Cyclopentenylcytosine triphosphate. Formation and inhibition of CTP synthetase.
Cyclopentenylcytosine (CPEC) is phosphorylated in L1210 cells with CPEC triphosphate as the major metabolite. Partially purified uridine-cytidine kinase catalyzes the initial phosphorylation of cyclopentenylcytosine with an apparent Km of 196 +/- 9 microM, and cyclopentenylcytosine is a competitive inhibitor of cytidine phosphorylation by this enzyme with a Ki value of 144 +/- 14 microM. Examination of the CTP synthetase activity in extracts of L1210 cells revealed a dose-dependent decrease on exposure of cells to CPEC. Synthesis of CPEC triphosphate by an enzymatic method permitted direct examination of the inhibition of partially purified CTP synthetase. CPEC triphosphate inhibited bovine CTP synthetase with a median inhibitory concentration of 6 microM, whereas CPEC mono- and diphosphates were ineffective. CTP synthetase showed a classical Michaelis-Menten hyperbolic plot of velocity and UTP concentration in the presence of saturating concentrations of ATP and glutamine, but CPEC triphosphate induced sigmoidal kinetic plots. The Hill coefficient was calculated to be 3.2.
Kang
G J
GJ
Laboratory of Pharmacology and Experimental Therapeutics, National Cancer Institute, Bethesda, Maryland 20892.
Cooney
D A
DA
Moyer
J D
JD
Kelley
J A
JA
Kim
H Y
HY
Marquez
V E
VE
Johns
D G
DG
D000818:Animals; D019731:Carbon-Nitrogen Ligases; D003562:Cytidine / Q000031:analogs & derivatives* / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000494:pharmacology*; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology; D007941:Leukemia P388 / Q000201:enzymology; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D013058:Mass Spectrometry; D051379:Mice; D010766:Phosphorylation
D003597:Cytosine Nucleotides; C058208:cyclopentenylcytosine 6-triphosphate; D003562:Cytidine; D003570:Cytidine Triphosphate; C045911:cyclopentenyl cytosine; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase