Enzymatic synthesis of unlabeled and beta-P-32-labeled beta-L-2 ',3 '-dideoxyadenosine-5 '-triphosphate as a potent inhibitor of adenylyl cyclases and its use as reversible binding ligand

beta-L-2',3'-Dideoxyadenosine-5'-triphosphate (beta-L-2',3'-dd-5'-ATP) was prepared enzymatically from the corresponding monophosphate by the use of a denylate kinase, creatine phosphate, and creatine kinase in a single step. The beta-P-32-labeled analog was prepared similarly, but in a two step reac tion. beta-L-2',3'-dd-5'-ATP inhibited adenylyl cyclase from rat brain comp etitively with respect to substrate (5'-ATP . Mn2+) and exhibited an IC50 s imilar to 24 nM. The labeled ligand was used in the development of a revers ible binding assay for adenylyl cyclases, Binding of beta-L-2',3'-dd-[beta- P-32]5'-ATP was saturable with increasing concentrations of ligand and incr eased in proportion to membrane protein, and was enhanced by Mn2+ to a grea ter extent than by Mg2+. Binding was displaced with adenine nucleotides kno wn to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatin e phosphate, or creatine kinase, Binding was rapid, with a half-time for th e 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 b inding suggest an interaction with adenylyl cyclase per se and to a configu ration of the enzyme consistent with. an interaction at the catalytic activ e site. The data suggest that this is a pretransition state inhibitor and c ontrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state noncom petitive inhibitor.