Inhibition of Bordetella pertussis and Bacillus anthracis adenylyl cyclases by polyadenylate and "P"-site agonists.
Inhibition of adenylyl cyclases from Bacillus anthrasis and Bordetella pertussis by polyadenylate and by the most potent "P"-site agonists was investigated. These bacterial adenylyl cyclases differed in their sensitivity to inhibition by nominal "P"-site agents and in the effect of divalent cations on this inhibition. The enzyme from Bordetella pertussis was relatively insensitive to inhibition by "P"-site agonists, exhibiting a rank order of potency of 2'd3'AMP greater than 3'-AMP greater than 2',5'-ddAdo approximately Ado approximately 2'-dAdo, with IC50 values for 2'd3'AMP and 3'-AMP of 1-3 mM. Inhibition by 2'd3'AMP, however, was not affected by divalent cation, making it distinct from "P"-site-mediated inhibition of most mammalian adenylyl cyclases. The sensitivity to these nucleosides was comparable with potency for inhibition of bovine sperm adenylyl cyclase but was 3 orders of magnitude less potent than for activated enzyme from bovine or rat brain. The Bordetella pertussis enzyme was similarly insensitive to inhibition by polyadenylate, with 16 microM inhibiting less than 20%. By comparison, Bacillus anthrasis adenylyl cyclase was more potently inhibited by 2'd3'AMP (IC50 approximately 85 microM) but not by the other nucleosides (less than 15% inhibition at 1 mM), and inhibition by 2'd3'AMP was optimally enhanced by 5-10 mM Mg2+ or Mn2+, as is typical for inhibition by "P"-site agonists. The Bacillus anthrasis enzyme was potently inhibited by polyadenylate (IC50 approximately 0.3 microM), comparable to inhibition of brain adenylyl cyclases. Sensitivity of Bacillus anthrasis adenylyl cyclase to poly(A) was diminished somewhat by Ca2+/calmodulin (to IC50 approximately 1 microM) although Ca2+/calmodulin was without effect on inhibition by 2'd3'AMP. In contrast to inhibition of mammalian adenylyl cyclases via the "P"-site, inhibition of both bacterial adenylyl cyclases by 2'd3'AMP was competitive with respect to substrate MgATP. The data indicate basic differences in susceptibilities of these bacterial adenylyl cyclases to inhibition by poly(A), by adenosine analogs, and the effects of divalent cations. Although the potency of 2'd3'AMP and the metal-dependent nature of inhibition of Bacillus anthrasis adenylyl cyclase shared characteristics of "P"-site-mediated inhibition, the fact that inhibition of both bacterial adenylyl cyclases was competitive with respect to substrate strongly suggests that this inhibition was at the catalytic site and that these bacterial enzymes do not contain a distinct "P"-site.
Johnson
R A
RA
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794-8661.
Shoshani
I
I
D000241:Adenosine / Q000494:pharmacology*; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000302:isolation & purification; D000818:Animals; D001408:Bacillus anthracis / Q000201:enzymology*; D001665:Binding Sites; D001886:Bordetella pertussis / Q000201:enzymology*; D002417:Cattle; D003839:Deoxyadenosines / Q000494:pharmacology; D016048:Dideoxyadenosine / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D011061:Poly A / Q000494:pharmacology*; D013094:Spermatozoa / Q000201:enzymology
D000067956:Adenylyl Cyclase Inhibitors; D003839:Deoxyadenosines; D011061:Poly A; D000249:Adenosine Monophosphate; D016048:Dideoxyadenosine; D000262:Adenylyl Cyclases; D000241:Adenosine