Effects of spermine and magnesium ions on the aminoacylation of yeast tRNA(Tyr).
Stimulatory effects of Mg2+ and spermine on the kinetics of the aminoacylation of tRNA(Tyr) were examined using purified yeast tRNA(Tyr) and tyrosyl-tRNA synthetase. The apparent Km for tRNA(Tyr) was the lowest at Mg2+ concentrations between 2 and 5 mM and was not influenced by spermine. In the absence of spermine, the apparent Vmax was the highest at Mg2+ concentrations of 5 mM or higher, whereas the presence of spermine strongly stimulated the reaction at lower Mg2+ concentrations. Spermine alone could not substitute for Mg2+, nor was it able, at any Mg2+ concentration, to increase the reaction rate above the level reached at high concentrations of Mg2+ alone. Calculations of the concentration of Mg3.tRNA(Tyr) complex as a function of initial Mg2+ concentration, using the binding constants derived from physical measurements, allow the conclusion that spermine exerts its stimulatory activity by creating strong binding sites for Mg2+; this would enable the tRNA to assume the conformation required for optimal aminoacylation. The conformational requirement for the first tRNA: synthetase encounter is obviously less stringent, since the apparent Km for tRNA(Tyr) is not influenced by spermine.
D002413:Cations, Divalent; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D008970:Molecular Weight; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D013096:Spermine / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism
D002413:Cations, Divalent; D012346:RNA, Transfer, Amino Acyl; D013096:Spermine; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase; D008274:Magnesium