The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2-[ 3 H]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 C 1 /C 2 heterodimer; the K d is 40 ؎ 3 M. Binding is observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate (PP i ). A substrate analog, Ap(CH 2 )pp (␣,-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 Psite 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.Adenosine and various analogs of the nucleoside have both stimulatory and inhibitory effects on adenylyl cyclase activity (reviewed in Ref. 1). Londos and Wolff (2) categorized these effects mechanistically, based on their structure-activity relationships. Two types of adenosine-reactive sites were identified: those with strict requirements for the ribose moiety, designated R sites, and those with strict structural constraints for interaction with the purine ring, designated P sites. R sites are the ligand-binding sites of adenosine-specific G protein 1 -coupled receptors, which can either stimulate or inhibit adenylyl cyclase activity indirectly, while P sites, whose occupancy inhibits cyclic AMP synthesis, are structural features of adenylyl cyclases themselves (2-7). The physiological significance of Psite inhibition is unclear, but concentrations of 3Ј-AMP found in vivo appear sufficient to inhibit adenylyl cyclase activity (8).P-site inhibition is typically noncompetitive or uncompetitive with respect to substrate ATP, depending on the divalent cation utilized in the assay (Mn 2ϩ usually yielding noncompetitive kinetics; Mg 2ϩ uncompetitive) (3, 4, 9 -11). Furthermore, the apparent potency of such inhibitors increases when adenylyl cyclase is activated (3-5, 7, 11). Representative P-site reagents, ordered by potency, include 2Ј,5Ј-dideoxy-3Ј-ATP Ͼ 2Ј,5Ј-dideoxy-3Ј-ADP Ͼ 2Ј,5Ј-dideoxy-3Ј-AMP Ͼ 2Ј-deoxy-3Ј-AMP Ͼ 3Ј-AMP Ͼ 2Ј-deoxyadenosine Ͼ adenosine (12, 13). Although several mechanisms for P-site inhibition have been proposed, there is as yet no conclusive evidence to support any particular hypothesis. Using engineered, soluble forms of mammalian adenylyl cyclase, we and others have shown that the conserved cytosolic domains of the enzymes contain the structural components necessary for G s␣ -and forskolin-stimulate...