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Gemcitabine is a Competitive Inhibitor of Cytidine‐5′‐triphosphate Synthase (CTPS) that Induces Enzyme Filament Polymerization
Author(s) -
McCluskey Gregory,
Bearne Stephen
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.722.7
Subject(s) - cytidine , gemcitabine , chemistry , enzyme , biochemistry , biology , cancer , genetics
CTP plays a central role in nucleotide and lipid biosynthesis and is synthesized de novo by the catalytic action of CTP synthase (CTPS). CTPS is an important modulator of lymphocyte proliferation and has been identified as a potential therapeutic target for the treatment of acute lymphoblastic leukemia. The cytidine analogue gemcitabine is used to treat various cancers by inhibiting DNA polymerase, and also depleting intracellular CTP levels. While depletion of CTP levels suggested that gemcitabine inhibits CTPS activity in vivo , in vitro characterization of the inhibition is lacking. We demonstrate that gemcitabine is a potent competitive inhibitor of CTPS, binding with an affinity that exceeds that exhibited for UTP by 100‐fold. Site‐directed mutagenesis (E149D) studies reveal that a conserved glutamate (E149) is necessary for gemcitabine recognition, and this residue is also required for catalyzing the conversion of UTP to CTP. CTPS polymerization is an additional mode of inhibition where filament formation apparently prevents conformational changes conducive to enzymatic activity. Using electron microscopy, we detected CTPS filaments in gemcitabine‐treated samples of wild‐type enzyme, but not in E149D CTPS which cannot bind gemcitabine. These studies highlight the critical role that E149 plays in catalysis and the conformational changes of the enzyme. Supported by the Canadian Institutes for Health Research