Mechanism of activation of cyclic nucleotide phosphodiesterase: requirement of the binding of four Ca2+ to calmodulin for activation.
Kinetic studies on the activation of cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) as a function of calmodulin and Ca2+ concentrations have been carried out. A general approach to analyzing the mechanism of activation, which takes into consideration the various interactions among phosphodiesterase and calmodulin liganded with Ca2+ to differing degrees, is presented. The method is applicable to other calmodulin-regulated enzyme systems. Our kinetic analysis reveals that all four Ca2+ must be bound to calmodulin for the protein to form an activated complex with phosphodiesterase. The mechanistic and regulatory advantages of having four Ca2+ sites on calmodulin can be briefly stated as follows. (i) With the enzyme--calmodulin--Ca4(2+) complex as the dominant active species, the activation of phosphodiesterase as a function of Ca2+ concentration is highly cooperative. This phenomenon serves as an effective on/off switch for phosphodiesterase activation. (ii) At normal cellular levels of Ca2+ (less than 0.1 microM), phosphodiesterase and calmodulin do not form a complex. Thus, the distribution of calmodulin among its various target enzymes is reshuffled for each Ca2+ surge. (iii) The affinity between the enzyme and the fully liganded calmodulin (0.1-1 mM) is 10(4)-10(5) times better than that in the absence of Ca2+ (greater than or equal to 10 microM). The tremendous increase in affinity can be achieved rather easily through a 10- to 20-fold increase in the affinity of Ca2+ for the enzyme-calmodulin complex in each of the four binding steps.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D008433:Mathematics
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium