Omburo G A, Torphy T J, Scott G, Jacobitz S, Colman R F, Colman R W
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Blood. 1997 Feb 1;89(3):1019-26.
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.
两种环磷酸腺苷（cAMP）类似物，8 - 和2 - [(4 - 溴 - 2,3 - 二氧代丁基)硫代]腺苷3',5'-环一磷酸（8 - 和2 - BDB - TcAMP）已被用于探究重组单核细胞cAMP特异性磷酸二酯酶（PDE4a）的催化位点。2 - BDB - TcAMP是PDE4a水解cAMP的可逆竞争性抑制剂（Ki = 5.5 μmol/L），8 - BDB - TcAMP以时间和浓度依赖性方式不可逆地使该酶失活，二级速率常数为0.022 mmol/L-1 min-1。底物cAMP以及特异性抑制剂咯利普兰和登布茶碱的存在会降低PDE4a的失活速率，但环磷酸鸟苷（cGMP）或腺苷一磷酸（AMP）则不会。用[3H]硼氢化钠还原酶 - 抑制剂复合物表明，每摩尔酶掺入了1.2摩尔亲和标记物。放射性标记的肽由10个氨基酸残基（697至706）组成，位于推测的催化结构域羧基末端附近。该肽（GPGHPPLPDK）有7个非极性脂肪族残基，其中4个是脯氨酸，赋予该肽高度结构化的构象。此肽是在参与底物识别的推测催化结构域中首个被鉴定出来的。
