Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase.
Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.
D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors*; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D001665:Binding Sites; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005227:Fatty Acids / Q000138:chemical synthesis / Q000494:pharmacology; D007532:Isoleucine / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D016712:Mupirocin; D011485:Protein Binding
D000900:Anti-Bacterial Agents; D011756:Diphosphates; D005227:Fatty Acids; D007532:Isoleucine; D016712:Mupirocin; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase