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Inactivation of recombinant monocyte cAMP-specific phosphodiesterase by
cAMP analog, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic
monophosphate
GA Omburo, TJ Torphy, G Scott, S Jacobitz, RF Colman and RW Colman
Sol Sherry Thrombosis Research Center, Temple University School of
Medicine, Philadelphia, PA 19140, USA.
Two cAMP analogs, 8- and 2- [(4-bromo-2,3-dioxobutyl)thio]adenosine
3',5'-cyclic monophosphate (8- and 2-BDB-TcAMP) have been used in probing
the catalytic site of recombinant monocyte cAMP-specific phosphodiesterase
(PDE4a). 2-BDB-TcAMP is a reversible and competitive inhibitor (Ki = 5.5
mumol/L) of cAMP hydrolysis by PDE4a, 8-BDB-TcAMP irreversibly inactivates
the enzyme in a time- and concentration- dependent manner with a second
order rate constant of 0.022 mmol/L-1 min-1. The rate of inactivation of
PDE4a is reduced by the presence of the substrate cAMP and specific
inhibitors, rolipram and denbufylline, but not by cGMP or AMP. Reduction of
the enzyme-inhibitor complex with sodium [3H]borohydride shows that 1.2 mol
of the affinity label/mol of enzyme was incorporated. The radiolabeled
peptide is composed of 10 amino acid residues (697 to 706) located near the
carboxyl end of the proposed catalytic domain. The peptide (GPGHPPLPDK) has
seven nonpolar and aliphatic residues, of which four are proline, giving
the peptide a highly structured conformation. This peptide is the first to
be identified in the putative catalytic domain involved in substrate
recognition.
Volume 89,
Issue 3,
pp. 1019-1026,
02/01/1997
Copyright © 1997 by The American Society of Hematology
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