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 APT-pyridoxal , which forms a covalent bond with the Lys residue located at or near the gamma -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.