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J Biol Chem, Vol. 275, Issue 7, 4555-4560, February 18, 2000

Fhit-nucleotide Specificity Probed with Novel Fluorescent and Fluorogenic Substrates*

Alexandra DraganescuDagger §, Santosh C. HodawadekarDagger , Kyle R. Gee, and Charles BrennerDagger ||

From the Dagger  Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and  Molecular Probes, Eugene, Oregon 97402

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 of Km 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.

* This work was supported by National Institutes of Health NCI Research Grant CA75954 (to C. B.), a National Institutes of Health NCI institutional training grant (to A. D.), and new investigator awards from the March of Dimes Birth Defects Foundation, the Burroughs Wellcome Fund, and the Arnold and Mabel Beckman Foundation (to C. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Unilever Research, Edgewater, NJ 07020.

|| To whom correspondence should be addressed: Kimmel Cancer Center, Thomas Jefferson University, 233 S. Tenth St., Rm. 826, Philadelphia, PA 19107. Tel.: 215-503-4573; Fax: 215-923-1696; E-mail: brenner@dada.jci.tju.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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