A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps
A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps
复制标题
DOI：
10.1021/bi9812672
复制
发表时间：
1998-09-08
期刊：
影响因子：
2.9
通讯作者：
Adams, JA
中科院分区：
文献类型：
--
作者：
Saylor, P;Wang, CQ;Adams, JA
发现癌蛋白v-Fps的激酶结构域的活性对镁离子浓度敏感。初始速度与游离镁浓度的曲线是双曲的，不能推断出ATP-Mg的起源，这表明酶上有两个金属螯合位点，第二个位点对催化作用不是必需的。第二种金属具有很强的活化作用，使用0.2 mM的ATP-Mg和1 mM的肽EAEIYEAIE，反应速率常数几乎增加了20倍，从0.5到8.3 s（-1）。这种速率的增加是由于在高镁浓度下ATP-Mg的表观亲和力大大增加。在游离Mg2+ 0.5和10 mM时，KATP-Mg分别为3.6和0.22 mM。根据观察到的ATP-Mg对零和无限游离金属的亲和力推断，在没有第二种金属的情况下，KATP-Mg的亲和力大于8mm，在第二种金属存在的情况下，KATP-Mg的亲和力大于0.1 mM，至少增强了80倍。相比之下，二价离子的游离水平不影响最大周转量（k(cat)），在0.5和20 mM游离Mg2+之间，对肽底物的k -m只有2倍的影响。粘度计研究表明，游离Mg2+不影响磷酰转移速率或0.5 mM以上的净产物释放，但直接影响ATP-Mg的解离常数。在第二金属离子存在和不存在的情况下，ATP-Mg的Kd分别为0.32和0.4 mM。在高镁浓度下，ATP-Mg和肽底物独立结合，而在低镁浓度（0.5 mM）下，存在显著的负结合协同作用，这表明第二金属可能有助于减少ATP-Mg和肽之间的电荷排斥。数据表明，第一个金属是足够的磷酰转移。虽然第二种金属可能对磷酰转移或产物结合有一定的影响，但它是一种专利激活剂，通过控制ATP-Mg结合发挥最小的作用。
The activity of the kinase domain of the oncoprotein v-Fps was found to be sensitive to the concentration of magnesium ions. Plots of initial velocity versus free magnesium concentration are hyperbolic and do not extrapolate to the origin at stoichiometric ATP-Mg, indicating that there are two sites for metal chelation on the enzyme and the second is nonessential for catalysis. The second metal is strongly activating and increases the reaction rate constant almost 20-fold from 0.5 to 8.3 s(-1) using 0.2 mM ATP-Mg and 1 mM peptide, EAEIYEAIE. This increase in rate is due to a large increase in the apparent affinity of ATP-Mg at high magnesium concentrations. At 0.5 and 10 mM free Mg2+, KATP-Mg is 3.6 and 0.22 mM, respectively. Extrapolation of the observed affinity of ATP-Mg to zero and infinite free metal indicates that KATP-Mg is greater than 8 mM in the absence of the second metal and 0.1 mM in the presence of the second metal, a minimum 80-fold enhancement. By comparison, free levels of the divalent ion do not influence maximum turnover (k(cat)) and have only a 2-fold effect on the K-m for the peptide substrate between 0.5 and 20 mM free Mg2+. Viscosometric studies indicate that free Mg2+ does not influence the rates of phosphoryl transfer or net product release above 0.5 mM but does affect directly the dissociation constant for ATP-Mg. The Kd for ATP-Mg in the absence and presence of the second metal ion is >32 and 0.4 mM, respectively. At high magnesium concentrations, ATP-Mg and the peptide substrate bind independently, while at lower concentrations (0.5 mM), there is significant negative binding synergism suggesting that the second metal may help to reduce charge repulsion between ATP-Mg and the peptide. The data indicate that the first metal is sufficient for phosphoryl transfer. While the second metal could have some influence on phosphoryl transfer or product binding, it is a patent activator that functions minimally by controlling ATP-Mg binding.