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Title: Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Authors:
; ;  [1];  [2]
  1. (UNC)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1234733
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 291; Journal Issue: (1) ; 01, 2016
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Williams, Tishan L., Yin, Yuhui W., Carter, Jr., Charles W., and Texas-MED). Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based. United States: N. p., 2016. Web. doi:10.1074/jbc.M115.690321.
Williams, Tishan L., Yin, Yuhui W., Carter, Jr., Charles W., & Texas-MED). Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based. United States. doi:10.1074/jbc.M115.690321.
Williams, Tishan L., Yin, Yuhui W., Carter, Jr., Charles W., and Texas-MED). 2016. "Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based". United States. doi:10.1074/jbc.M115.690321.
@article{osti_1234733,
title = {Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based},
author = {Williams, Tishan L. and Yin, Yuhui W. and Carter, Jr., Charles W. and Texas-MED)},
abstractNote = {},
doi = {10.1074/jbc.M115.690321},
journal = {Journal of Biological Chemistry},
number = (1) ; 01, 2016,
volume = 291,
place = {United States},
year = 2016,
month = 7
}
  • The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flowed method. The transfer of tryptophan to tRNA/sup Trp/ was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-(/sup 14/C)tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-(/sup 14/C)tryptophan were used. The authors measured either the formation of a single labeled aminoacyl-tRNA/sup Trp/ per enzyme subunit or the turnover of labeled aminoacyl-tRNA/sup Trp/ synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a singlemore » active subunit (subunits presenting absolute half-of-the-sites reactivity); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNA/sup Trp/ under each labeling conditions were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate if aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.« less
  • The purified high molecular weight form of HeLa cell DNA polymerase ..cap alpha.. (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) was shown to associate tightly with several aminoacyl-tRNA synthetase activities. Fractionation of the high molecular weight enzyme on hexylagarose followed by gel filtration, chromatography on phosphocellulose, or polyacrylamide gel electrophoresis under nondenaturing conditions demonstrated copurification of only tryptophanyl-tRNA synthetase (L-tryptophan:tRNA/sup Trp/ ligase (AMP-forming), EC 6.1.1.2) along with DNA polymerase ..cap alpha... The high molecular weight (660,000) and low molecular weight (145,000) forms of DNA polymerase ..cap alpha.. were shown to possess a highly specific, noncovalent, diadenosine 5',5'''-P/sup 1/,P/sup 4/-tetraphosphate (Ap/sub 4/A) bindingmore » activity. The dissociation constants were determined to be 16 and 22 ..mu..M, respectively, by utilization of a charcoal adsorption procedure. No high-affinity binding of ATP could be detected. These findings suggest a link between the amino acid activation process and DNA replication in mammalian cells.« less