The GTP Mechanism

Early work on the GTP mechanism showed that the silyl groups on chain ends rapidly exchange in the presence of anionic catalysts [33, 34]. Without catalyst no exchange occurs [35]. No exchange occurred in the bifluoride catalyzed polymerization of MMA with dimethylphenylsilyl ketene acetal (Scheme 19a) in the presence of dimethyltolylsilyl fluoride [1]. However, in a similar experiment with trimethylsilyl acetate, TBA Ac, and dimethylphenylsilyl ketene acetal, complete exchange occurred within 5 min [36] (Scheme 19b). 

Certain monomers provide evidence relating to the GTP mechanism problem. Ober has shown that [3-(methacryloxy)propyl]pentamethyldisiloxane, (Scheme 23a) polymerizes very slowly under GTP conditions (100 h). Molecular weight control and MWD, however, were excellent [44]. A dissociative mechanism can explain this unusual property. The tethered siloxane group is stabilizing the enolate ends by forming a cyclic complex (Scheme 23). No mention has been made of a similar stabilizing effect for 2-trimethylsily-loxyethyl methacrylate (Scheme 23b). 

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,... 

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. 

Finally, it may be possible to obtain some limited information on the mechanisms of action of agonists from the shapes of binding curves. For example, as discussed later, the binding of some agonists is affected by guanosine triphosphate (GTP), immediately suggesting the involvement of G-proteins in the transduction mechanism. 

MAPK kinase (MAPKK). MAPK kinase itself is activated by phosphorylation by still another protein kinase, termed MAPK kinase kinase (MAPKKK). MAPK kinase kinase is activated upon interaction with a member of the Ras superfamily of small G proteins, which are bound to the plasma membrane (see Ch. 19). The exact mechanism of activation remains unknown, but it is believed that Ras and related proteins, in the activated GTP-bound form, can bind MAPK kinase kinase and thereby draw the kinase to the plasmalemma, where it is activated by as yet unknown factors, perhaps even an additional kinase, MAPK kinase kinase kinase (MAPKKKK). The mechanism governing the activation of Ras and related proteins by extracellular signals is quite complex and involves numerous Tinker proteins, for example She, Grb and Sos, that couple Ras to a variety of plasmalemma-associated growth factor-protein tyrosine kinase receptors (see Chs 20,24 and 27). 

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 ... 

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... 

This phosphotransferase [EC 2.7.2.1] catalyzes the thermodynamically favored phosphorylation of ADP to form ATP Aeq = [ATP][acetate]/ [acetyl phosphate] [ADP] = 3000). GDP is also an effective phosphoryl group acceptor. This enzyme is easily cold-denatured, and one must use glycerol to maintain full catalytic activity. Initial kinetic evidence, as well as borohydride reduction experiments, suggested the formation of an enzyme-bound acyl-phosphate intermediate, but later kinetic and stereochemicaT data indicate that the kinetic mechanism is sequential and that there is direct in-line phosphoryl transfer. Incidental generation of a metaphosphate anion during catalysis may explain the formation of an enzyme-bound acyl-phosphate. Acetate kinase is ideally suited for the regeneration of ATP or GTP from ADP or GDP, respectively. 

In the second control mechanism, exerted at a later stage, an excess of GMP in the cell inhibits formation of xanthylate from inosinate by IMP dehydrogenase, without affecting the formation of AMP (Fig. 22-35). Conversely, an accumulation of adenylate inhibits formation of adenylosuccinate by adenylosuccinate synthetase, without affecting the biosynthesis of GMP. In the third mechanism, GTP is required in the conversion of IMP to AMP (Fig. 22-34, step (T)), whereas ATP is required for conversion of IMP to GMP (step (4)), a reciprocal arrangement that tends to balance the synthesis of the two ribonucleotides. 

It has also been shown that mung beans, peas, and other plants contain a pyrophosphorylase which forms GDP-D-glucose from a-D-glucose 1-P and GTP. Based on the data obtained with enzymic plant preparations, we proposed the following mechanism for cellulose synthesis ... 

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