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

Research output: Contribution to journalArticle

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Abstract

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-sulfonicacid-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 × 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 × 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
Volume267
Issue number8
StatePublished - Mar 15 1992

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Skeletal Muscle Myosins
Myosin-Light-Chain Kinase
Calmodulin
Chemical activation
Rabbits
Kinetics
Isomerization
Peptides
Phosphotransferases
Association reactions
Phosphorylation
Spinacia oleracea
Sulfonic Acids
Substrates
Amides
Fluorescence
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin. / Bowman, Bonita F.; Peterson, Julian A.; Stull, James T.

In: Journal of Biological Chemistry, Vol. 267, No. 8, 15.03.1992, p. 5346-5354.

Research output: Contribution to journalArticle

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