ARCHIVES OFBIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 2, May, pp. 481-485, 1979
Binding
of Zn 2+ to Rabbit Muscle Fructose 1,6-Bisphosphatase Its Effect on the Catalytic Properties
S. PONTREMOLI,*
B. SPARATORE,* F. SALAMINO,” B. L. HORECKER?
*Institute of Biological Chemistry, tRoche Institute of Molecular
E. MELLONI,”
University of Genoa, Genoa, Biology, Nutley, New Jersey
Italy, 07110
and
AND
and
Received October 24, 1978; revised January 4, 197!3 At low concentrations (2 mM) that would fill both sets of sites (Species I). However, this form of the enzyme is unlikely to exist in the presence of traces of Zn2+, which compete with Mg*+ for binding to the first set of sites. Under normal physiological conditions, when traces of Zn2+ are likely to be present, unphysiological concentrations of Mg*+ would be required and Zn*+ is therefore more likely to serve as the activator. On the other hand, in fasting animals, where the concentrations of physiological chelators such as histidine or methyl histidine (8) may be elevated, Mg2+ may serve as activator. It should be noted that Ca*+ has also been reported to be a potent inhibitor of muscle Fru-P,ase activity (2) and all of these metal cations may function in the regulation of Fru-Pzase activity in muscle in viva. Recently Benkovic et al. (9) concluded
METAL
ION-RABBIT
MUSCLE
FRUCTOSE
that rabbit liver Fru-P,ases possess 8 binding sites for Zn2+, rather than 12 binding sites as reported by us for both the rat liver (4) and rabbit liver (5) enzymes. The reason for this difference remains undefined. However, their binding experiments were carried out in the presence of a substrate analog, rather than the substrate Fru-P,, and furthermore they observed binding of 8 eq/mol in equilibrium dialysis experiments carried out in the absence of substrate and at lower concentrations of enzyme. They attributed this to nonspecific binding of Zn’+ to denatured enzyme formed during the dialysis, but there is no reason to expect that the denatured enzyme would bind more Zn2+ than the native form. The results reported by us for both the rat liver and rabbit liver enzymes support the conclusion that these enzymes bind 12 eqlmol; as reported here the same methods identify the presence of only 8 binding sites in the muscle enzyme. ACKNOWLEDGMENT The Institute of Biological Chemistry, University of Genoa, acknowledges support from the Italian CNR.
1,6-BISPHOSPHATASE
485
INTERACTIONS REFERENCES
1. BLACK, W. J., VAN TOL, A., FERNANDO, J., AND HORECKER, B. I,. (1972) Arch. Biochem. Biophys. 151, 576-690. 2. VAN TOL, A., BLACK, B. L. (19’72) A&L. 591-596.
W. J., AND HORECKER, Biochem. Biophys. 151,
3. TEJWANI, G. A., PEDROSA, F. O., PONTREMOLI, S., AND HORECKER, B. L. (1976) Proc. Nat. Acad. Sci. USA 73, 2692-2695. 4. PEDROSA, F. O., PONTREMOLI, RECKER, B. L. (1977) f’roc. USA 74, 2742-2745.
S., AND Nat. Acad.
Hosci.
5. PONTREMOLI, S., :MELLONI, E., SALAMINO, F., SPARATORE, B., AND HORECKER, B. L. (1978) Arch. Biochem. Biophys. 188, 90-97. 6. TASHIMA, &em.
Y., AND YOSHIMURA, 78, 1161-1169.
N. (1975)
J. Bio-
7. HUMMEL, chim.
J. P., AND DREYER, W. J. (1962) Biophys. .4&a 63, 530-532.
Bio-
8. PONTREMOLI, S., MELLONI, E., DE FLORA, A., AND HORECKER, B. L. (1974) Proc. Nat. Acad. Sci. USA 71, 2166-2168. 9. BENKOVIC, P. A., C:APERELLI, M., DE MAINE, M., AND BENKOVIC, S. J. (1978) Proc. Nat. Acad. Sci. USA 75, 21(35-2189.
