The Role of Mg2+ Cofactor in the Guanine Nucleotide Exchange and GTP Hydrolysis Reactions of Rho Family GTP-binding Proteins

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg²⁺ cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg²⁺ for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg²⁺ similarly, with dissociation constants in the submicromolar concentration. The presence of Mg²⁺, however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg²⁺, and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg²⁺, suggesting that one role of GEF is to displace bound Mg²⁺ from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg²⁺, indicating that the GEF-catalyzed nucleotide exchange involves a Mg²⁺-independent as well as a Mg²⁺-dependent mechanism. Although Mg²⁺ is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4–10-fold upon binding to Mg²⁺, and<i>k</i> _cat values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg²⁺ was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg²⁺ cofactor. These studies directly demonstrate a role of Mg²⁺ in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg²⁺, whereas RhoGAP utilizes the Mg²⁺ cofactor to achieve high catalytic efficiency and specificity.