title: A new nonhydrolyzable reactive cAMP analog, (Sp)-adenosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate irreversibly inactivates human platelet cGMP-inhibited cAMP phosphodiesterase.
abstract: Levels of cAMP that control critical platelet functions are regulated by cGMP-inhibited cAMP phosphodiesterase (PDE3A). We previously showed that millimolar concentrations of the hydrolyzable 8-[(4-bromo-2,3-dioxobutyl)thioadenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP) inactivate PDE3A. We have now synthesized a nonhydrolyzable affinity label to probe the active site of PDE3A. The nonhydrolyzable adenosine 3',5'-cyclic monophosphorothioates, Sp-cAMPS and Rp-cAMPS, function as competitive inhibitors of PDE3A with K(i) = 47.6 and 4400 microM, respectively. We therefore coupled Sp-cAMPS with 1,4-dibromobutanedione to yield (Sp)-adenosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, [Sp-cAMPS-(BDB)]. Sp-cAMPS-(BDB) inactivates PDE3A in a time-dependent, irreversible reaction with k(max) = 0.0116 min(-1) and K(I) = 10.1 microM. The order of effectiveness of protectants in decreasing the rate of inactivation (with K(d) in microM) is: Sp-cAMPS (24) > Rp-cGMPS (1360), Sp-cGMPS (1460) > GMP (4250), AMP (10600), Rp-cAMPS (22170). These results suggest that the inactivation of PDE3A by Sp-cAMPS-(BDB) is a consequence of reaction at the overlap of the cAMP and cGMP binding regions in the active site.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
authors.forename: Su H
authors.initials: SH
authors.lastname: Hung
authors.forename: K S
authors.initials: KS
authors.lastname: Madhusoodanan
authors.forename: James A
authors.initials: JA
authors.lastname: Beres
authors.forename: Robert L
authors.initials: RL
authors.lastname: Boyd
authors.forename: James L
authors.initials: JL
authors.lastname: Baldwin
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Robert W
authors.initials: RW
authors.lastname: Colman
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006152:Cyclic GMP / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008958:Models, Molecular; D009946:Organothiophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D009946:Organothiophosphorus Compounds; C455844:adenosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP