Özet:
Elongation factor Tu is an essential enzyme responsible for delivering aminoacyltRNA's to ribosomal A-site during protein synthesis. In its active GTP-bound state, it forms an EF-Tu · GTP · aminoacyl-tRNA ternary complex whereas it is inactive in the GDP-bound state. One very unstable and dynamic structural element in the nucleotide binding domain of EF-Tu is the Switch I region, whose structure changes from a β-sheet to an α-helix during the transition from the GTP- to GDP-bound state of EF-Tu. In this thesis, the role of the universally conserved Asp50 residue in the Switch I region in Escherichia coli EF-Tu was studied. Mutants of Asp50 to alanine and asparagine were made in EF-Tu and mutant EF-Tu’s were studied in their interactions with GDP, GTP and aminoacyl- tRNA, as well as in their activity in poly(Phe) synthesis, GTP hydrolysis and dipeptide formation using in vitro kinetic assays. D50A and D50N mutants of EF-Tu were comparable to wild-type EF-Tu with respect to GDP binding and GDP/GTP nucleotide exchange rates. However, the Asp50 mutants have significantly reduced affinity for aminoacyl- tRNA. When analyzed in the presence of high excess of aminoacyl-tRNA, Asp50 mutants were comparable to wild-type EF-Tu in their activity in poly(Phe) synthesis. On the other hand, pre-steady state measurements of GTP hydrolysis and peptide bond formation indicated that the amount of GTP hydrolyzed by the mutants are significantly reduced compared to wild type EF-Tu, even though the rate of GTP hydrolysis was not altered. The results obtained from this work suggest that Asp50 is crucial for a tight GTP conformation of EF-Tu. When Asp50 is mutated, the enzyme easily switches to GDP conformation, explaining the reduced affinity of the Asp50 mutants in aminoacyl-tRNA binding. On the other hand, when ternary complexes with Asp50 mutants reach to the A-site on the ribosomes, some mutant EF-Tu's may switch to their GDP-like conformation, therefore, releasing the aminoacyl-tRNA, which proceed to peptide bond formation without GTP hydrolysis.
