The Gly42 → Val mutant of G(iα1) was characterized structurally and biochemically to elucidate two important features of G(iα1)-catalyzed GTP hydrolysis. The crystal structure of the GTPγS-bound G42VG(iα1) protein demonstrates that the steric bulk of Val42 pushes the Gln204 residue into a catalytically incompetent conformation, providing a rationale for the diminished GTPase activity of this mutant. The same phenomenon may also account for the diminished GTPase activity of the homologous transforming Gly42 → Val mutation in p21(ras). Similarly, the steric bulk of the unique Ser42 residue in G(zα) may account for the comparatively slower rate of GTP hydrolysis by this G(α) subunit. The G42VG(iα1) subunit was also characterized structurally in its GDP · P(i)- and GDP-bound states, providing a unique opportunity to view three 'snapshots' of GTP hydrolysis. Hydrolysis of GTP to a transient GDP · P(i)-bound intermediate is associated with substantial conformational changes in the switch II segment of the protein. Eventual release of P(i) results in further removal of switch I from the active site and a highly mobile switch II segment. Despite their disparate biochemical properties, the structural similarity of G42VG(iα1) to the G203AG(iα1) mutant in the GDP · P(i)-bound form suggests that both mutations stabilize a conformation of the GDP-P(i)-bound protein that occurs only transiently in the wild-type protein. The structures of the GDP- bound forms of the wild-type and mutant proteins are similar.