Catalysis of Tyrosyl-Adenylate Formation by the Human Tyrosyl-tRNA Synthetase

Although the active site residues in theBacillus stearothermophilus and human tyrosyl-tRNA synthetases are largely conserved, several differences exist between the two enzymes. In particular, three amino acids that stabilize the transition state for the activation of tyrosine in B. stearothermophilu...

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Bibliographic Details
Published in:The Journal of biological chemistry 2002-04, Vol.277 (17), p.14812-14820
Main Authors: Austin, Joseph, First, Eric A.
Format: Article
Language:English
Subjects:
Adenosine Monophosphate - analogs & derivatives
Adenosine Monophosphate - biosynthesis
Adenosine Monophosphate - metabolism
Amino Acid Sequence
Bacillus stearothermophilus
Binding Sites
Catalysis
Geobacillus stearothermophilus - enzymology
Humans
Kinetics
Molecular Sequence Data
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Spectrometry, Fluorescence
Tyrosine - analogs & derivatives
Tyrosine - biosynthesis
Tyrosine - metabolism
Tyrosine-tRNA Ligase - chemistry
Tyrosine-tRNA Ligase - metabolism
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Summary:Although the active site residues in theBacillus stearothermophilus and human tyrosyl-tRNA synthetases are largely conserved, several differences exist between the two enzymes. In particular, three amino acids that stabilize the transition state for the activation of tyrosine in B. stearothermophilus tyrosyl-tRNA synthetase (Cys-35, His-48, and Lys-233) are not present in the human enzyme. This raises the question of whether the activation energy for the tyrosine activation step is higher for the human tyrosyl-tRNA synthetase than for the B. stearothermophilus enzyme. In this paper, we demonstrate that intrinsic fluorescence changes can be used to monitor the pre-steady state kinetics of human tyrosyl-tRNA synthetase. In contrast to theB. stearothermophilus enzyme, catalysis of the tyrosine activation step is potassium-dependent in the human tyrosyl-tRNA synthetase. Specifically, potassium increases the forward rate constant for tyrosine activation 260-fold in the human tyrosyl-tRNA synthetase. Comparison of the forward rate constants for catalysis of tyrosine activation by the human and B. stearothermophilus enzymes indicates that despite differences in their active sites and the potassium requirement of the human enzyme, the activation energies for tyrosine activation are identical for the two enzymes. The results of these investigations suggest that differences exist between the active sites of the bacterial and human tyrosyl-tRNA synthetases that could be exploited to design antimicrobials that target the bacterial enzyme.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M103396200