DnaA protein Lys-415 is close to the ATP-binding site: ATP-pyridoxal affinity labeling

Toshio Kubota, Yuji Ito, Kazuhisa Sekimizu, Mitsuo Tagaya, Tsutomu Katayama

研究成果: ジャーナルへの寄稿学術誌査読

4 被引用数 (Scopus)


Binding of ATP, but not of ADP, activates Escherichia coli DnaA protein for replicational initiation of the chromosome. To elucidate this switching mechanism, we used the affinity-labeling agent ATP-pyridoxal, which forms a covalent bond with the Lys residue located at or near the γ-phosphate of ATP. ATP-pyridoxal inhibited the ATP binding for DnaA protein, with a competitive mode. Binding stoichiometry was 0.28 ATP-pyridoxal/DnaA molecule, a value consistent with that of ATP. Thus, ATP-pyridoxal was a potent antagonist for the DnaA ATP-binding site. The labeled DnaA protein was inactive for minichromosome replication in vitro, suggesting that conformation of the region is important for DnaA activity. Isolation of the labeled, tryptic fragment and the Edman degradation revealed that ATP-pyridoxal modified Lys-415. Thus, this residue is likely close to the bound ATP. Since Lys-415 is located in the DNA-binding domain, these findings imply internal interaction between the domains for ATP binding and DNA binding.

ジャーナルBiochemical and Biophysical Research Communications
出版ステータス出版済み - 11月 16 2001

!!!All Science Journal Classification (ASJC) codes

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学


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