Biosynthesis and characterization of 4-fluorotryptophan-labeled Escherichia coli arginyl-tRNA synthetase

Qing-Shuo Zhang, Li Shen, En Duo Wang, Ying Lai Wang

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4- fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan- substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher K(m) values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of 19F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.

Original languageEnglish (US)
Pages (from-to)187-192
Number of pages6
JournalJournal of Protein Chemistry
Volume18
Issue number2
DOIs
StatePublished - 1999
Externally publishedYes

Fingerprint

Arginine-tRNA Ligase
Biosynthesis
Escherichia coli
Enzymes
Tryptophan
Hot Temperature
Enzyme Stability
4-fluorotryptophan
Transfer RNA
Nuclear magnetic resonance spectroscopy
Rate constants
Thermodynamic stability
Substitution reactions
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance

Keywords

  • 19F NMR
  • 4-fluorotryptophan
  • Arginyl-tRNA synthetase
  • HPLC

ASJC Scopus subject areas

  • Biochemistry

Cite this

Biosynthesis and characterization of 4-fluorotryptophan-labeled Escherichia coli arginyl-tRNA synthetase. / Zhang, Qing-Shuo; Shen, Li; Wang, En Duo; Wang, Ying Lai.

In: Journal of Protein Chemistry, Vol. 18, No. 2, 1999, p. 187-192.

Research output: Contribution to journalArticle

Zhang, Qing-Shuo ; Shen, Li ; Wang, En Duo ; Wang, Ying Lai. / Biosynthesis and characterization of 4-fluorotryptophan-labeled Escherichia coli arginyl-tRNA synthetase. In: Journal of Protein Chemistry. 1999 ; Vol. 18, No. 2. pp. 187-192.
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