May 28, 1976 - ... Jersey 07110; and t Institute of Biological Chemistry, University of Genoa, Genoa, Italy ..... Colowick, S. P. & Womack, F. C. (1969) J. Biol.
Proc. Natl. Acad. Sci. USA
Vol. 73, No. 8, pp. 2692-2695, August 1976 Biochemistry
Dual role of Zn2+ as inhibitor and activator of fructose 1,6bisphosphatase of rat liver (histidine/divalent cations/cobalt/binding sites/gluconeogenesis)
GoPi A. TEJWANI* f, FABIO 0. PEDROSA* §, S. PONTREMOLIt, AND B. L. HORECKER* *
Roche Institute of Molecular Biology, Nutley, New Jersey 07110; and t Institute of Biological Chemistry, University of Genoa, Genoa, Italy
Contributed by B. L. Horecker, May 28, 1976
ABSTRACT At neutral pH, Zn2+ is a potent and specific inhibitor of rat liver fructose 1,6-bisphosphatase (EC 3.1.3.11; D-fructose-1,6-bisphosphate 1-phosphohydrolase). Inhibition by Zn2+ is uncompetitive with respect to the activating cations Mg2+ and Mn2+, and the kinetic data suggest that the enzyme possesses a distinct high-affinity binding site for Zn2+, with Kg of approximately 0.3 MM. At higher concentrations (about 10M) Zn2+, and to a lesser extent Co2+, function as activating cations. Binding studies show that the enzyme binds two equivalents of Zn2+ per subunit; one equivalent is partially displaced by Mg2+ and is presumably bound to the site for activating cations. A second equivalent binds to the high-affinity site, presumably identical to the inhibitory site. The results suggest that Zn2+ functions as an allosteric regulator, and that the commonly observed activation of fructose 1,6-bisphosphatase at neutral pH by EDTA, histidine, and other chelators is due to removal of endogenous Zn2+ by these agents.
MATERIALS AND METHODS Fru-P2ase was purified from livers of Sprague-Dawley rats by heat treatment, adsorption on carboxymethyl-cellulose, and elution with substrate (15). The specific activity of the purified enzyme preparation was 28 ,mol of fructose 1,6-bisphosphate (Fru-P2) hydrolyzed per min/mg of protein at 250 in a coupled spectrophotometric assay system based on the reduction of NADP (16). The assay mixture (1 ml) contained 20 mM triethanolamine, 20 mM diethanolamine (pH 7.5), 2 mM MgCl2, 40 mM (NH4)2SO4, 0.1 mM Fru-P2 (Sigma-Na4 salt), 0.2 mM NADP (Sigma), 0.1 mM EDTA, 2 Mug of glucose 6phosphate dehydrogenase (Boehringer, EC 1.1.1.49) and 2Mug of phosphoglucose isomerase (Boehringer, EC 5.3.1.9). For the inhibition studies we used an assay procedure based on the formation of inorganic phosphate (17, 18), in order to avoid complications arising from inhibition of the coupling enzymes. The assay mixture was identical to that described above, except that NADP, (NH4)2SO4, and the coupling enzymes were omitted. Metal ions, EDTA, and other chelators were added as indicated. The reaction was stopped by addition of 0.5 ml of the color-developing reagent (18). After 15 min the absorbance was measured at 650 nm with a Gilford Spectrophotometer model 240, and compared to values obtained with Pi standards. Radioactive 65ZnCl2 was purchased from New England Nuclear, Boston, Mass., and aliquots were added to Aquasol (New England Nuclear) for measurement of radioactivity in a Beckman LS250 Counter.
Fructose 1,6-bisphosphatase (Fru-P2ase, EC 3.1.3.11; D-fructose-1,6-bisphosphate 1-phosphohydrolase) has been shown to be activated by a variety of chelating agents, including EDTA (1-10), imidazole (1, 10, 11), histidine (9-12), and citrate (9, 13). In earlier publications from this laboratory, the activation by these agents was attributed to the formation of metal-chelate complexes, which were presumed to be necessary, along with the free cations Mg2+ or Mn2+, for maximum activity in the neutral pH region (8, 9). This interpretation was questioned by Nimmo and Tipton (10), who reported that the same activation could be achieved by treating the enzyme solutions and assay mixtures with Chelex 100. However, the inhibitory metal ion or ions remained unidentified, as did the possible physiological significance of activation by the natural chelating agents.
RESULTS Effects of Zn2+ and other heavy metal ions Of a number of heavy metal ions tested, only Zn2+ was found to be an effective inhibitor of neutral Fru-P2ase activity at low concentrations (Table 1). No inhibition was observed with Fe2+ or Fe3+, and Cu2+ was inhibitory only at much higher con-
In the present report we present evidence that the effects of the various chelators may be related to the presence of Zn2+, which is a potent inhibitor of Fru-P2ase in the neutral pH range. The effects of Zn2+ are completely reversed by the addition of EDTA or histidine. In the alkaline pH range, Zn2+ is not inhibitory, and EDTA and histidine are not activators. The inhibition by Zn2+ is not overcome by increasing the concentration of the activating cations, Mg2+ or Mn2 , suggesting that inhibition is due to the binding of Zn2+ to a different site on the enzyme. At higher concentrations, however, Zn2+ can partially replace Mg2+ or Mn2+. We have also confirmed the earlier report by Kirtley and Dix (14) that Co2+ can serve as the activating cation.
centrations. Some inhibition was observed with low concentrations of Co2 , but at higher concentrations this was not sig-
nificantly increased. The inhibition by Zn2+ was affected only slightly by increasing the concentration of Mg2+ from 2 to 5 mM, but it was completely reversed by the addition of 1 mM histidine (Fig. 1). In the presence of Mg2+, 50% inhibition was observed at a Zn2+ concentration of approximately 0.3 MM. When the enzyme was assayed with Mn2+ as the activating cation, the results were
Abbreviations: Fru-P2, fructose 1,6-bisphosphate; Fru-P2ase, fructose 1,6-bisphosphatase. * Present address: Radiation Research Division, Ohio State University Hospitals, 410 West 10th Avenue, N-212, Columbus, Ohio 43210. § On leave from: Department of Biochemistry, Federal University of Parana, Curitiba, Brazil. Predoctoral trainee, Department of Biochemistry, Cornell University, Graduate School of Medical Sciences, New York, N.Y. 10021.
similar (Fig. 2), and in other experiments (data not shown) the inhibition by Zn2+ was found to be uncompetitive with respect to Mn2+. Activation by Zn2+ and Co2+ When the concentration of Zn2+ was increased beyond the levels shown in Fig. 1, the extent of inhibition was found to 2692
Biochemistry: Tejwani et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
2693
Table 1. Effect of metal ions on rat liver Fru-P2ase % Inhibition*
Addition
1 jiM
20 jM
ZnSO4 CoCl2 Pb(N03)2 Cd(N03)2 SrCl2 CuS04 FeSO4 Fe2(SO4)3 BaCl2
83 23 21 11 11 0 4 0 0
40 77 57 19 51 10 0 14
460
*Assayed by the Pi-release method in the presence of
2 mM MgCl2 as described in Materials and Methods with 0.6 fig of Fru-P2ase. The control activity in the absence of other additions was 4.7 Mmol of Pi formed per min/mg of enzyme.
decrease, particularly when the assay was carried out with suboptimal concentrations of Mg2+ (4 P:
~~~~MgOI2
0
CDo
ZnSO4 2
ZnSO4 (PM)
FIG. 1. Inhibition of Mg2+-activated Fru-P2ase by Zn2+ and reversal by L-histidine. Fru-P2ase )0.6 Ag) was assayed at pH 7.5 by the Pi release method as described in Materials and Methods. The concentrations of MgCl2 used were 2 mM, with (A) or without (-) 1 mM histidine, or 5 mM without histidine (0). Histidine also abolished the inhibition by Zn2+ in the presence of 5 mM Mg2+ (data not shown).
5
0
q
~
0.02
g
0.04
0.10
MnCI2, ZnSO4, COCI2 (mM) FIG. 3. Activation of Fru-P2ase by divalent cations. The conditions were as described in the legend to Fig. 1, except that 0.3 Mug of enzyme was added and the activating cations were as follows: MnCl2 (0), MgCl2 (A), ZnSO4 (0), and CoCl2 (3). Note the difference in the concentration scale for Mg2+. No activation by Zn2+ was observed when the enzyme was assayed at pH 9.2.
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Biochemistry: Tejwani et al.
