Formycin triphosphate-terbium complex: a novel spectroscopic probe for phosphoryl transfer enzymes

The conditions under which the fluorescent pyrazolopyrimidine nucleotide formycin A triphosphate (7-amino-3-(β-d-(5′-tripolyphosphate)ribofuranosyl)pyrazolo[4 ,3-d]pyrimidine, FTP) forms a 1:1 complex in solution with Tb³⁺ have been characterized. The complex has a dissociation constant of approx. 10^-7 M. Within the complex, the luminescence of Tb³⁺ is dramatically sensitized by energy transfer from formycin. The value for 50% transfer efficiency, Förster's R_o (Förster, T. (1964) in Modern Quantum Chemistry (Sinanoglu, O., ed.), pp 93-137, Academic Press, New York) was determined to be 3.34 ± 0.4 A ̊, and the effective distance between the donor and acceptor transition dipoles, R, in the complex was estimated to be 6.6 ± 1.0 A ̊. The quantum yield of Tb³⁺ in the complex is sensitive to the number of O-H oscillators bound to the Tb³⁺, which allows determination of the number of waters bound to it (approx. 4). Preliminary results show that the complex binds to the phosphoryl transfer enzyme hexokinase in the presence of the glucose analogs N-acetylglucosamine, frucose and xylose, which are not phosphorylated by the enzyme. The binding occurs with a loss of energy efficiency consistent with a new distance from the effective transition dipole of formycin to that of terbium of approx. 9.6 Å. The FTP-terbium complex can be used as both a spectroscopic and an X-ray diffraction probe. Studies with this compound should be most valuable for correlating solution and crystallographic data.