Hydrolysis of GTP, bound to members of the Gprotein superfamily, terminates their downstream signaling activity. A conserved glutamine serves a critical role in this pivotal guanosine triphosphatase (GTPase) reaction. However, the role of the catalytic glutamine in GTP hydrolysis is still not well understood. We have employed substrate-assisted catalysis to probe the catalytic mechanism of Gscx using GTP analogues. These GTP analogues, each having different functional groups, were designed to support or refute particular putative GTPase mechanisms. We have found that a hydrogen donor group, in close proximity to the T-phosphate of GTP, is necessary and sufficient to substitute for the function of the catalytic glntamine in the GTPase reaction.