5a-formylbicyclomycin: studies on the bicyclomycin-Rho interaction.
Bicyclomycin (1) is a commercial antibiotic whose primary site of action is the rho 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 I(50) value for 3 was 35 microM, showing that 3 was a more effective inhibitor of rho poly C-dependent ATPase activity than 1 (I(50) = 60 microM). 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 microM). 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 NaBH(4). 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.
Department of Chemistry, University of Houston, Texas 77204-5641, USA.
F
F
Vincent
W R
WR
Widger
M
M
Openshaw
S J
SJ
Gaskell
H
H
Kohn
D000227:Adenine Nucleotides / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000737:chemistry*; D001665:Binding Sites; D001894:Borohydrides / Q000737:chemistry; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000138:chemical synthesis / Q000737:chemistry*; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry; D007097:Imines / Q000737:chemistry; D046911:Macromolecular Substances; D011066:Poly C / Q000737:chemistry; D012234:Rho Factor / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013329:Structure-Activity Relationship
D000227:Adenine Nucleotides; D000588:Amines; D000900:Anti-Bacterial Agents; D001894:Borohydrides; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004791:Enzyme Inhibitors; D007097:Imines; D046911:Macromolecular Substances; D012234:Rho Factor; D011066:Poly C; C025364:sodium borohydride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C031715:rho-ATPase; C024504:bicozamycin