Mechanism of GTP hydrolysis

It is generally assumed that hydrolysis of the y-phosphate bond proceeds via an Sn2 mechanism, as shown in Fig. 5.17. The hydrolysis proceeds by an in-line attack of a water molecule on the y-phosphate, in which the GDP residue is displaced from the y-phosphate. 

In the postulated transition state, the y-phosphorus atom is penta-coordinated, whereby the ligands are configmed in the form of a triagonal bipyramid. Mg is indispensable for the catalysis it is needed for binding of substrate and product, as well as for the catalysis itself. Activation of the water molecule for nucleophilic attack at the y-phosphate requires involvement of side groups of the protein in the sense of a general base catalysis. 

For the structural determination of the activated form of the a-subunit, use of A1F4 as a ligand was of great importance. AIF4 is an activator of GDP-bound a-sub-units and due to this characteristic — in addition to the bacterial toxins mentioned above — is often used for detection of G-proteins and for their structural characterization. In the presence of A1F4 , permanent activation of the G-protein is observed G GDP is fixed by binding of AIF4 in a conformation that permits activation of the effector molecule. 

In comparison to the Ras protein (see Chapter 9) and bacterial EF-Tu, there are differences in the details of the residues involved. The general hydrolysis mechanism, as formulated in Fig. 5.17, also applies to these proteins, however. 

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The slow intrinsic GTP-hydrolysis by Ras can be accelerated by orders of magnitudes upon interaction with its GTPase activating proteins (GAPs). The cytosolic RasGAPs pl20RasGAP and NF1 (neurofibromin) are the main factors, which ensure that cellular Ras exists predominantly in its inactive GDP-complexed state [26]. The mechanism of GTP-hydrolysis and its stimulation has been the object of controversial disputes for over a decade. 

Time-resolved X-ray crystallography has brought further insight into the mechanism of GTP hydrolysis and has confirmed former conclusions. With this method it was possible to obtain the structure of Ras bound to GTP, rather than non-hydrolyzable analogs like GppNHp or GppCH2p, and, moreover, to follow the structural changes in Ras due to GTP hydrolysis [203]. Initially, Ras is bound... 

This is taken as further evidence for an associative (SN2-like) mechanism of GTP hydrolysis. More details about this debate are reviewed by [210]. 

IV. Signal Termination The Mechanism of GTP Hydrolysis and Conformational Deactivation... 

Ting, T. D., and Ho, Y.-K. (1991). Molecular mechanism of GTP hydrolysis by bovine transducin Pre-steady-state kinetic analyses. Biochemistry 30, 8996-9007. 

A great deal of structural information about G proteins is known from x-ray crystallographic studies, providing insight into GTP-mediated conformational changes in Ga, subunit interactions with effector proteins, and the mechanism of GTP hydrolysis. By contrast, relatively little structural information is known about the interaction between the receptor and G protein and how this interaction leads to GDP release. After an overview of the structure of heptahelical receptors and heterotrimeric G proteins, this chapter will discuss the current models of the receptor-G protein complex and proposed mechanisms for receptor-catalyzed nucleotide exchange. 

The observation that AlFx complexes activate G-proteins has been therefore useful for the study of the mechanism of G-protein activation, for understanding the biochemical mechanism of GTP hydrolysis, and for the elucidation of three-dimensional structures of several GTPases, including the discovery of the GTPase-activating proteins. 

Sprang SR. Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. Science 1994 265 1405-1412. 

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