engleski

Characterization of yeast seryl-tRNA synthetase active site mutants with improved discrimination against substrate analogues

The involvement of amino acids within the motif 2 loop of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) in serine and ATP binding has been previously demonstrated (B.Lenhard et. al., J. Biol. Chem. 272 (1997) 1136-1141.). In our attempt to analyze the structural basis for the substrate specificity and to explore further the catalytic mechanism employed by S. cerevisiae SerRS, we tested the catalytic effects of amino acid replacement at positions Lys287, Asp288 and Ala289 with purified wild-type and mutant seryl-tRNA synthetases. The alteration of these semi-conserved amino acids interferes with tRNA-dependent optimization of serine recognition. Additionally, mutated enzymes SerRS11 (K287T, D288Y, A289V) and SerRS12 (K287R) are less sensitive to inhibition by two competitive inhibitors: serine hydroxamate (serHX), an analogue of serine, and 5´-O-[N-(L-seryl)-sulfamoyl]adenosine (serAMS), a stable analogue of aminoacyl-adenylate, than the wild type enzyme. SerRS mutants also display different activation kinetics for serine and serine hydroxamate, indicating that specificity toward the substrates is modulated by amino acid replacement in motif 2 loop.

seryl-tRNA synthetase; substrate analogues; inhibition

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