Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin

Bonita F. Bowman, Julian A. Peterson, James T. Stull

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36 Scopus citations


Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4- maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS- amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 107 M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 106 M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process.

Original languageEnglish (US)
Pages (from-to)5346-5354
Number of pages9
JournalJournal of Biological Chemistry
Issue number8
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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