Catalysis of tyrosyl-adenylate formation by the human tyrosyl-tRNA synthetase.
Although the active site residues in the Bacillus 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 the B. 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.
Austin
Joseph
J
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport, Shreveport, Louisiana 71130, USA.
First
Eric A
EA
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism*; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D014443:Tyrosine; C019586:tyrosinyl-5'-AMP; D014448:Tyrosine-tRNA Ligase