Mechanism of activation of cyclic nucleotide phosphodiesterase: requirement of the binding of four Ca2+ to calmodulin for activation.

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Abstract
Kinetic studies on the activation of [bovine brain] cyclic nucleotide phosphodiesterase (3'',5''-cyclic-nucleotide 5''-nucleotidohydrolase) as a function of calmodulin and Ca2+ concentrations were conducted. 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. Kinetic analysis reveals that all 4 Ca2+ must be bound to calmodulin for the protein to form an activated complex with phosphodiesterase. The mechanistic and regulatory advantages of having 4 Ca2+ sites on calmodulin can be briefly stated as follows. With the enzyme-calmodulin-Ca42+ 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. At normal cellular levels of Ca2+ (< 0.1 .mu.M), phosphodiesterase and calmodulin do not form a complex. The distribution of calmodulin among its various target enzymes is reshuffled for each Ca2+ surge. The affinity between the enzyme and the fully liganded calmodulin (0.1-1 mM) is 104-105 times better than that in the absence of Ca2+ (.gtoreq. 10 .mu.M). 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 4 binding steps.