Abstract
As a possible probe for metal activation of calcineurin, Tb3+ was tested for effects on calcineurin activity. Calcineurin was activated by Tb3+ with the following kinetic parameters estimated: k cat = 0.78 ± 0.02 sec−1, K m(pNPP) = 32.6 ± 1.8 mM, and K act(Tb3+) = 0.08 ± 0.03 mM. Terbium luminescence was demonstrated in the presence of the heterodimer of calcineurin and exploited to localize the binding of exogenous metal to the enzyme active site. Exogenous Mn2+ reduced luminescence, although the affinity of calcineurin for Tb3+ seemed to be greater. Putative active-site ligands, such as para-nitrophenol and a synthetic peptide from the autoinhibitory region, reduced the luminescence of terbium. Collectively, these data suggested that Tb3+ was binding directly at the active site of calcineurin, with the corollary that exogenous activating metal (Mn2+) binds at the active site of the enzyme. These data support the hypothesis that activating, exogenous divalent metal participates directly in catalysis.
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Martin, B.L., Jurado, L.A. Activation of Calcineurin by the Trivalent Metal Terbium. J Protein Chem 17, 473–478 (1998). https://doi.org/10.1023/A:1022574719239
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DOI: https://doi.org/10.1023/A:1022574719239