Kinetic control of the dissociation pathway of calmodulin-peptide complexes.
The mechanism of dissociation reactions induced by calcium chelators has been studied for complexes of Drosophila calmodulin with target peptides, including four derived from the skeletal muscle myosin light chain kinase target sequence. Reactions were monitored by fluorescence stopped-flow techniques using a variety of intrinsic probes and the indicator Quin2. For most of the complexes, apparently biphasic kinetics were observed in several fluorescence parameters. The absence of any obvious relationship between dissociation rates and peptide affinities implies kinetic control of the dissociation pathway. A general mechanism for calcium and peptide dissociation was formulated and used in numerical simulation of the experimental data. Unexpectedly, the rate of the slowest step decreases with increasing [peptide]/[calmodulin] ratio. Numerical simulation shows this step could contain a substantial contribution from a reversible relaxation process (involving the species Ca2-calmodulin-peptide), convolved with the following step (loss of C-terminal calcium ions). The results indicate the potentially key kinetic role of the partially calcium-saturated intermediate species. They show that subtle changes in the peptide sequence can have significant effects on both the dissociation rates and also the dissociation pathway. Both effects could contribute to the variety of regulatory behavior shown by calmodulin with different target enzymes.
Brown
S E
SE
Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Martin
S R
SR
Bayley
P M
PM
D000634:Aminoquinolines; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004331:Drosophila melanogaster; D004533:Egtazic Acid; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014862:Wasp Venoms / Q000737:chemistry / Q000378:metabolism
D000634:Aminoquinolines; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; C065738:MLCK peptide; D004533:Egtazic Acid; C037500:mastoparan; C071123:mastoparan X; C035739:Quin2; D002118:Calcium