The role of polyamines in the aminoacyl transfer ribonucleic acid synthetase reactions. Demonstration of the requirement for magnesium ion and a secondary stimulatory effect of spermine.
Spermine and related polyamines have been reported to substitute for Mg2+ in the aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases, but not in the ATP-PP-i exchange reaction. Such observations have led some workers to propose that these reactions proceed via a concerted mechanism rather than the usual two-step mechanism involving an aminoacyladenylate intermediate. In an attempt to elucidate the mechanism of the spermine effect on acylation and exchange, both reactions were re-examined using isoleucyl-tRNA synthetase from Escherichia coli. In the absence of added Mg2+ untreated tRNA was acylated in the presence of spermine, but tRNA from which Mg2+ had been scrupulously removed was not. ATP-PP-i exchange was not observed when spermine was used in place of Mg2+; however, if tRNA possessing sequestered Mg2+ was added, the exchange reaction was observed. These data suggest that a primary effect of spermine is to displace bound Mg2+ from tRNA in quantities sufficient to promote both the ATP-PP-i exchange and esterification of tRNA. The previously reported stimulatory effects of polyamines on these reactions are believed to be artifacts due to Mg2+ contamination of tRNA. Providing trace levels of Mg2+ are present, spermine exerts a secondary stimulation of the rate of aminoacylation, the mechanism of which is unknown. The results presented refute arguments that these enzymes proceed by a concerted mechansim and support the intermediacy of aminoacyladenylates.
D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D011756:Diphosphates; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D012329:RNA, Bacterial; D012343:RNA, Transfer; D013096:Spermine / Q000494:pharmacology*
D011756:Diphosphates; D012329:RNA, Bacterial; D013096:Spermine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase; D008274:Magnesium