Enzyme hyperspecificity. Rejection of threonine by the valyl-tRNA synthetase by misacylation and hydrolytic editing.
Valyl-tRNA synthetase from Bacillus stearothermophilus activates thereonine and forms a 1:1 complex with threonyl adenylate, but it does not catalyze the net formation of threonyl-tRNAVal at pH 7.78 and 25  degrees  C in the quenched flow apparatus it decomposes at a rate constant of 36s-1. During this process there is a transient formation of Thr-tRNAVal reaching a maximum at 25 ms and rapidly falling to zero after 150 ms. At the peak, 22% of the (14C) threonine from the complex is present as (14C) Thr-tRNA. The reaction may be quenched with phenol and the partially mischarged tRNA isolated. The enzyme catalyzes its hydrolysis with a rate constant of 40s-1. The data fit a kinetic scheme in which 62% of the threonine from the threonyl adenylate is transferred to the tRNA. This may be compared with the rate constant of 12s-1 at which 84% of the valine is transferred to tRNAVal from the enzyme-bound valyl adenylate, and the rate constant of 0.015s-1 for the subsequent hydrolysis of Val-tRNAVal. Inhibition studies indicate a distinct second site for hydrolysis. The translocation of the aminoacyl moiety between the two sites could be mediated by a transfer between the 2'-and 3'-OH groups of the terminal adenosine fo the tRNA. The hyperspecificity of the enzyme is based on discriminating between the two competing substrates twice: once against the undesired substrate in the synthetic step, and once against the desired substrate in the destructive step.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D011756:Diphosphates / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D010636:Phenols / Q000494:pharmacology; D012343:RNA, Transfer / Q000378:metabolism; D013696:Temperature; D013912:Threonine / Q000378:metabolism*; D046249:Transfer RNA Aminoacylation; D014633:Valine / Q000378:metabolism; D014637:Valine-tRNA Ligase / Q000378:metabolism*
D011756:Diphosphates; D010636:Phenols; D013912:Threonine; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D014637:Valine-tRNA Ligase; D014633:Valine