SIGNIFICANCE OF THR182 IN THE NUCLEOTIDE-EXCHANGE AND GTP-HYDROLYSIS REACTIONS OF THE ALPHA-SUBUNIT OF GTP-BINDING PROTEIN G(I2)

The crystal structures of the GTP- and GDP-bound alpha subunits of het erotrimeric GTP-binding proteins were recently determined, and a conse rved Thr residue in the G2 (linker 2) region of the alpha subunits, wh ich corresponds to Thr182 in G(12)alpha, was deduced to interact with the gamma-phosphate of GTP and Mg2+, To investigate biochemically the significance of the Thr residue, we produced a mutant G(12)alpha, in w hich Thr182 was substituted for Ala (T182A), in Escherichia coli, The rate of guanosine 5'-(gamma-thio)tri-phosphate (GTP gamma S) binding t o T182A was higher than that to the wild-type G(12)alpha, especially w ith a high concentration (10 mM) of Mg2+. The rate of dissociation of bound GDP from T182A was also much faster than that from the wild-type with the high Mg2+ concentration, Moreover, T182A had much lower GTPa se activity than the wild-type, like the gip mutant (R179C) of G(12)al pha found in human endocrine tumors, The ability of T182A to interact with beta gamma subunits and membrane-bound receptors was the same as that of the wild-type alpha subunit, T182A could take on a GTP-bound a ctive conformation, as judged from its sensitivity to tryptic digestio n, These results indicated that Thr182 plays an important role not onl y in the Mg2+-sensitive GDP-GTP exchange reaction but also in the GTPa se activity of G(12)alpha. The T182A mutant of G(12)alpha, characteriz ed by the faster GDP release and the slower GTP hydrolysis, would be a novel mutant that retains the ability to interact with receptors and beta gamma subunits.