Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate. 216 Feb 72

We previously showed that 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate inactivates cAMP phosphodiesterase (PDE3A); however, millimolar concentrations were needed to inactivate PDE3A because of ongoing hydrolysis. We have now synthesized a nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate (S(p)-8-BDB-TcAMPSMe). S(p)-8-BDB-TcAMPSMe inactivates PDE3A in a time-dependent, irreversible manner, exhibiting saturation kinetics with a k(max) of (19.5 +/- 0.3) x 10(-3) min(-1) and a K(I) of 3.5 +/- 0.3 muM. To ascertain whether S(p)-8-BDB-TcAMPSMe reacts in the active site, nonhydrolyzable analogues of the substrate cAMP, or the competitive inhibitor cGMP, were included to protect against the inactivation of PDE3A. The order of effectiveness of protectants in decreasing the rate of inactivation (with K(d) values in micromolar) is as follows: S(p)-cAMPS (18) > R(p)-cGMPS (560) and S(p)-cGMPS (1260) > 5'-AMP (17 660), R(p)-cAMPS (30 110), and 5'-GMP (42 170). We docked S(p)-8-BDB-TcAMPSMe into PDE3A, based on the structural model of PDE3A-cAMP and the kinetic data from site-directed mutants. The S(p)-8-BDB-TcAMPSMe fits into the active site in the model. These results suggest that inactivation of PDE3A by the affinity reagent is a consequence of reaction at the overlap between cAMP and cGMP binding regions in the active site. S(p)-8-BDB-TcAMPSMe has proven to be an effective active site-directed irreversible cAMP affinity label for platelet PDE3A and can be used to identify amino acids in the active site of PDE3A as well as in other cAMP phosphodiesterases.
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PMID:A nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate, irreversibly inactivates human platelet cGMP-inhibited cAMP phosphodiesterase at micromolar concentrations. 1186 34