Robertson J G, Villafranca J J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1993 Apr 13;32(14):3769-77. doi: 10.1021/bi00065a032.
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)
为了区分游离金属离子结合和核苷酸复合离子结合所产生的影响，我们重新开始了对CTP合成酶的金属离子激活作用和特异性的研究。通过用5 mM EDTA透析制备脱辅基CTP合成酶。用原子吸收光谱法对脱辅基酶进行分析表明，所有结合的金属离子都可以被去除。因此，脱辅基酶中未检测到Mg2+、Mn2+、Cu2+、Zn2+、Co2+、Ni2+或Fe2+。在2 mM核苷酸（ATP加UTP）的总浓度下，Mg(2+)依赖性酶激活的半饱和值约为2.6 mM。这些数据表明，与仅仅使核苷酸底物络合相比，该酶需要更多的Mg2+才能达到完全催化活性。速度对[Mg2+]游离浓度的分析表明，活性取决于[Mg2+]游离浓度。在NH4(+)依赖性和谷氨酰胺依赖性测定中，[Mg2+]游离浓度的半饱和值分别为660和280 microM。在NH4(+)依赖性测定中，[Mn2+]游离浓度和[Co2+]游离浓度的半饱和值约为2.6和3.8 microM，在谷氨酰胺依赖性测定中分别为2.8和4.7 microM。这些结果与该酶上存在游离金属离子的单独结合位点一致。在0.1 - 10 mM范围内，Cu2+、Zn2+、Ni2+和Ca2+均未激活该酶。此外，在浓度<50 microM且不存在二硫苏糖醇的情况下，Cu2+和Zn2+都是CTP合成酶的有效抑制剂。Zn2+的抑制作用可被EDTA逆转，而Cu2+的抑制作用则不能。在存在二硫苏糖醇的情况下，Zn2+、Co2+和Ni2+在金属离子浓度低于200 microM时抑制该酶。（摘要截短于250字）
