金属离子对GTP结合蛋白Cdc42Hs的内源性GTP酶活性的影响

EFFECTS OF METAL IONS ON THE INTRINSIC GTP-HYDROLYSIS ACTIVITIES OF SMALL GTPase Cdc42Hs

不同的金属离子会对GTP结合蛋白Cdc42Hs的内源性GTP水解酶活性产生不同的影响。相对于生理条件下的辅基Mg2 + 而言 ,Mn2 + 对Cdc42Hs酶活力有所激活 ,表现在饱和浓度时 ,实验曲线指数项的表观速率常数kobs 有2倍左右的提高 ,而其稳态反应速度要低于Mg2 +。Mg2 +和Mn2 +的实验曲线在本质上没有什么差别 ,都是一个指数项和一个一次项的叠加 ,说明Mn2 + 和Mg2 + 以相似的机制结合于Cdc42Hs。在Ca2 + 存在时 ,实验曲线无明显的指数项出现 ,Ca2 + 的存在仅使稳态反应速度有所降低 ,说明Ca2 + 以不同于Mg2 + 和Mn2 + 的机理与Cdc42Hs结合。随着Mg2 +和Mn2 +离子浓度的增大 ,指数项的表观速率常数kobs 逐步升高 ,稳态反应速度υs 逐渐降低。进一步的动力学模型分析得到了这一反应的微观动力学常数和Mg2 +、Mn2 +与蛋白的结合常数。

Different metal ions have different effects on the intrinsic GTP-hydrolysis activity of Cdc42Hs. Compared with Mg2+, the physiological cofactor, Mn2+ has a slight activation effect on GTP-hydrolysis activity. The kobs of the exponential phase of the experiment curve of Mn2+ is two-times higher than that of Mg2+ under saturated condition. And the velocity of the steady state is lower than that of Mg2+. Essentially, there is no difference between the experiment curves of Mn2+ and Mg2+. Both of them have an exponential phase and linear phase, which indicate that Mn2+ and Mg2+ adopt the same mechanism in binding to Cdc42Hs. In the case of Ca2+, there is no detectable exponential phase in the experiment curve. The presence of Ca2+ only slows down the velocity of the steady state. This indicates that the binding mechanism of Ca2+ to Cdc42Hs is different from that of Mn2+ and Mg2+. With the increasing of Mn2+ and Mg2+ concentration, the kobs of the exponential phase increase, and the velocity of the steady state decrease. A detail kinetic analysis deduces the microscopic kinetic constants of the hydrolysis reaction and the disassociate constants of the metal ions with the protein.