Catalytic mechanism of valyl-tRNA synthetase from baker's yeast. Reaction pathway and rate-determining step in the aminoacylation of tRNAVal.
The catalytic mechanism of valyl-tRNA synthetase from baker's yeast has been investigated by pre-steady-state and steady-state kinetic measurements and end product dissociation studies. The pre-steady-state kinetics show a lag period during the early time when the reaction is started with free enzyme. The preincubation of the synthetase with tRNAVal and/or valine or preformation of Val approximately AMP leads to a progressive suppression of the lag. This lag probably reflects conformational transitions of the enzyme-substrate complex necessary for the transfer. At low pH or at a low ionic strength, the tRNAVal charging occurs much faster at the pre steady state than at the steady state. We show that after the fast transfer of valine from adenylate to tRNAVal, followed by the fast dissociation of AMP and PPi, a new adenylate is synthesized which promotes the dissociation of the nascent Val-tRNAVal. This dissociation occurs in a multistep process. First ATP and magnesium promote the ejection of the valine moiety of Val-tRNAVal from the adenylate site. A new adenylate is then synthesized which promotes, in the presence of magnesium, several state changes of the end product complex. A complex is finally generated in which the enzyme-bound Val-tRNAVal is able to exchange rapidly with a tRNAVal molecule. The free tRNAVal plays an active role in this exchange. Depending upon the experimental conditions, one of these steps can determine the steady-state rate of tRNAVal charging. The dissociations of enzyme-bound uncharged tRNAVal or aa-tRNAs substituted on the amino acid or on the tRNA parts by noncognate parts as well as the effect of the replacement of the adenylates by wrong adenylates have been investigated. It is shown that the valine and the tRNA moieties of Val-tRNAVal and the valine moiety of the adenylate are involved in this mechanism of dissociation. Finally, the rate-determining step of the reversal of tRNAVal charging at the steady-state has been investigated. It is shown that this step is the dissociation of the deacylated tRNAVal from enzyme.
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D012343:RNA, Transfer; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D046249:Transfer RNA Aminoacylation; D014637:Valine-tRNA Ligase / Q000378:metabolism*
D015636:Magnesium Chloride; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D014637:Valine-tRNA Ligase; D008274:Magnesium