18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase.
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.
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D002417:Cattle; D006152:Cyclic GMP; D006150:Guanine Nucleotides / Q000032:analysis*; D006157:Guanosine Monophosphate / Q000032:analysis*; D006801:Humans; D007553:Isotope Labeling; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D010103:Oxygen Isotopes
D006150:Guanine Nucleotides; D010103:Oxygen Isotopes; D006157:Guanosine Monophosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP