Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin.
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 10(7) 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 10(6) 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.
Bowman
B F
BF
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
Peterson
J A
JA
Stull
J T
JT
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D005456:Fluorescent Dyes; D007700:Kinetics; D008297:Male; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010944:Plants / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013379:Substrate Specificity; D013737:Testis / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D002147:Calmodulin; D005456:Fluorescent Dyes; D010455:Peptides; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D009219:Myosin-Light-Chain Kinase; D002118:Calcium