Characterization of metal ion activation and inhibition of CTP synthetase.
A reinvestigation of the metal ion activation and specificity of CTP synthetase was begun in order to separate effects due to binding of free metal ions and binding of nucleotide-complexed ions. Apo-CTP synthetase was prepared by dialysis against 5 mM EDTA. Analysis of apo-enzyme by atomic absorption spectroscopy revealed that all bound metal ions could be removed. Thus, apo-enzyme contained no detectable amounts of Mg2+, Mn2+, Cu2+, Zn2+, Co2+, Ni2+, or Fe2+. The half-saturation value of Mg(2+)-dependent enzyme activation was approximately 2.6 mM at a total concentration of 2 mM nucleotides (ATP plus UTP). These data suggest that the enzyme requires more Mg2+ for full catalytic activity than required simply to complex the nucleotide substrates. Analysis of velocity versus [Mg2+]free demonstrated that activity depends on [Mg2+]free. The half-saturation values for [Mg2+]free were 660 and 280 microM in the NH4(+)- and glutamine-dependent assays, respectively. The half-saturation values for [Mn2+]free and [Co2+]free were approximately 2.6 and 3.8 microM in the NH4(+)-dependent assay and 2.8 and 4.7 microM in the glutamine-dependent assay. These results are consistent with the presence of a separate binding site for free metal ion on the enzyme. Over the range of 0.1-10 mM, neither Cu2+, Zn2+, Ni2+, nor Ca2+ activated the enzyme. Also, both Cu2+ and Zn2+ were effective inhibitors of CTP synthetase in the absence of dithiothreitol at concentrations < 50 microM. Inhibition by Zn2+ was reversed by EDTA, whereas inhibition by Cu2+ was not. In the presence of dithiothreitol, Zn2+, Co2+ and Ni2+ inhibited the enzyme at less than 200 microM metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)
Robertson
J G
JG
Department of Chemistry, Pennsylvania State University, University Park 16802.
Villafranca
J J
JJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000378:metabolism / Q000494:pharmacology*; D009532:Nickel / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014544:Uridine Triphosphate / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D001051:Apoenzymes; D008670:Metals; D003035:Cobalt; D008345:Manganese; D003300:Copper; D009532:Nickel; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D008274:Magnesium; D015032:Zinc; D014544:Uridine Triphosphate