Binding
of Zn 2+ to Rabbit Muscle Fructose 1,6-Bisphosphatase Its Effect on the Catalytic Properties
S. PONTREMOLI,*
B. SPARATORE,* F. SALAMINO,” B. L. HORECKER?
*Institute of Biological Chemistry, tRoche Institute of Molecular
E. MELLONI,”
University of Genoa, Genoa, Biology, Nutley, New Jersey
Italy, 07110
and
AND
and
Received October 24, 1978; revised January 4, 197!3 At low concentrations (2 mM) that would fill both sets of sites (Species I). However, this form of the enzyme is unlikely to exist in the presence of traces of Zn2+, which compete with Mg*+ for binding to the first set of sites. Under normal physiological conditions, when traces of Zn2+ are likely to be present, unphysiological concentrations of Mg*+ would be required and Zn*+ is therefore more likely to serve as the activator. On the other hand, in fasting animals, where the concentrations of physiological chelators such as histidine or methyl histidine (8) may be elevated, Mg2+ may serve as activator. It should be noted that Ca*+ has also been reported to be a potent inhibitor of muscle Fru-P,ase activity (2) and all of these metal cations may function in the regulation of Fru-Pzase activity in muscle in viva. Recently Benkovic et al. (9) concluded
METAL
ION-RABBIT
MUSCLE
FRUCTOSE
that rabbit liver Fru-P,ases possess 8 binding sites for Zn2+, rather than 12 binding sites as reported by us for both the rat liver (4) and rabbit liver (5) enzymes. The reason for this difference remains undefined. However, their binding experiments were carried out in the presence of a substrate analog, rather than the substrate Fru-P,, and furthermore they observed binding of 8 eq/mol in equilibrium dialysis experiments carried out in the absence of substrate and at lower concentrations of enzyme. They attributed this to nonspecific binding of Zn’+ to denatured enzyme formed during the dialysis, but there is no reason to expect that the denatured enzyme would bind more Zn2+ than the native form. The results reported by us for both the rat liver and rabbit liver enzymes support the conclusion that these enzymes bind 12 eqlmol; as reported here the same methods identify the presence of only 8 binding sites in the muscle enzyme. ACKNOWLEDGMENT The Institute of Biological Chemistry, University of Genoa, acknowledges support from the Italian CNR.
1,6-BISPHOSPHATASE
485
INTERACTIONS REFERENCES
1. BLACK, W. J., VAN TOL, A., FERNANDO, J., AND HORECKER, B. I,. (1972) Arch. Biochem. Biophys. 151, 576-690. 2. VAN TOL, A., BLACK, B. L. (19’72) A&L. 591-596.
W. J., AND HORECKER, Biochem. Biophys. 151,
3. TEJWANI, G. A., PEDROSA, F. O., PONTREMOLI, S., AND HORECKER, B. L. (1976) Proc. Nat. Acad. Sci. USA 73, 2692-2695. 4. PEDROSA, F. O., PONTREMOLI, RECKER, B. L. (1977) f’roc. USA 74, 2742-2745.
S., AND Nat. Acad.
Hosci.
5. PONTREMOLI, S., :MELLONI, E., SALAMINO, F., SPARATORE, B., AND HORECKER, B. L. (1978) Arch. Biochem. Biophys. 188, 90-97. 6. TASHIMA, &em.
Y., AND YOSHIMURA, 78, 1161-1169.
N. (1975)
J. Bio-
7. HUMMEL, chim.
J. P., AND DREYER, W. J. (1962) Biophys. .4&a 63, 530-532.
Bio-
8. PONTREMOLI, S., MELLONI, E., DE FLORA, A., AND HORECKER, B. L. (1974) Proc. Nat. Acad. Sci. USA 71, 2166-2168. 9. BENKOVIC, P. A., C:APERELLI, M., DE MAINE, M., AND BENKOVIC, S. J. (1978) Proc. Nat. Acad. Sci. USA 75, 21(35-2189.
