Fhit-nucleotide Specificity Probed with Novel Fluorescent and Fluorogenic Substrates

• Published in: The Journal of biological chemistry Vol. 275; no. 7; pp. 4555 - 4560
• Main Authors: Draganescu, Alexandra, Hodawadekar, Santosh C., Gee, Kyle R., Brenner, Charles
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18-02-2000; American Society for Biochemistry and Molecular Biology
• Subjects: Acid Anhydride Hydrolases; Adenine Nucleotides - metabolism; Boron Compounds; diadenosine polyphosphates; Fhit protein; Fluorescent Dyes; Guanosine 5'-O-(3-Thiotriphosphate) - analogs & derivatives; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Humans; Kinetics; Magnetic Resonance Spectroscopy; Neoplasm Proteins; Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.7.4555

Fhit, a member of the histidine triad superfamily of nucleotide-binding proteins, binds and cleaves diadenosine polyphosphates and functions as a tumor suppressor in human epithelial cancers. Function of Fhit in tumor suppression does not require diadenosine polyphosphate cleavage but correlates with the ability to form substrate complexes. As diadenosine polyphosphates are at lower cellular concentrations than mononucleotides, we sought to quantify interactions between Fhit and competitive inhibitors with the use of diadenosine polyphosphate analogs containing fluorophores in place of one nucleoside. Appp-S-(7-diethylamino-4-methyl-3-(4-succinimidylphenyl)) coumarin (ApppAMC), Appp-S-(4-4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacine-3-yl) methylaminoacetyl (ApppBODIPY), and GpppBODIPY, synthesized in high yield, are effective Fhit substrates, producing AMP or GMP plus fluorophore diphosphates. GpppBODIPY cleavage is accompanied by a 5.4-fold increase in fluorescence because BODIPY fluorescence is quenched by stacking with guanine. Titration of unlabeled diadenosine polyphosphates, inorganic pyrophosphate, mononucleotides, and inorganic phosphate into fluorescent assays provided values ofKm and KI as competitive inhibitors. The data indicate that Fhit discriminates between good substrates via kcat and against cellular competitors in equilibrium binding terms. Surprisingly, pyrophosphate competes better than purine mononucleotides.