Different mechanisms for Ca2+ dissociation from complexes of calmodulin with nitric oxide synthase or myosin light chain kinase.
We have determined the stoichiometry and rate constants for the dissociation of Ca2+ ion from calmodulin (CaM) complexes with rabbit skeletal muscle myosin light chain kinase (skMLCK), rat brain nitric oxide synthase (nNOS) or with the respective peptides (skPEP and nPEP) representing the CaM-binding domains in these enzymes. Ca2+ dissociation kinetics determined by stopped-flow fluorescence using the Ca2+ chelator quin-2 MF are as follows. 1) Two sites in the CaM-nNOS and CaM-nPEP complexes have a rate constant of 1 s-1. 2) The remaining two sites have a rate constant of 18 s-1 for CaM-nPEP and > 1000 s-1 for CaM-nNOS. 3) Three sites have a rate constant of 1.6 s-1 for CaM-skMLCK and 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 of these rate constants with those determined for complexes between the peptides and tryptic fragments representing the C- or N-terminal lobes of CaM indicate a mechanism for Ca2+ dissociation from CaM-nNOS of 2C slow + 2N fast and from CaM-skMLCK of (2C + 1N) slow + 1N fast. Ca2+ removal inactivates CaM-nNOS and CaM-skMLCK activities with respective rate constants of > 10 s-1 and 1 s-1. CaM-nNOS inactivation is fit by a model in which rapid Ca2+ dissociation from the N-terminal lobe of CaM is coupled to enzyme inactivation and slower Ca2+ dissociation 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 Ca(2+)-binding sites are coupled to both dissociation of the complex and enzyme inactivation.
Department of Physiology, University of Rochester Medical Center, New York 14642, USA.
A
A
Persechini
H D
HD
White
K J
KJ
Gansz
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011817:Rabbits; D051381:Rats
D002147:Calmodulin; D019001:Nitric Oxide Synthase; D009219:Myosin-Light-Chain Kinase; D002118:Calcium