Graham, Deborah Louise; (1999) The catalytic mechanism of Rho GTPase-activating protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Rho guanine nucleotide binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). Rho-GAPs contain a single conserved arginine residue within their catalytic domain, that has been proposed to be involved in the catalytic GTP hydrolysis mechanism. The role of this arginine in RhoGAP has been elucidated by mutagenesis followed by determination of catalytic and equilibrium binding constants. The turnover numbers for wild-type, R282A and R282K RhoGAPs were 5s-1 0.025s-1 and 0.010s-1 respectively. Thus, the function of this arginine could not be replaced by lysine or alanine. Nevertheless, the R282A mutation had a minimal effect on binding affinity of RhoGAP to Rho.GMPPNP, which confirms the importance of the arginine residue for catalysis as opposed to formation of the protein:protein complex. Aluminium fluoride enhances binding of the GDP-bound form of small G proteins to their GAPs. These complexes have been interpreted as being analogues of the transition state of the hydrolytic reaction. The catalytic arginine has been shown to have a role in coordinating and stabilising these analogues. In the presence of aluminium fluoride, R282A RhoGAP binds almost as well as wild-type to Rho.GDP, demonstrating that Arg 282 is not required for the interaction. In addition, the affinity of wild-type RhoGAP for the triphosphate form is similar to that for Rho.GDP with aluminium fluoride. These observations suggest that proposed transition state analogue formed in solution does not have the anticipated properties of the true transition state. Additionally, it has been observed that, in the absence of aluminium, magnesium can support fluoride-mediated complex formation between Rho.GDP and RhoGAP. Quantitative information on the requirements for both aluminium- and magnesium-dependent fluoride-mediated effects has been obtained. This has led to a high resolution crystal structure of Rho.GDP complexed with RhoGAP and magnesium fluoride, with the magnesium adopting an unusual configuration.

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