Enzymatic synthesis of unlabeled and beta-(32)P-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 adenylate kinase, creatine phosphate, and creatine kinase in a single step. The beta-(32)P-labeled analog was prepared similarly, but in a two step reaction. beta-L-2', 3'-dd-5'-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5'-ATP.Mn(2+)) and exhibited an IC(50) approximately 24 nM. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of beta-L-2',3'-dd-[beta-(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-3'ATP, a post-transition state noncompetitive inhibitor.
Shoshani
I
I
Department of Physiology, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8661, USA.
Boudou
V
V
Pierra
C
C
Gosselin
G
G
Johnson
R A
RA
D000255:Adenosine Triphosphate / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology*; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007536:Isomerism; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D051381:Rats
D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases