Activation mechanism of rabbit skeletal muscle myosin light chain kinase. 5'-p-fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme.

5'-p-fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3 = -0.040 min-1 and -0.038 min-1, and Ki = 0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.