Proc. Nati. Acad. Sci. USA 73 (1976)
Table 2. Effect of chelators on rat liver Fru-P2ase* pH 7.5
pH 9.2
Additions
No Zn2+
1 JIM Zn2+
No Zn2+
1 JIM Zn2+
None EDTA(0.1 mM) L-Histidine (1 mM) Citrate (3 mM) o-Phenanthroline (50 MM)
2.7 9.0 8.7 9.1 8.5
0.6 8.8 8.6 3.8 7.9
2.0 2.3 2.3 4.8 1.7
2.1 2.0 1.8 3.5 1.9
* The activity was assayed by the Pi-release method as described in Materials and Methods. Simifar results were obtained with purified Fru-P2ase from rabbit liver. Values are Amol of Pi released per min/mg.
tivation by chelating agents was not observed. Chelators that activate at neutral pH, such as EDTA, histidine, and o-phen-
anthroline, also reverse the inhibition by added Zn2+. The Ki for Zn2+ inhibition is 3 X 10-7 M. At this concentration other heavy metals, such as Pb2+, Cd2+, Cu2+, Fe2+, and Fe3+, inhibit only slightly or not at all. The evidence suggests that the enzyme contains a specific site for Zn2+, to which it binds with high affinity. At higher concentrations (about 10-5 M), Zn2+ also appears to bind to the site for activating cations, and at these concentrations Zn2+ will replace Mg2+ or Mn2+. A curious finding is that with Zn2+ as an activator, the inhibitory effects of Zn2+ are abolished, although the binding studies show that it still binds to the high-affinity site. The results suggest that the allosteric properties of the enzyme depend on the nature of the cation bound to the activating site. We have previously shown that Ca2+ is a potent inhibitor of the rat liver enzyme (15) as well as the enzyme purified from rabbit muscle (7). However, inhibition by Ca2+ is observed at both alkaline and neutral pH and is competitive with Mg2+ or Mn2+; in this respect its mechanism of inhibition is fundamentally different from that of Zn2+, which does not show competitive inhibition with respect to Mg2+ or Mn2+, and which functions as an activator, rather than inhibitor, when it binds to the Mg2+ (Mn2+) site. The binding studies confirm that each subunit binds two equivalents of Zn2+, one of which is partially displaced by Mg2+ and a second that binds to a high affinity site, consistent with the low Ki for inhibition by
20E
10
Ln
0
20 40
60
80 100 120
FRACTION NUMBER
FIG. 5. The number of binding sites for Zn2+, determined in the presence of excess Zn2+. Binding measurements were carried out by the rate-dialysis procedure of Colowick and Womack (20), using a 2.5-ml stirring flow-dialysis cell (Technilab Instruments, Inc., Pequannock, N.J.) with the lower chamber modified to contain 1.5 ml. The upper chamber contained 0.96 ml of 40 mM diethanolamine-40 mM triethanolamine buffer, pH 7.5. At the points indicated by the arrows the following were added: 40 Ml of 1 mM 65ZnC12 (specific activity = 2.96 mCi/mg); 30 Ml (375 Mg, 2.68 nmol) of Fru-P2ase; 10 Ml, 10 Ml, and 20 Ml of 1 M MgCl2; and 100 Ml of 10 mM ZnSO4. Fractions (2.1 ml) were collected at a flow rate of 7.0 ml/min, and 1.8-ml aliquots were transferred to 10 ml of Aquasol for determination of radioactivity in a Beckman Liquid Scintillation Counter. The number of equivalents of Zn2+ bound per Fru-P2ase subunit was calculated from the radioactivity in tubes number 40 and 85 and the expected radioactivity, estimated from the broken line, and was found to be 2.16 and 1.62, respectively.
to be established. The fact that it is reversed by concentrations of histidine that are found in livers of fasting animals (12) suggests that inhibition by Zn2+ and its reversal by histidine may play a role in the regulation of gluconeogenesis. While this manuscript was in preparation, we became aware I I.I... I I.I... I
65zn2+ Fru-P2ase 6.0 5.0 x
Zn2+
-
O
I
4.0
Zn2+.
The physiological significance of inhibition by Zn2+ remains
20 0.
0
50
100
FRACTION NUMBER
MgCI 2 (mM)
FIG. 4. Effect of increasing Mg2+ concentration in the presence and absence of citrate. The conditions were as described in the legend to Fig. 1, except that assays were carried out at pH 9.2. (0) Citrate was not added; (A, 0) activity in the presence of 3 mM citrate. (-) Data corrected for the concentration of free Mg2+, using a value of 4 X 103 for the stability constant for the Mg2+-citrate complex (19).
