Initiators for GTP

Ester enolates operate as both catalysts and initiators for GTP... 

Binding of the ligand-receptor complex initiates a conformational change in the G protein that allows exchange of GDP for GTP on its a subunit, leading to dissociation of the a and Py subunits of the G protein (Figure 14-1). 

Figure 14-1. Signaling via G protein-coupled receptors. Ligand binding to its cell-surface receptor initiates interaction of the receptor with the heterotrimeric G protein for which it is specific. A conformational change in the G protein brought about by binding of the ligand-receptor complex promotes exchange of GDP for GTP. The activated Gd-GTP dissociates from the Gp complex and both can interact with effectors, which carry on the signal to the mechanism that implements the cellular response.

Regulation of protein synthesis in the rabbit reticulocyte. The vast majority of the protein synthesized in the rabbit reticulocyte is hemoglobin. The gross rate of protein synthesis in the reticulocyte is controlled indirectly by the concentration of heme. Heme inactivates a kinase that would otherwise inactivate the initiation complex involving eIF-2 and eIF-2B. The kinase phosphorylates the eIF-2 factor, making it impossible for the eIF-2-eIF-2B complex to exchange GDP for GTP. 

Acrylates polymerize two orders of magnitude faster than methacrylates by anion catalyzed GTP however, the polymerization dies at about 10,000 MW. During the anion catalyzed polymerization of acrylates the silyl ketene acetal end groups migrate to internal positions. These ketene acetals are too hindered to act as initiators for branch formation [9]. 

To obtain polymer with low MWDs in a living polymerization the rate of initiation must be faster or similar to the rate of propagation. This can suitably be accomplished if the structure of the initiator is the same as that of the growing chain end. For GTP this is a silyl ketene acetal (Scheme 9). The... 

Ester Enolates Operate as Both Initiators and Catalysts for GTP... 

In a study that in itself almost lays to rest the associative mechanism for GTP, Quirk generated an ester enolate by addition of TBA 9-methylfluo-renide to MMA and used it as a catalyst for GTP of MMA initiated by MTS at 50 °C. The conversion was quantitative, the MWD was in the 1.2 range, and the molecular weight was controlled by the ratio of MTS to monomer [6]. In similar experiments without the MTS, conversions of only 14-52% were obtained, the MWD was broad and molecular weight control was lost. 

The fact that known anionic initiators for MMA can act as catalysts for GTP and the need for low amounts of catalysts in itself nearly puts to rest the associative mechanism. Seven of the other factors support the dissociative process. Except for the low temperature exchange studies, none supports the associative mechanism. Based on the lack of exchange of added silyl fluoride with silyl ketene acetal ends it looks like fluoride and bifluoride catalysts operate by irreversible generation of ester enolate chain ends [1] (Scheme 19b). On the other hand carboxylate catalysts appear to operate by reversible generation of ester enolate ends as evidenced by rapid exchange of silyl acetate with silyl ketene acetal ends [36] (Scheme 19c). 

In addition to the enzyme the reaction mixture contained Hepes buffer, IMP, GTP, MgCl, and creatine phosphate and phosphocreatine kinase (a regeneration system for GTP). The reaction was initiated by adding aspartic acid. Samples were removed at intervals, and the reactions were terminated by direct injection onto the HPLC column. Figure 9.113 shows chromatograms of samples removed at 0,5, and 10 minutes of incubation. The disappearance of IMP and GTP and the appearance of GTP and sAMP can be noted. 

Silyl ketene acetals are relatively stable species and require activation by a catalyst in order to initiate polymerization of a,3-unsaturated monomers. Numerous catalysts for GTP polymerizations have been examined " and these studies have revealed that bifluorides and bioxyanions such as tris(dimethylamino) sulfonium bifluoride (TASHF2) and tetra-n-butyl ammonium bibenzoate (TBABB), respectively, afford optimum polymerization characteristics. Note. Bifluoride based catalysts are not soluble in tetrahydrofuran (THF) and thus acetonitrile is used as the solvent in these cases in general TBABB is the optimum catalyst as it is readily soluble in THF and affords better control of molecular weight, conversion, and polydispersity.) GTP has been shown to be robust < 1.1) and is compatible with... 

The possible role of guanosine 3 -pyrophosphate-5 -triphosphate (30) in protein synthesis in E. coli has been examined. Although it can substitute for GTP reactions catalysed by initiation factor (IF)2 and elongation factor... 

Once correct positioning occurs, and the match is made between the anticodon of the met-tRNA and the start codon, the GTP molecule bound to eIF-2 is hydrolyzed in a reaction promoted by eIF-5. The physical nature of this reaction remains controversial. There are thought to be two forms of eIF-5 with molecular masses of 125 kDa and 60 kDa without, however, any differences in their biological properties (Hershey, 1991 Merrick, 1992). The hydrolysis of GTP causes the release of the initiation factors from the surface of the 40S ribosomal subunit, and allows attachment of the 60S subunit by triggering the release of eIF-6 from it. The formation of the SOS initiation complex culminates in the formation of the first peptide bond at the ribosomal P site. The initiation factor eIF-4D is required for the formation of the first peptide bond. eIF-4D is a small protein (about 16 kDa), and has a unique posttranslational modification of its lysine-50 residue by the action of a polyamine, spermidine, to form a hypusine residue essential for its activity (Hershey, 1991 Merrick, 1992). Furthermore, in order to allow efficient and catalytic use of eIF-2 after GTP hydrolysis and its release from the complex, another factor, eIF-2B, facilitates the exchange of eIF-2 bound GDP for GTP. 

In the initial screen, one compound (given the name GTP Green, Fig. 6-8) displayed fluorescence enhancement ( tum-on ) upon GTP binding and was favorably photostable compared to other candidates [76], Prior to the discovery of GTP Green, the only other GTP sensor (rationally designed) was a turn-off sensor [78], GTP Green displayed a red-shifted 80-fold fluorescence enhancement (Tern = 540 nm) in the presence of GTP and only < two-fold increases with the other nucleotide triphosphates ATP, TTP, and UTP. This compound was also found to be selective for GTP over the mono- and diphosphate homologs. 

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