Inactivation of platelet PDE2 by an affinity label: 8-[(4-bromo-2, 3-dioxobutyl)thio]cAMP

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase (PDE2) is the second most abundant of this class of enzymes in platelets. PDE2 probably plays an important role in the regulation of elevated intracellular concentrations of cAMP and cGMP in platelets inhibited by prostacyclin and/or nitric oxide. The cAMP and cGMP PDEs have catalytic domains with 28-40% identity, but vary in their substrate specificity and affinity. As a first step toward the goal of identifying important amino acids in the substrate binding site pocket, we have employed the affinity analog 8-[(4-bromo-2, 3-dioxobutyl)thio]adenosine-3'5' cyclic monophosphate (8-BDB-TcAMP) to inactivate PDE2 and observe the pattern of protection by substrates and their products. Incubation of purified platelet PDE2 with 8-BDB-TcAMP (2-10 mM) resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.013 min(-1) mM(-1). Both substrates, cAMP and cGMP, as well as the products of hydrolysis by PDE2, AMP and GMP, exhibited concentration-dependent protection against inhibition by 8-BDB-TcAMP, but no protection was noted with ADP or ATP, which are not hydrolyzed by the enzyme. This compound, 8-BDB-TcAMP, and similar affinity reagents should prove useful in delineating amino acids in the active site of PDE2.