FIG. 6. The dissociation constant for the high-affinity binding site. The binding measurements were carried out as described in the legend to Fig. 5, except that the concentration of 65Zn2+ was 10 MM, instead of 40 AM, and the amount of Fru-P2ase added was 280 Ag (2 nmol). At the third arrow 10 Ml of 10 mM ZnSO4 was added. The dissociation constant, calculated from the data in tube number 65, was 0.17 MM. A correction, estimated from the height of the spike after addition of nonradioactive Zn2+, was made for binding of 65Zn2+ to the walls of the chamber. This extent of binding was confirmed in a control experiment without enzyme. This correction was negligible in the experiment in Fig. 5, where the concentration of 65Zn2+ was 4-fold greater.
Biochemistry: Tejwani et al. of an abstract submitted to the American Society of Biological Chemists 67th Annual Meeting by W. A. Frey, C. A. Caperelli, and S. J. Benkovic [(1976) Fed. Proc. 35, 1705] in which they describe activation of rabbit liver Fru-P2ase by Zn2 , and evidence for the presence of this metal in purified enzyme preparations. 1. McGilvery, R. W. (1964) in Fructose 1,6-Diphosphatase and Its Role in Gluconeogenesis, eds. McGilvery, R. W. & Pogell, B. M. (American Institute of Biological Sciences, Washington, D.C.), pp. 3-13. 2. Krebs, H. A. & Woodford, M. (1965) Biochem. J. 94,436-445. 3. Underwood, A. H. & Newsholme, E. A. (1965) Blochem. J. 95, 767-774. 4. Rosen, 0. M., Rosen, S. M. & Horecker, B. L. (1965) Arch. Biochem. Biophys. 112,411-420. 5. Pogell, B. M., Tanaka, A. & Siddons, R. C. (1968) J. Biol. Chem. 243, 1356-1367. 6. Traniello, S., Melloni, E., Pontremoli, S., Sia, C. L. & Horecker, B. L. (1972) Arch. Biochem. Biophys. 149,222-231. 7. Van Tol, A., Black, W. J. & Horecker, B.. (1972) Arch. Biochem. Biophys. 151,591-596. 8. Rosenberg, J. S., Tashima, Y., Horecker, B. L. & Pontremoli, S. (1973) Arch. Biochem. Biophys. 154,283-291. 9. Datta, A. G., Abrams, B., Sasaki, T., van den Berg, J. W. O.,
Proc. Natl. Acad. Sci. USA 73 (1976)
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Pontremoli, S. & Horecker, B. L. (1974) Arch. Biochem. Biophys. 165, 641-645. 10. Nimmo, H. G. & Tipton, K. F. (1975) Biochem. J. 145, 323334. 11. Hers, H. G. & Eggermont, E. (1964) in Fructose 1,6-Diphosphatase and Its Role in Gluconeogenesis, eds. McGilvery, R. W. & Pogell, B. M. (American Institute of Biological Sciences, Washington, D.C.), pp. 14-19. 12. Pontremoli, S., Melloni, E., De Flora, A. & Horecker, B. L. (1974) Proc. Natl. Acad. Sci. USA 71, 2166-2168. 13. Fu, J. Y. & Kemp, R. G. (1973) J. Biol. Chem. 248, 11241125. 14. Kirtley, M. E. & Dix, J. C. (1971) Arch. Biochem. Biophys. 147, 647-652. 15. Tejwani, G. A., Pedrosa, F. O., Pontremoli, S. & Horecker, B. L. (19761 Arch. Biochem. Biophys., in press. 16. Pontremoli, S., Traniello, S., Luppis, B. & Wood, W. A. (1965) J. Biol. Chem. 240,3459-3463. 17. Itaya, K. & Ui, M. (1966) Clin. Chim. Acta 14,361-366. 18. Tashima, Y. & Yoshimura, N. (1975) J. Biochem. 78, 11611169. 19. Stability Constants of Metal Ion Complexes, The Chemical Society (1971) Special Publication no. 25 (compiled by Sillen, L. E. & Martell, A. E.) (Burlington House, London), p. 413. 20. Colowick, S. P. & Womack, F. C. (1969) J. Biol. Chem. 244, 774-777.
