On the roles of magnesium and spermidine in the isoleucyl-tRNA synthetase reaction. Analysis of the reaction mechanism by total rate equations.
The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PPi exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine.ATP complex. Spermidine.ATP is a weaker substrate. The role of spermidine.ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.
Airas
R K
RK
Department of Biochemistry, University of Turku, Finland.
D004926:Escherichia coli / Q000201:enzymology*; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008433:Mathematics; D008962:Models, Theoretical; D012355:RNA, Transfer, Ile / Q000378:metabolism; D013095:Spermidine / Q000494:pharmacology*
D012355:RNA, Transfer, Ile; D007533:Isoleucine-tRNA Ligase; D008274:Magnesium; D013095:Spermidine