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Involvement of a divalent cation in the binding of fructose 6-phosphate to Trypanosoma cruzi phosphofructokinase: kinetic and magnetic resonance studies.
Arch Biochem Biophys. 1990 Apr; 278(1):187-94.AB

Abstract

When Mg2+ ions were replaced by Mn2+ in the assay of Trypanosoma (Schizotrypanum) cruzi phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) the Km for D-fructose 6-phosphate (F6P) was reduced threefold while the corresponding constant for ATP was essentially unaffected. A detailed kinetic investigation showed that the apparent Km for F6P decreased monotonically with increasing free Mn2+ concentrations, from a limiting value of 5.7 mM in its absence to a limiting value of 1.1 mM in the presence of saturating concentrations of the ion; the Vmax of the enzyme was, on the other hand, not affected by the concentration of Mn2+. Conversely, it was shown that the apparent Km for Mn2+ at fixed MnATP concentrations decreased with increasing F6P concentrations, from a limiting value of 30 microM in the absence of the sugar phosphate to 9 microM at saturating concentrations of the substrate, while the apparent Vmax increased monotonically from zero to its limiting value. Both electron paramagnetic resonance and water proton longitudinal relaxation studies showed binding of one Mn2+ ion per 18,000 Da catalytic subunit of enzyme in the absence of F6P, with a dissociation constant of 57 +/- 4 microM, comparable to the apparent Km for the ion in the absence of F6P. The presence of saturating level of F6P decreases the value of the dissociation constant of Mn2+ to a limiting value of 7.9 microM in agreement with the results of the kinetic analysis. The substrate F6P decreases the enhancement of the water proton longitudinal relaxation rate in a saturable fashion, suggesting displacement of water molecules coordinated to the enzyme-bound Mn2+ ion by the sugar phosphate. Computer fitting of the several dissociation constants and relaxation enhancements for binary and ternary complexes gives a value of 7.9 mM for the dissociation constant of the enzyme-F6P complex in the absence of Mn2+ and 1.1 mM in the presence of saturating concentrations of the ion, in excellent agreement with the respective Km values of F6P extrapolated to zero and saturating Mn2+, respectively. Studies of the frequency dependence of the water proton longitudinal relaxation rate enhancements in the presence of both binary (enzyme-Mn2+) and ternary (enzyme-Mn2(+)-F6P) complexes, are most simply explained by assuming two exchangeable water molecules in the coordination sphere of the enzyme-bound Mn2+ in the binary complex, while in the ternary complex the data are consistent with the displacement of one of the water molecule from the coordination sphere with no significant alteration of the correlation time. Overall, the kinetic and binding data are consistent with the formation of an enzyme-metal-F6P bridge complex at the active site of T. cruzi phosphofructokinase, a coordination scheme which is unique among the phosphofructokinases.

Authors+Show Affiliations

Centro de Biología Celular, Escuela de Biología, Facultad de Ciencias, Universidad Central de Venezuela, Caracas.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

2138869

Citation

Urbina, J A., et al. "Involvement of a Divalent Cation in the Binding of Fructose 6-phosphate to Trypanosoma Cruzi Phosphofructokinase: Kinetic and Magnetic Resonance Studies." Archives of Biochemistry and Biophysics, vol. 278, no. 1, 1990, pp. 187-94.
Urbina JA, Ysern X, Mildvan AS. Involvement of a divalent cation in the binding of fructose 6-phosphate to Trypanosoma cruzi phosphofructokinase: kinetic and magnetic resonance studies. Arch Biochem Biophys. 1990;278(1):187-94.
Urbina, J. A., Ysern, X., & Mildvan, A. S. (1990). Involvement of a divalent cation in the binding of fructose 6-phosphate to Trypanosoma cruzi phosphofructokinase: kinetic and magnetic resonance studies. Archives of Biochemistry and Biophysics, 278(1), 187-94.
Urbina JA, Ysern X, Mildvan AS. Involvement of a Divalent Cation in the Binding of Fructose 6-phosphate to Trypanosoma Cruzi Phosphofructokinase: Kinetic and Magnetic Resonance Studies. Arch Biochem Biophys. 1990;278(1):187-94. PubMed PMID: 2138869.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Involvement of a divalent cation in the binding of fructose 6-phosphate to Trypanosoma cruzi phosphofructokinase: kinetic and magnetic resonance studies. AU - Urbina,J A, AU - Ysern,X, AU - Mildvan,A S, PY - 1990/4/1/pubmed PY - 1990/4/1/medline PY - 1990/4/1/entrez SP - 187 EP - 94 JF - Archives of biochemistry and biophysics JO - Arch. Biochem. Biophys. VL - 278 IS - 1 N2 - When Mg2+ ions were replaced by Mn2+ in the assay of Trypanosoma (Schizotrypanum) cruzi phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) the Km for D-fructose 6-phosphate (F6P) was reduced threefold while the corresponding constant for ATP was essentially unaffected. A detailed kinetic investigation showed that the apparent Km for F6P decreased monotonically with increasing free Mn2+ concentrations, from a limiting value of 5.7 mM in its absence to a limiting value of 1.1 mM in the presence of saturating concentrations of the ion; the Vmax of the enzyme was, on the other hand, not affected by the concentration of Mn2+. Conversely, it was shown that the apparent Km for Mn2+ at fixed MnATP concentrations decreased with increasing F6P concentrations, from a limiting value of 30 microM in the absence of the sugar phosphate to 9 microM at saturating concentrations of the substrate, while the apparent Vmax increased monotonically from zero to its limiting value. Both electron paramagnetic resonance and water proton longitudinal relaxation studies showed binding of one Mn2+ ion per 18,000 Da catalytic subunit of enzyme in the absence of F6P, with a dissociation constant of 57 +/- 4 microM, comparable to the apparent Km for the ion in the absence of F6P. The presence of saturating level of F6P decreases the value of the dissociation constant of Mn2+ to a limiting value of 7.9 microM in agreement with the results of the kinetic analysis. The substrate F6P decreases the enhancement of the water proton longitudinal relaxation rate in a saturable fashion, suggesting displacement of water molecules coordinated to the enzyme-bound Mn2+ ion by the sugar phosphate. Computer fitting of the several dissociation constants and relaxation enhancements for binary and ternary complexes gives a value of 7.9 mM for the dissociation constant of the enzyme-F6P complex in the absence of Mn2+ and 1.1 mM in the presence of saturating concentrations of the ion, in excellent agreement with the respective Km values of F6P extrapolated to zero and saturating Mn2+, respectively. Studies of the frequency dependence of the water proton longitudinal relaxation rate enhancements in the presence of both binary (enzyme-Mn2+) and ternary (enzyme-Mn2(+)-F6P) complexes, are most simply explained by assuming two exchangeable water molecules in the coordination sphere of the enzyme-bound Mn2+ in the binary complex, while in the ternary complex the data are consistent with the displacement of one of the water molecule from the coordination sphere with no significant alteration of the correlation time. Overall, the kinetic and binding data are consistent with the formation of an enzyme-metal-F6P bridge complex at the active site of T. cruzi phosphofructokinase, a coordination scheme which is unique among the phosphofructokinases. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/2138869/Involvement_of_a_divalent_cation_in_the_binding_of_fructose_6_phosphate_to_Trypanosoma_cruzi_phosphofructokinase:_kinetic_and_magnetic_resonance_studies_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0003-9861(90)90247-V DB - PRIME DP - Unbound Medicine ER -