The role of the metal ion in the p21 ras catalysed GTP-hydrolysis: Mn 2+versus Mg 2+

Abstract

GTP and ATP hydrolysing proteins have an absolute requirement for a divalent cation, which is usually Mg 2+, as a cofactor in the enzymatic reaction. Other phosphoryl transfer enzymes employ more than one divalent ion for the enzymatic reaction. It is shown here for p21 ras, a well studied example of GTP hydrolysing proteins, that the GTP-hydrolysis rate is significantly faster if Mg 2+ is replaced by Mn 2+, both in the presence or absence of its GTPase-activating protein Ras-GAP. This effect is not due to a different stoichiometry of metal ion binding, since one metal ion is sufficient for full enzymatic activity. To determine the role of the metal ion, the crystal structure of p21(G12P)·GppCp complexed with Mn 2+ was determined and shown to be very similar to the corresponding p21(G12P)·GppCp·Mg 2+ structure. Especially the coordination sphere around the metal ions is very similar, and no second metal ion binding site could be detected, consistent with the assumption that one metal ion is sufficient for GTP hydrolysis. In order to explain the biochemical differences, we analysed the GTPase reaction mechanism with a linear free energy relationships approach. The result suggests that the reaction mechanism is not changed with Mn 2+ but that the transition metal ion Mn 2+ shifts the p K a of the γ-phosphate by almost half a unit and increases the reaction rate due to an increase in the basicity of GTP acting as the general base. This suggests that the intrinsic GTPase reaction could be an attractive target for anti-cancer drug design. By using Rap1A and Ran, we show that the acceleration of the GTPase by Mn 2+ appears to be a general phenomenon of GTP-binding proteins.