A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A.
The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3',5'-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K(I)=43.4+/-7.2muM and k(cart)=0.007+/-0.0006 min(-1)). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K(d)=72 microM)>Sp-cGMPS (124), Sp-cAMPS (182)>GMP (1517), Rp-cAMPS (3762), AMP (4370 microM). NAD(+), neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.
Hung
Su H
SH
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, OMS 418, Philadelphia, PA 19140, USA.
Liu
Andy H
AH
Pixley
Robin A
RA
Francis
Penelope
P
Williams
LaTeeka D
LD
Matsko
Christopher M
CM
Barnes
Karine D
KD
Sivendran
Sharmila
S
Colman
Roberta F
RF
Colman
Robert W
RW
D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D001665:Binding Sites; D001792:Blood Platelets; D006152:Cyclic GMP / Q000031:analogs & derivatives*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D058987:Phosphodiesterase 3 Inhibitors; D011485:Protein Binding
D000345:Affinity Labels; D004791:Enzyme Inhibitors; D058987:Phosphodiesterase 3 Inhibitors; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; D006152:Cyclic GMP