Eckstein F, Karpen J W, Critchfield J M, Stryer L
Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Gottingen, Federal Republic of Germany.
J Biol Chem. 1988 Oct 5;263(28):14080-5.
The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.
确定了牛视网膜杆状外段环鸟苷酸磷酸二酯酶催化水解的立体化学过程。鸟苷3',5'-环一磷酸硫代酯的Sp非对映异构体在H2(18)O中被环鸟苷酸磷酸二酯酶水解，生成[16O,18O]鸟苷5'-单磷酸硫代酯。该同位素异构体与二苯基磷酰氯反应，形成P1-(5'-鸟苷基)P2-(二苯基)1-硫代二磷酸的两种非对映异构体。这种非对映异构体混合物的31P NMR光谱与由蛇毒磷酸二酯酶水解鸟苷5'-对硝基苯基硫代磷酸酯的Rp非对映异构体得到的[16O,18O]鸟苷5'-单磷酸硫代酯的光谱相同。这一发现表明，18O在P1-(5'-鸟苷基)P2-(二苯基)1-硫代二磷酸的Rp非对映异构体中起桥连作用，而在Sp非对映异构体中不起桥连作用。由于已知蛇毒磷酸二酯酶反应是构型保留的，因此视网膜杆状环鸟苷酸磷酸二酯酶催化水解过程中磷原子的构型发生翻转。
