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␤-L-2,3-Dideoxyadenosine-5-triphosphate (␤-L-2,3-dd-5-ATP) was prepared enzymatically from the corresponding monophosphate by the use of adenylate kinase, creatine phosphate, and creatine kinase in a single step. The ␤-32 P-labeled analog was prepared similarly, but in a two step reaction. ␤-L-2,3-dd-5-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5-ATP⅐Mn 2؉ ) and exhibited an IC 50 ϳ24 nM. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of ␤-L-2,3-dd-[␤-32 P]5-ATP was saturable with increasing concentrations of ligand and increased in proportion to membrane protein, and was enhanced by Mn 2؉ to a greater extent than by Mg 2؉ . Binding was displaced with adenine nucleotides known to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatine phosphate, or creatine kinase. Binding was rapid, with a half-time for the on-rate <1.8 min and for the off-rate <0.8 min. The potency and mechanism of the inhibition of this ligand and the pattern of agents that displace binding suggest an interaction with adenylyl cyclase per se and to a configuration of the enzyme consistent with an interaction at the catalytic active site. The data suggest that this is a pretransition state inhibitor and contrasts with the equipotent 2,5-dd-3ATP, a post-transition state noncompetitive inhibitor.Adenylyl cyclases (ATP-pyrophosphate lyase (cyclizing); E.C. 4.6.1.1.) are a family of membrane-bound enzymes that catalyze the formation of cAMP from 5Ј-ATP. From several lines of evidence, it has become clear that the adenylyl cyclase catalytic site exhibits specificity for the adenine moiety, enhanced binding via substrate and inhibitor phosphate groups, and tolerance of modifications to the ribose (1-8). The impact of the phosphate groups, in terms of structure and electronic character, to interaction of nucleotides with adenylyl cyclases is evident in the observed catalytic efficacy of known substrates, 2 the competitive but weak inhibition by 5Ј-AP(CH 2 )PP (6), and the enhanced inhibition via the so-called P-site 3 of adenine nucleosides with progressively more phosphates added at the 3Ј-ribosyl position (1-4, 9 -11). That the enzyme tolerates modifications at the ribose was evident in the earliest comparisons of inhibitors (1, 9 -11) and was developed further in a characterization of P-site-mediated inhibition of several adenylyl cyclase isozymes (5). The adenine nucleoside 3Ј-polyphosphates are the most potent P-site ligands (1-4) and inhibit via a dead-end noncompetitive mechanism implying that they bind to the enzyme in the post-transition state configuration for and at the leaving site(s) of the products, cAMP and inorganic metal⅐PP i (12)(13)(14). Whereas inhibition by P-site ligands has been well characterized biochemically and pharmacologically and potent and specific inhibitors of the enzyme have been synthesized, potent agents targeted to the pre...