the preparation and be induced by small changes in preparation conditions. The occurrence of two different enzyme forms would explain why very careful investigations of different laboratories have given conflicting results as regards the number of zinc ions needed for full activity. It can be seen from these results that there is no contribution to the 35C1-relaxation from the minimum amount of the Zn2+ ions needed for full activity. This applies to both forms of the enzyme. There seemsthus to be no chloride or phosphate (cf. Ref. (22)) binding to the Zn2+ ions associated with the enzymatic activity. In order to further characterize the anion binding at the functional site, some 35C1relaxation time
NORNE ET AL.
554
(T, and T2) measurements were performed. For a 0.263 IIIM enzyme solution (with 4 ZrP per protein dimer) containing 0.5 M KC1 the reduction in the relaxation rates on addition of a stoichiometric amount of orthophosphate was
and
0 = 28 s-l
(at 28°C).
These can be analyzed to give an apparent correlation time of 8 ns and an apparent quadrupole coupling constant of 1.4 MHz (cf. Refs. (28) and (29)). Comparison ofthese values with the overall reorientation correlation time of the enzyme and known values of the quadrupole coupling constant for Cl- binding to positively charged amino acid side chains and for Cl- coordination to metal ions (25, 28, 30) gives the following important conclusions about the functional anion binding site in alkaline phosphatase. (1) The quadrupole coupling constant is much smaller than that characteristic of metal-coordinative binding but corresponds
Ratio CZd+l /[Enzyme1 FIG. 1. Titration of apoalkalme phosphatase with Zn*+, as obtained by previous enzyme preparation. (0) Percentage enzyme activity as a function of added Zn*+. The solution contained 15 pM enzyme in 0.01 M Tris/Cl- buffer at pH 8.0; 100% enzyme activity corresponds to a specific activity of 2,200 U/mg protein. (V) Excess 35C1linewidth as a function of added ZnZ+. The solution contained 0.22 mM enzyme in 0.5 M KCl, 0.05 M Tris/Cl- at pH 7.7. The right-hand scale pertains to enzyme activity and the left-hand scale to the excess linewidth. The lines are drawn for visual aid.
’
6 a Ratio CZn “I / [Enzyme1 2
4
on
FIG. 2. Titration of apoalkaline phosphatase with Znzf, as obtained by recent enzyme preparation. (0) Percentage enzyme activity as a function of added Zn2+. The solution contained 15 pM enzyme in 0.01 M Tris/Cl- buffer at pH 8.0; 100% enzyme activity corresponds to a specific activity of 1950 U/mg protein. (V) Excess 35C1linewidth as a function of added Zn2+. The solution contained 0.11 mM enzyme in 0.5 M KCl, 0.05 M Tris/Cl- at pH 8.3. The right-hand scale pertains to enzyme activity and the left-hand scale to the excess linewidth. The lines are drawn for visual aid.
closely to that for interaction with a positive amino acid residue. (A second sphere enzyme Zn(H,O)Cl- complex may not be excluded on the basis of these data but is not consistent with the Zn titrations.) (2) There is a very rapid (characteristic time < 10Pgs) internal motion of the anion binding group (28). In order to shed further light on the anion binding characteristics of alkaline phosphatase, variable temperature (in the range 4-30°C) 35C1linewidth studies were performed at different zinc contents. Below 4 Zn per enzyme molecule the relaxation rate decreases (Arrhenius’ activation energy 23 kJ/mol) with increasing temperature at all Zn contents pointing to rapid exchange conditions for Cl- (Cf. Ref. (25)). On the other hand, the Zn-induced relaxation enhancement obtained at higher Zn contents corresponds to slow-exchange conditions. Thus this relaxation contribution increases strongly with increasing temperature (activation energy about 39 kJ/mol). One would expect that the lifetime of a bound Cl- ion is much longer for metal-coordinative binding than for binding to an amino acid residue and indeed most proteins with
Zn AND Cl BINDING TOE. coli ALKALINE
32 -
22.
dtio
C3n2+l&nzy4ne] . (dwithout
5 i!n*+
(42.0$$g&m (43.6
--^-
PHOSPHATASE
555
Zn contents. From Fig. 3 it can be inferred that the presence of Mg2+ does not alter the main features of the titration curves, i.e., Zn stoichiometry and the magnitude of the relaxation rate. However, it can be seen that addition of Mgz+ to the metal-free enzyme causes a small increase (ea. 40%) in the 35C1 excess relaxation. Furthermore, in the presence of Mg2+ (1.5 per enzyme molecule) the increase in relaxation rate in the range of O-2 Zn*+ is suppressed and the relaxation rate is nearly constant in the whole range O-4 Zn*+. Vallee and co-workers (12) found a significant coupling between Zn*+ and Mg*+ binding and the present data support this view. Particularly significant is the decrease in relaxation rate on addition of Mg*+ to the 2 Zn-enzyme (Fig. 3). REFERENCES 1. PLOCKE, D. J., LEVINTHAL, C., AND VALLEE, B. L. (1962) Biochemistry 1, 373-378.
FIG. 3. Titration of apoalkaline phosphatase with Zn*+ and Mg2+ followed by the excess 35C1linewidth. The solutions contained 0.2 M KC1 and 0.05 M Tris/Clat pH 8.1. The enzyme concentration was always within 20% of 0.31 mM and has been normalized to this value. Upper: The W- linewidth as a function of added Zn2+ without Mg2+ (0) and with 1.5 mol M$+/mol enzyme (W). Lower: The 35C1-linewidth as a function of added Mgl+ without Zn2+ (V), and in a solution containing 2.0 mol Zn2+/mol enzyme (0) and 3.6 mol Zn2+/mol enzyme (W). The curves are drawn as a visual aid.
metal-coordinative Cl- binding seem to be characterized by slow Cl- exchange while in general rapid exchange seems to apply in the other case (25, 31, 32). Thus the variable temperature studies indicate that Cl- coordinates to Zn only above the minimum amount of Zn needed for full activity. In view of the interesting findings (11, 12) recently that there is a considerable intrinsic content of magnesium in alkaline phosphatase and that magnesium affects the binding of other metal ions as well as enzyme activity it was important to investigate in detail the effect of Mg2+ concentration on the 35C1relaxation enhancement due to alkaline phosphatase at different
2. DAEMEN, F. J. M., AND RIORDAN, J. M. (1974) Biochemistry 13, 2865-2871. 3. LAZDUNSKI, C., PETITCLERC, C., AND LAZDUNSKI, M. (1969) Eur. J. Biochem. 8, 510-517. 4. APPLEBURY, M. L., JOHNSON, B. P., AND COLEMAN, J. E. (197O)J.Biol. Ch.em. 245,4968-4976. 5. SZAJN, H., AND CSOPAK, H. (1977) Biochem. Biophys. Acta 480, 143-153. 6. HARRIS, M. J., AND COLEMAN, J. E. (1968) J. Biol. Chem. 243, 5063-5073. 7. SIMPSON, R. T., VALLEE, B. L., AND TAIT, G. H. (1968) Biochemistry ‘7, 4336-4342. 8. CSOPAK, H., GARELLICK, G., AND HALLBERG, B. (1972) Acta Chem. Stand. 26, 2401-2411. 9. REID, T. W., AND WILSON, J. B. (1971) in The Enzymes (Boyer, P., ed.), Vol. 4, pp. 373-415,
Academic Press, New York. 10. REYNOLDS, J. A., AND SCHLESINGER, M. J. (1968) Biochemistry
11. ANDERSON,
12.
13.
14.
15.
R. B.
‘7, 2080-2085. A., KENNEDY, F. S., L. (1976) Biochemistry
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