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Biophysical and biochemical characterization of yeast tRNA nucleotidyltransferase variants


Biophysical and biochemical characterization of yeast tRNA nucleotidyltransferase variants

Rahman, Mohammed Samiur (2017) Biophysical and biochemical characterization of yeast tRNA nucleotidyltransferase variants. Masters thesis, Concordia University.

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The enzyme ATP(CTP):tRNA-specific tRNA nucleotidyltransferase adds cytidine-cytidine-adenosine (CCA) to the 3’ end of eukaryotic tRNAs during their maturation. This CCA sequence plays a vital role in aminoacylation and hence in protein synthesis. In yeast, this enzyme is defined as a Class II tRNA nucleotidyltransferase due to the presence of five conserved N-terminal motifs (A to E). Based on the available crystal structures of related tRNA nucleotidyltransferases, specific functions have been assigned to each of these motifs. We previously have shown that mutations in motif C that reduce enzyme activity can be overcome by a mutation in motif A that restores this activity. Here we explore the roles of two acidic residues (glutamate 189 and aspartate 190) found within motif C and one residue (arginine 64) found in motif A to understand better the role of motif C and the potential interactions between motifs A and C.

Site-directed mutagenesis was used to change arginine 64 (to tryptophan), or glutamate 189 (to glutamine, lysine, alanine or phenylalanine) or aspartate 190 (to alanine or phenylalanine) alone, or in combination with the arginine 64 tryptophan substitution and the effects of these amino acid alterations on enzyme structure and function were studied. Biophysical analyses (circular dichroism and fluorescence spectroscopy and thermal denaturation experiments) suggest no major changes in structure in almost all of the variants tested. Kinetic analysis revealed no alterations in substrate binding (Km), but a large drop in turnover number (kcat) for the 189 and 190 variants (but not the arginine 64 variant). The reduced activity in the 189 and 190 variants is alleviated when accompanied by the change of arginine 64 to tryptophan, which also suppresses the temperature-sensitive phenotype. Taken together these data suggest that arginine 64 is not required for enzyme activity unlike glutamate 189 and aspartate 190. Moreover, they suggest an interaction between motifs A and C, and that motif C plays a role in accommodating and orienting the substrates to promote catalysis involving motif A.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Rahman, Mohammed Samiur
Institution:Concordia University
Degree Name:M. Sc.
Date:December 2017
Thesis Supervisor(s):Joyce, Paul
ID Code:983427
Deposited On:11 Jun 2018 03:45
Last Modified:23 Jan 2020 01:00


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