Goldberg N D, Walseth T F, Stephenson J H, Krick T P, Graff G
J Biol Chem. 1980 Nov 10;255(21):10344-7.
The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.
在[18O]水中进行磷酸二酯酶催化的cGMP水解反应，以确定键断裂的位点以及18O掺入5'-GMP中的化学计量。研究了三种不同形式的磷酸二酯酶，包括处于基础状态和激活状态的钙调蛋白-钙依赖性酶。每种形式的磷酸二酯酶催化的cGMP水解反应进行时，在生成的每个5'-GMP分子的磷酸部分中可回收1个18O原子。未检测到来自生成的5'-GMP的含有两个或三个18O的磷酸分子种类。这些结果表明，磷酸二酯酶催化的cGMP水解通过磷上的亲核取代进行，导致P-O键断裂。18O掺入的化学计量表明，当水解反应由基础状态或激活状态的多种形式的磷酸二酯酶催化时，反应进行过程中没有磷酸-水氧交换。当反应在具有预定18O富集度的介质中进行时，对磷酸二酯酶反应的这些考虑有助于确定将cGMP酶催化水解速率作为5'-GMP磷酸18O标记速率的函数进行监测的有效性。
