Novel guanine nucleotide analogues have been used to investigate the role of Mg(2+) in nucleotide release and binding with the small G protein rac. The fluorescent analogues have 7-(ethylamino)-8-bromocoumarin-3-carboxylic acid attached to the 3'-position of the ribose via an ethylenediamine linker. This modification has only small effects on the interaction with rac. There are large fluorescence changes on binding of the triphosphate to rac, on hydrolysis, and then on release of the diphosphate. Furthermore, the fluorescence is sensitive to the presence of Mg(2+) in the active site. Using this signal, it was shown that, for a variety of conditions, the nucleotides dissociate by a two-step mechanism. Mg(2+) is released first followed by the nucleotide. With the diphosphate, Mg(2+) is fast and nucleotide release slow. For the fluorescent GMPPNP analogue, the rate of dissociation is limited by Mg(2+) release. In the latter case, Mg(2+) binds tightly with a K(d) of 61 nM, whereas for the diphosphate the K(d) is 11 microM (30 degrees C, pH 7.6).