Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates

Fhit, a member of the histidine triad superfamily of nucleotide-binding pro teins, binds and cleaves diadenosine polyphosphates and functions as a tumo r suppressor in human epithelial cancers. Function of Fhit in tumor suppres sion does not require diadenosine polyphosphate cleavage but correlates wit h the ability to form substrate complexes. As diadenosine polyphosphates ar e at lower cellular concentrations than mononucleotides, we sought to quant ify interactions between Fhit and competitive inhibitors with the use of di adenosine polyphosphate analogs containing fluorophores in place of one nuc leoside. Appp-S-(7-diethylamino-4-methyl-3-(4-succinimidylphenyl)) coumarin (ApppAMC), Appp-S-(4-4-difluoFo-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 GIMP plus fluorophor e diphosphates. GpppBODIPY cleavage is accompanied by a 5.4-fold increase i n fluorescence because BODIPY fluorescence is quenched by stacking with gua nine. Titration of unlabeled diadenosine polyphosphates, inorganic pyrophos phate, mononucleotides, and inorganic phosphate into fluorescent assays pro vided values of K-m and K-I as competitive inhibitors. The data indicate th at Fhit discriminates between good substrates via k(cat) and against cellul ar competitors in equilibrium binding terms. Surprisingly, pyrophosphate co mpetes better than purine mononucleotides.