Small GTPases function as binary switches, taking on different functional states depending on whether they are bound to GTP or GDP. Maintenance of these nucleotide-dependent states at a cellular level is accomplished through the action of different regulatory proteins, which function to either increase nucleotide exchange, suppress nucleotide exchange, or catalyze GTP hydrolysis. For the small GTPases KRAS, NRAS, and HRAS (collectively RAS), several somatic mutations have been identified that alter this GTPase cycle to favor GTP binding, resulting in the functional activation of these proteins. Many of these mutations cause KRAS, NRAS, and HRAS to become deficient in GTP hydrolysis, promote enhanced rates of nucleotide exchange, or promote a combination of both. Here, however, we examine in detail the chemical mechanism of an active site mutation that converts RAS into a GTP-dependent transferase, allowing RAS proteins to undergo autophosphorylation. Contrary to our expectations, and in contrast to GTP hydrolysis, autophosphorylation appears dependent on active site opening to dynamically optimize positioning of Thr59 for nucleophilic attack against γ-phosphate of GTP. We argue that this reaction, allowed by the A59T mutation, has important implications for the evolution of GTPase-regulated GTP hydrolysis.
Johnson et al. (Mon,) studied this question.