Abstract：
The wildtype H-Ras protein functions as a molecular switch in a variety of cell signaling pathways, and mutations to key residues result in a constitutively active oncoprotein. However, there is some debate regarding the mechanism of the intrinsic GTPase activity of H-Ras. It has been hypothesized that ordered water molecules are coordinated at the active site by Q61, a highly transforming amino acid site, and Y32, a position that has not previously been investigated. Here, we examine the electrostatic contribution of the Y32 position to GTP hydrolysis by comparing the rate of GTP hydrolysis of Y32X mutants to the vibrational energy shift of each mutation measured by a nearby thiocyanate vibrational probe to estimate changes in the electrostatic environment caused by changes at the Y32 position. We further compared vibrational energy shifts for each mutation to the hydration potential of the respective side chain and demonstrated that Y32 is less critical for recruiting water molecules into the active site to promote hydrolysis than Q61. Our results show a clear interplay between a steric contribution from Y32 and an electrostatic contribution from Q61 that are both critical for intrinsic GTP hydrolysis.
摘要：
野生型H-Ras蛋白在多种细胞信号通路中起分子开关的作用,和关键残基的突变导致组成型活性癌蛋白。然而,关于H-Ras的固有GTP酶活性的机制存在一些争论。据推测，有序水分子在活性位点被Q61协调，Q61是一个高度转化的氨基酸位点，和Y32，这个职位以前没有被调查过。这里,我们通过将Y32X突变体的GTP水解速率与附近的硫氰酸盐振动探针测量的每个突变的振动能移进行比较，以估计由Y32位置的变化引起的静电环境的变化，来检查Y32位置对GTP水解的静电贡献。我们进一步比较了每个突变的振动能移与相应侧链的水合潜力，并证明了Y32对于将水分子募集到活性位点以促进水解的重要性不如Q61。我们的结果表明，Y32的空间贡献和Q61的静电贡献之间存在明显的相互作用，这两者对于固有的GTP水解都至关重要。
