Molecular mechanism of GTP hydrolysis by bovine transducin: pre-steady-state kinetic analyses.

Abstract

During the visual transduction process in rod photoreceptor cells, transducin (T) mediates the flow of information from photoexcited rhodopsin (R*) to the cGMP phosphodiesterase (PDE) via a cycle of GTP binding and hydrolysis. The pre-steady-state kinetics of GTP hydrolysis by T was studied by rapid quenching and filtration techniques in a reconstituted system containing purified R* and T. Kinetic analyses have shown that the turnover of T-bound GTP can be dissected into four partial reactions: (1) the R*-catalyzed GTP binding via a GDP/GTP exchange reaction, (2) the on-site hydrolysis of bound GTP, which leads to the formation of a T-GDP.Pi complex, (3) the release of the tightly bound inorganic phosphate (Pi) from T-GDP.Pi, and (4) the recycling of T-GDP. The R*-catalyzed GTP binding was estimated to occur in less than 1 s. In rapid acid quenching experiments, the rate of Pi formation due to GTP hydrolysis exhibited biphasic characteristics. An initial burst of Pi formation occurred between 1 and 4 s, which was followed by a slow steady-state rate. Increasing T concentration yielded a proportional increase in the burst and steady-state rate. The addition of Gpp(NH)p decreased both parameters. D2O decreased the rise of the initial burst with a kinetic isotope effect of approximately 1.7 but has no effect on the steady-state rate of Pi formation. These results indicate that the burst represents the fast hydrolysis of GTP at the binding site of T, which results in the accumulation of T-GDP.Pi complexes. The steady-state rate represents the slow release of Pi. This finding was further supported by rapid filtration experiments that monitored the formation of free Pi in solution. An initial lag time in the formation of free Pi was observed before a steady-state rate was established, indicating that the initially formed Pi was tightly bound to T. Finally, the release of GDP from T-GDP.Pi was not detected. This suggests that another cycle of GTP exchange catalyzed by R* should occur before the release of bound GDP. The rate of Pi release from T-GDP.Pi was measured under single-turnover conditions and had a half life of approximately 20 s, which was identical with the rate of deactivation of the PDE due to GTP hydrolysis by T.(ABSTRACT TRUNCATED AT 400 WORDS)