Interactions of the ras-like protein p25rab 3A with Mg2+ and guanine nucleotides

Abstract

The rab3A gene product is a 25 kDa guanine-nucleotide-binding protein which is expressed at high levels in neural tissue and has about 30% sequence similarity to ras. Purified p25rab3A has been used as substrate to examine its kinetics of nucleotide binding and hydrolysis, and to study the effects of Mg2+ on these processes. p25rab3A binds GDP and GTP similarly well, with nanomolar affinity. Mg2+ increases the affinity between p25rab3A and guanine nucleotides by 3- and 7-fold for GTP and GDP respectively, primarily by drastically decreasing the nucleotide off-rates. The Mg2+ binding affinity to p25rab3A. [alpha 32P]GDP was determined to be about 4 microns using entrapment of [alpha-32P]GDP as a measure of Mg2+ binding. At a Mg2+ concentration of 11 mM. GTPase activity was rate-limited by the GDP off-rate. Surprisingly, at a Mg2+ concentration of 80 nM. GTPase activity was comparable with that in the presence of excess Mg2+. In this case, kcat. was rate-limiting. At Mg2+ concentrations below 10 nM there was no detectable GTPase activity, indicating that Mg2+ is required for the GTPase activity of p25rab3A.

The Role of Mg2+ Cofactor in the Guanine Nucleotide Exchange and GTP Hydrolysis Reactions of Rho Family GTP-binding Proteins

“…2B), and the presence of excess EDTA (ϳ1 mM) in the binding assay had no effect on the result (data not shown), again suggesting that Mg 2ϩ cofactor is not required for the nucleotide binding to Rac1. This is in contrast to the previous observations made for other members of Ras superfamily GTPases such as Ras (19), Rab3A (20), Sec4p (21), and RalA (39), in which cases removal of bound Mg 2ϩ by EDTA treatment was found to completely abolish their nucleotide binding capabilities. We conclude that the Rho family GTPases behave differently from other Ras-related molecules in this aspect.…”

“…In the presence of Mg 2ϩ , Ras exhibits an extremely high binding affinity to the guanine nucleotides with a dissociation constant on the order of subnanomolar concentration (18,19,40). In the cases of Rab3A and related Sec4 GTPases, removal of Mg 2ϩ by EDTA treatment drastically increases the off rate of bound nucleotides and completely abolishes GTP␥S binding capability (20,21). The intrinsic GTPase activity of Ras and Rab3A became undetectable when Mg 2ϩ cofactor was removed (19,20), indicating that the bivalent ion is absolutely required for GTP hydrolysis in the respective GTPase reactions.…”

“…In the cases of Rab3A and related Sec4 GTPases, removal of Mg 2ϩ by EDTA treatment drastically increases the off rate of bound nucleotides and completely abolishes GTP␥S binding capability (20,21). The intrinsic GTPase activity of Ras and Rab3A became undetectable when Mg 2ϩ cofactor was removed (19,20), indicating that the bivalent ion is absolutely required for GTP hydrolysis in the respective GTPase reactions. In the guanine nucleotide exchange reactions of Ras and ARF, the specific GEFs Sos and ARNO appear to promote the dissociation of GDP in part through destabilizing bound Mg 2ϩ from the respective GTPases (22)(23)(24).…”

RhoB can adopt a Mg2+ free conformation prior to GEF binding

“…3(A)]. In the absence of Mg 2þ , GTP hydrolysis was detected albeit at a comparatively slow rate of 0.067 min 21 . In the presence of excess Mg 2þ , the release of gPi increased with a turnover rate of 0.514 min 21 .…”

“…In the absence of Mg 2þ , GTP hydrolysis was detected albeit at a comparatively slow rate of 0.067 min 21 . In the presence of excess Mg 2þ , the release of gPi increased with a turnover rate of 0.514 min 21 . The determined rate of RhoB hydrolysis is similar to RhoA with very slow hydrolysis under steady-state conditions in the absence of GAP proteins, unlike the high rates of hydrolysis determined for Cdc42 which is believed to be enhanced by an internal arginine finger.…”