Kinetic Studies of the Activation of Adenylate Cyclase by Calmodulin.

Adenylate cyclase toxin (ACT) is a virulence factor secreted by and plays a causative role in whooping cough. After ACT attaches to lung phagocytes, the adenylate cyclase (AC) domain of the toxin is transported into the cytoplasm where it is activated by calmodulin (CaM) to cyclize ATP into 3',5'-cyclic adenosine monophosphate (cAMP). Production of high concentrations of cAMP disrupts immune functions of phagocytes. To better understand the mechanism of activation of AC by CaM, the studies reported herein were conducted. Major observations are as follows: (1) dependence of steady-state velocities on CaM and ATP concentrations suggests that CaM and ATP bind to AC in a random fashion. (2) A pre-steady-state lag phase is observed when AC is added to solutions of CaM and ATP, reflecting the association of AC and CaM. Analysis of pre-steady-state data indicates that CaM binds to AC and AC:ATP with second-order rate constants of 30 and 60 μM s, respectively, and that CaM dissociates from the resultant complexes with a first-order rate constant of 0.002 s. (3) A biphasic dependence of steady-state velocities on CaM concentration is observed: the first phase extending from 0.01 to 1 nM CaM ( ∼ 0.06 nM) and the second phase from 1 to 2000 nM CaM ( ∼ 60 nM). These results suggest that AC exists in at least two conformations, with each conformation exhibiting distinct binding affinity for CaM and distinct potential for activation.