Is there a rate-limiting step before GTP cleavage by H-ras p21?

Abstract

A slow fluorescence change of the complex between ras p21 and the fluorescent GTP analogue 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-triphosphate (mGTP) has been postulated to be a signal arising from a step which is rate limiting and precedes the actual GTP hydrolysis reaction [Neal, S. E., Eccleston, J. F., & Webb, M. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565]. We have now shown that the rate of the fluorescence change is accelerated by GTPase-activating protein (GAP) in the same manner as that of the GTP cleavage reaction. In contrast, a faster fluorescence change of smaller amplitude seen in the complex between p21 and the uncleavable 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-O-(beta,gamma-imidotriphosphate) (mGppNHp) is not affected by GAP. The corresponding fluorescent derivative of guanosine 5'-O-(gamma-thiotriphosphate) (mGTP gamma S) shows a very slow fluorescence change after binding to p21, and this rate is also accelerated significantly by GAP. Hydrolysis of GTP gamma S is similarly slow, and it is accelerated by GAP in a similar manner to the fluorescence change. The results are interpreted to indicate that the fluorescence change occurs either at the hydrolysis step or on release of inorganic phosphate or thiophosphate but does not occur in a rate-limiting step preceding hydrolysis.

Crystal Structure of the Gtr1pGTP-Gtr2pGDP Protein Complex Reveals Large Structural Rearrangements Triggered by GTP-to-GDP Conversion

“…This finding is consistent with a previous observation that Mg 2ϩ is not required for GDP binding to RagC (4). Biochemical studies suggested that the absence of Mg 2ϩ ion can increase the rate of GDP dissociation up to 1000-fold (11,12). Apparently, the GDP binding affinity of Gtr2p is less tight than the GTP binding affinity of Gtr1p, as supported by the relatively weak electron density map and high B-factor values for GDP.…”

An Open Conformation of Switch I Revealed by the Crystal Structure of a Mg2+-free Form of RHOA Complexed with GDP

“…In the case of Rab (7), the removal of Mg 2ϩ leads to a significant increase (ϳ1000-fold) in the dissociation rate of the bound nucleotide, whereas the association rate does not change significantly (24). This phenomenon coincides with the decreased contacts observed in the current Mg 2ϩ -free form.…”

Rap-specific GTPase Activating Protein follows an Alternative Mechanism

“…to follow the kinetics of the RasGAP-catalyzed reaction (49,50). However, we did not find sufficiently large fluorescent changes using either mant-GTP or tryptophan fluorescence of a Y32W mutant of Rap1A (data not shown).…”