GTP hydrolysis Mechanism

Figure 4 Residues within Ga that are critical to the GTP hydrolysis mechanism include arginine-178 and threonine-181 from switch I and glutamine-204 from switch II (colored as in Fig. 2 and numbered as in Ga i coordinates are from PDB record IGFI). Magnesium ion is highlighted in yellow. The planar anion aluminum tetrafluoride, which mimics the y-phosphate leaving group in the hydrolysis reaction, is depicted in metallic red. Note the position of serine-47, the target of phosphorylation by the Y. pestis protein kinase YpkA.

All the residues involved in important functions in the catalytic mechanism are strictly conserved in all homologous GTPases with one notable exception. Ras does not have the arginine in the switch 1 region that stabilizes the transition state. The assumption that the lack of this catalytically important residue was one reason for the slow rate of GTP hydrolysis by Ras was confirmed when the group of Alfred Wittinghofer, Max-Planck Institute,... 

Figure 13.12 A concerted mechanism for GTP hydrolysis by Ga in which transfer of a proton to the y phosphate is coupled to deprotonation of the attacking water by Gin 200. (Adapted from J. Sondek et al., Nature 372 276-279, 1994.)...

In summary, structural studies of Ras and Gq with GTP-yS and a transition state analog have illuminated the catalytic mechanism of their GTPase activity, as well as the mechanism by which GTP hydrolysis is stimulated by GAP and RGS. In addition, these structural studies have shown how tumor-causing mutations affect the function of Ras and Gq. 

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]. 

Here biophysical methods contributed a reductionistic approach. By analyzing a limited number of actors under well defined conditions, the mechanisms of nucleotide exchange, intrinsic and stimulated GTP-hydrolysis, effector binding, and membrane attachment have been elaborated to present a comprehensive model. 

The purpose of the study was theoretical investigation, at the atomic level, of the mechanism of the GTP hydrolysis catalyzed by the Cdc42-GAP enzymatic complex ... 

Figure 5 Kinetic mechanism of aminoacyl-tRNA selection by the ribosome. The aminoacyl-tRNAs are delivered to the ribosome in the form of a ternary complex with EF-Tu-GTP. Incorrect aminoacyl-tRNAs can either dissociate as a ternary complex in the initial selection phase or later as free aminoacyl-tRNA in the proofreading phase. The two selection phases are separated through the irreversible GTP hydrolysis by EF-Tu. Discrimination against incorrect tRNAs is achieved by increased dissociation rate constants (/r 2 and kj) as well as decreased forward rate constants (ks and ks) compared to cognate tRNAs.

The kinetic mechanism of aminoacyl-tRNA selection demonstrates that the forward steps of EF-Tu GTPase activation and accommodation are crucial for high fidelity. How can this observation be explained on a structural level. Unfortunately, we have high-resolution structures only of the ribosome prior to A-site binding and of the tRNAs bound to the ribosome after accommodation. Of the intermediate states, only the low-resolution structure of a ternary complex EF-Tu—GTP—aminoacyl-tRNA stalled after GTP hydrolysis has been determined by cryo-EM (Figure These studies revealed that EF-Tu interacts with the... 

Figure 8 EF-G-catalyzed translocation of the tRNA-mRNA complex within the ribosome, (a) Hybrid state formation and intersubunit rotation. Upon peptide bond formation, the ribosome fluctuates between the classical state and a hybrid state. In the classical state, the tRNAs are bound to the A and P site on both the 308 and 508 subunit. In the hybrid state, the anticodons remain in the A and P site on the 308 subunit whereas the acceptor ends move into the P and E site on the 508 subunit, respectively. 8imultaneously to hybrid state formation, the 308 subunit rotates relative to the 508 subunit as shown on the right site, (b) Kinetic mechanism of EF-G-catalyzed translocation. Upon GTP hydrolysis, unlocking occurs through a ribosomal rearrangement. Only subsequently, tRNA and mRNA movement as well as dissociation of the inorganic phosphate from EF-G take place.

MICROTUBULE ASSEMBLY/DISASSEMBLY KINETICS. Cellular microtubules must undergo turnover, and nucleotide hydrolysis appears to play a central role in priming microtubules for their eventual disassembly. Two fundamentally different assembly/disassembly mechanisms persist during what has been termed steady-state polymerization both rely on GTP hydrolysis to provide a source of Gibbs free energy to sustain the steady-state condition . ... 

Fig. 5.17. In-line attack in GTP hydrolysis. Hydrolysis of GTP takes place via an in-tine attack of a water molecnle at the y-phosphate. The reaction passes through a pentavalent transition state in which the ligands of the y-phosphate adopt a trigonal bipyramidal configuration. The mechanism by which the water molecule is activated for the attack on the y-phosphate is not shown in the figure. Possible mechanisms are presented in Fig. 5.18.

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