Binding of 3'-anthraniloyl-2'-deoxy-ATP to calmodulin-activated adenylate cyclase from Bordetella pertussis and Bacillus anthracis.
3'-Anthraniloyl-2'-deoxyadenosine 5'-triphosphate (Ant-dATP), a fluorescent analogue of ATP, was tested as a probe for the nucleotide-binding site of calmodulin (CaM)-activated adenylate cyclases from Bordetella pertussis (BPCYA47) and Bacillus anthracis (BACYA62). Ant-dATP competitively inhibited both bacterial enzymes expressed in Escherichia coli (ki approximately 10 microM). Binding of the analogue to adenylate cyclase was monitored by equilibrium dialysis and by an increase in its fluorescence emission at 420 nm upon excitation at 330 nm. Whereas the fluorescence of Ant-dATP was little influenced by divalent cations, CaM, or adenylate cyclase alone, the Ca2+.CaM.cyclase complex increased up to 4 times the quantum yield of Ant-dATP. Binding of the analogue to the catalytic site of BPCYA47 and BACYA62 was specific as shown by its displacement with ATP or 3'-dATP. Our results substantiate the role of CaM in favoring substrate binding to CaM-activated enzymes.
Sarfati
R S
RS
Unités de Chimie Organique, Institut Pasteur, Paris, France.
Kansal
V K
VK
Munier
H
H
Glaser
P
P
Gilles
A M
AM
Labruyère
E
E
Mock
M
M
Danchin
A
A
Bârzu
O
O
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D001408:Bacillus anthracis / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D001886:Bordetella pertussis / Q000201:enzymology*; D002147:Calmodulin / Q000494:pharmacology*; D003903:Deuterium; D017666:Deuterium Oxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology; D014867:Water; D062367:ortho-Aminobenzoates / Q000378:metabolism* / Q000494:pharmacology
D000067956:Adenylyl Cyclase Inhibitors; D002147:Calmodulin; D062367:ortho-Aminobenzoates; D014867:Water; C066294:3'-anthraniloyl-2'-deoxy-ATP; D000255:Adenosine Triphosphate; D014508:Urea; D003903:Deuterium; D000262:Adenylyl Cyclases; D017666:Deuterium Oxide