Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates

Alexandra Draganescu; Santosh C Hodawadekar; Kyle R Gee; Charles Brenner

doi:10.1074/jbc.275.7.4555

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Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates

Journal article

Open access

Peer reviewed

Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates

Alexandra Draganescu, Santosh C Hodawadekar, Kyle R Gee and Charles Brenner

The Journal of biological chemistry, Vol.275(7), pp.4555-4560

02/18/2000

DOI: 10.1074/jbc.275.7.4555

PMCID: PMC2556043

PMID: 10671479

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Abstract

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 K(m) and K(I) as competitive inhibitors. The data indicate that Fhit discriminates between good substrates via k(cat) and against cellular competitors in equilibrium binding terms. Surprisingly, pyrophosphate competes better than purine mononucleotides.

Guanosine 5'-O-(3-Thiotriphosphate) - metabolism

Proteins - metabolism

Acid Anhydride Hydrolases

Magnetic Resonance Spectroscopy

Adenine Nucleotides - metabolism

Humans

Neoplasm Proteins

Kinetics

Boron Compounds

Fluorescent Dyes

Guanosine 5'-O-(3-Thiotriphosphate) - analogs & derivatives

Details

Title: Subtitle: Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates
Creators: Alexandra Draganescu - Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
Santosh C Hodawadekar
Kyle R Gee
Charles Brenner
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.275(7), pp.4555-4560
DOI: 10.1074/jbc.275.7.4555
PMID: 10671479
PMCID: PMC2556043
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: R01 CA075954 / NCI NIH HHS R01 CA075954-03 / NCI NIH HHS CA75954 / NCI NIH HHS
Language: English
Date published: 02/18/2000
Academic Unit: Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9983788597002771

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