The Catalytic Mechanism of Mammalian Adenylyl Cyclase

Abstract

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...

Human recombinant soluble guanylyl cyclase: Expression, purification, and regulation

“…Both compounds followed first-order kinetics, indicating that only one molecule of PPi or cGMP is bound per sGC heterodimer. The cooperative inhibition of sGC by both reaction products suggests that a reverse reaction might occur, as was demonstrated in similar conditions for adenylyl cyclase (42).…”

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

“…These compounds are also referred to as P-site inhibitors. P-site inhibitors are noncompetitive or uncompetitive AC inhibitors that bind to the AC-PP i conformation (26,27). The most potent P-site inhibitors presently available are 2Ј,5Ј-dd-3Ј-ATP (IC 50 for rat brain AC, 40 nM) and 2Ј,5Ј-dd-3Ј-A 4 P (IC 50 for rat brain AC, 7.4 nM) (24,25).…”

Covalent Labeling of Adenylyl Cyclase Cytosolic Domains with γ-Methylimidazole-2′,5′-dideoxy-[γ-32P]3′-ATP and the Mechanism for P-site-mediated Inhibition

“…This would explain the lack of competition between 2Ј,5Ј-dd-3Ј-ATP and substrate under steady state kinetic conditions. Moreover, it suggests that binding of this ligand is a pseudo-irreversible process and the noncompetitive inhibition observed with it (12,22) likely occurs by a somewhat different mechanism than does the post-transition state, dead-end inhibition previously observed with 2Ј-d-3Ј-AMP, which requires bound metal-PP i for inhibition (5,8,11). The lack of competition between substrate and 2Ј,5Ј-dd-3Ј-ATP was clearly evident both in the experiments on associationdissociation of VC 1 and IIC 2 (Figs.…”

“…When the C 1 domain of the type V isozyme (VC 1 ) and the C 2 domain of the type II isozyme (IIC 2 ) are expressed separately in Escherichia coli and then recombined, a functional adenylyl cyclase is formed upon their association in solution (2). This truncated chimeric construct exhibits stimulation by G s ␣ or forskolin and inhibition by P-site ligands, 1 properties that are characteristic of the native enzyme (2)(3)(4)(5). Available evidence suggests that both catalysis and inhibition by P-site ligands occur at a common site within a cleft formed at the interface of the two cytosolic domains (6,7).…”