The molecular basis of leucine auxotrophy of quinone-treated Escherichia coli. Active site-directed modification of leucyl-tRNA synthetase by 6-amino-7-chloro-5,8-dioxoquinoline.
Leucyl-tRNA synthetase from Escherichia coli is rapidly inactivated by 6-amino-7-chloro-5,8-dioxoquinoline (quinone), a model substance for cytostatic quinones. Loss of activity follows pseudo-first order kinetics. The quinone masks essential--SH groups that are reactive with N-ethylmaleimide. Specific protection of the enzyme by leucine provides evidence for active site-directed modification. Half-maximal protection is found at a concentration of 150 micron which is identical with the dissociation constant of the enzyme.substrate complex. The competitive inhibitor leucinol also protects the enzyme from inactivation by the quinone. MgATP enhances the protective effect of leucinol about 250-fold, thus substantiating recently published findings on synergistic coupling of ligands to aminoacyl-tRNA synthetases. The results support the assumption that the bacteriostatic quinone directly interferes with leucyl-tRNA synthetase in growing cells. Active-site-directed inhibition of the enzyme could adequately explain the phenotypically observed auxotrophy for leucine of quinone-treated E. coli.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000634:Aminoquinolines / Q000494:pharmacology*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000378:metabolism*; D007935:Leucine-tRNA Ligase / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D012343:RNA, Transfer
D000634:Aminoquinolines; C018817:6-amino-7-chloro-5,8-dioxoquinoline; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007935:Leucine-tRNA Ligase; D007930:Leucine; D008274:Magnesium; D005033:Ethylmaleimide