engleski

Yeast seryl-tRNA synthetase: tRNA-dependent amino acid discrimination and anticooperative binding of tRNASer

Aminoacyl-tRNA synthetases catalyze formation of the ester bond between cognate pair of amino acid and transfer RNA (tRNA). The aminoacylation of tRNA is achieved in a two-step reaction. ATP is firstly used to activate the amino acid via enzyme-bound aminoacyl-adenylate intermediate, followed by the transfer of activated acyl group to 3'-end of tRNA. Presence of tRNASer is not prerequisite for seryl-adenylate formation in the activation step catalyzed by seryl-tRNA synthetase (SerRS) from yeast Saccharomyces cerevisiae. We have previously observed that yeast SerRS displays a higher affinity toward serine in the overall aminoacylation assay than in the amino acid activation reaction lacking tRNA. Here we present direct kinetic proof that binding of tRNASer induces local conformational change of SerRS leading to the optimized serine binding site, but without influence on ATP binding. Moreover, SerRS mischarges theronine to lesser extent in the presence of tRNASer. Our kinetic experiments show that equimolar concentration of tRNASer per dimeric enzyme induces a complete rearrangement of amino acid binding site, as judged by full range change of the Km value for serine. The stoichiometry of tRNASer binding on SerRS was explored by native polyacrylamide electrophoresis performed under the equilibrium and non-equilibrium conditions. SerRS:tRNASer noncovalent complex was detected by both electrophoretic methods and evaluated Kd value was about 0.04 mM. SerRS:tRNASer2 complex, previously detected by MALDI-MS, was electrophoretically detected only after stabilization by covalent cross-linking. It seems that binding of the first tRNASer induces conformational optimization of SerRS for efficient and accurate synthesis of seryl-adenylate on the account of the affinity for the second tRNA. Given results may suggest that SerRS with one bound tRNASer is physiologically active complex in seryl-tRNASer formation.

aminoacyl-tRNA synthetases; amino acid discrimination; tRNA

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