DIFFERENT MECHANISMS FOR CA2-OXIDE SYNTHASE OR MYOSIN LIGHT-CHAIN KINASE( DISSOCIATION FROM COMPLEXES OF CALMODULIN WITH NITRIC)

We have determined the stoichiometry and rate constants for the dissoc iation of Ca2+ ion from calmodulin (CaM) complexed with rabbit skeleta l muscle myosin light chain kinase (skMLCK), rat brain nitric oxide sy nthase (nNOS) or with the respective peptides (skPEP and nPEP) represe nting the CaM-binding domains in these enzymes. Ca2+ dissociation kine tics determined by stopped-flow fluorescence using the Ca2+ chelator q uin-2 MF are as follows. 1) Two sites in the CaM-nNOS and CaM-nPEP com plexes have a rate constant of 1 s(-1). 2) The remaining two sites hav e a rate constant of 18 s(-1) for CaM-nPEP and > 1000 s(-1) for CaM-nN OS. 3) Three sites have a rate constant of 1.6 s(-1) for CaM-skMLCK an d 0.15 s(-1) for CaM-skPEP. 4) The remaining site has a rate constant of 2 s(-1) for CaM-skPEP and > 1000 s(-1) for CaM-skMLCK. Comparison o f these rate constants with those determined for complexes between the peptides and tryptic fragments representing the C- or N-terminal lobe s of CaM indicate a mechanism for Ca2+ dissociation from CaM-nNOS of 2 C slow + 2N fast and from CaM-skMLCK of (2C + 1N) slow + 1N fast, Ca2 removal inactivates CaM-nNOS and CaM-skMLCK activities with respectiv e rate constants of > 10 s(-1) and 1 s(-1). CaM-nNOS inactivation is f it by a model in which rapid Ca2+ dissociation from the N-terminal lob e of CaM is coupled to enzyme inactivation and slower Ca2+ dissociatio n from the C-terminal lobe is coupled to dissociation of the CaM-nNOS complex. CaM-skMLCK inactivation is fit by a model in which the three slowly dissociating Ca2+-binding sites are coupled to both dissociatio n of the complex and enzyme inactivation.