5a-formylbicyclomycin: Studies on the bicyclomycin-rho interaction

Bicyclomycin (1) is a commercial antibiotic whose primary site of action is the Pho transcription termination factor. A new bicyclomycin irreversible inactivator, 5a-formylbicyclomycin (3), was prepared to provide information concerning the bicyclomycin-rho inactivation process and the drug's binding pocket within rho. The apparent Iso value for 3 was 35 mu M, showing that 3 was a more effective inhibitor of rho poly C-dependent ATPase activity than 1 (I-50 = 60 mu M). Mechanistic studies demonstrated that 3 inhibited poly C-dependent ATP hydrolysis, in part, by a reversible, noncompetitive pathway with respect to ATP (K-i = 62 rho rho M) Incubation of 3 with rho led to efficient imine formation. Adding excess 1 to solutions containing 3 and rho prevented imine formation, demonstrating that 1 and 3 bind to the same active site in the protein. The 3-rho imine was stabilized by either ATP or ADP or by both, and was converted to the nonreversible 3-rho amine adduct upon treatment with NaBH4. Mass spectrometric analysis of the amine provided a stoichiometry of approximately five bound 3 per rho hexamer indicating the number of bicyclomycin binding sites for the rho hexamer is between five and six. Monomer exchange experiments using modified 3-rho amine and wild type rho demonstrated that no more than two modified subunits per rho hexamer are sufficient to halt poly C-dependent rho ATPase activity.