The catalytic mechanism of mammalian adenylyl cyclase: Equilibrium binding and kinetic analysis of P-site inhibition

The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2'-[³H]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the forward and reverse reactions (i.e. cyclic AMP and ATP synthesis, respectively). There is one binding site for 2'-deoxyadenosine per C₁/C₂ heterodimer; the K(d) is 40 ± 3 μM. Binding is observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate (PP(i)). A substrate analog, Ap(CH₂)pp (α,β-methylene adenosine 5'-triphosphate), and cyclic AMP compete for the P-site in the presence of PP(i), but P-site analogs do not compete for substrate binding (in the absence of PP(i)). Kinetic analysis indicates that release of products from the enzyme is random. These facts permit formulation of a model for the adenylyl cyclase reaction for which we provide substantial kinetic support. We propose that P-site analogs act as dead-end inhibitors of product release, stabilizing an enzyme-product (E- PP(i)) complex by binding at the active site. Although product release is random, cyclic AMP dissociates from the enzyme preferentially. Release of PP(i) is slow and partially rate-limiting.