Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme

Peter J. Kennelly; Joel C. Colburn; James Lorenzen; Arthur M. Edelman; James T. Stull; Edwin G. Krebs

doi:10.1016/0014-5793(91)80977-B

Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme

Peter J. Kennelly, Joel C. Colburn, James Lorenzen, Arthur M. Edelman, James T. Stull, Edwin G. Krebs

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

5′-p-Fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [¹⁴C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k₃= -0.040 min^-1 and -0.038 min^t-1, and K_i=0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.

Original language	English (US)
Pages (from-to)	217-220
Number of pages	4
Journal	FEBS Letters
Volume	286
Issue number	1-2
DOIs	https://doi.org/10.1016/0014-5793(91)80977-B
State	Published - Jul 29 1991

Keywords

5′-p-Fluorosulfonylbenzoyl adenosine
Autoinhibition
Calmodulin
Myosin light chain kinase
Pseudosubstrate

ASJC Scopus subject areas

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

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10.1016/0014-5793(91)80977-B

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Kennelly, P. J., Colburn, J. C., Lorenzen, J., Edelman, A. M., Stull, J. T., & Krebs, E. G. (1991). Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme. FEBS Letters, 286(1-2), 217-220. https://doi.org/10.1016/0014-5793(91)80977-B

Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme. / Kennelly, Peter J.; Colburn, Joel C.; Lorenzen, James et al.
In: FEBS Letters, Vol. 286, No. 1-2, 29.07.1991, p. 217-220.

Research output: Contribution to journal › Article › peer-review

Kennelly, PJ, Colburn, JC, Lorenzen, J, Edelman, AM, Stull, JT & Krebs, EG 1991, 'Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme', FEBS Letters, vol. 286, no. 1-2, pp. 217-220. https://doi.org/10.1016/0014-5793(91)80977-B

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abstract = "5′-p-Fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3= -0.040 min-1 and -0.038 mint-1, and Ki=0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.",

keywords = "5′-p-Fluorosulfonylbenzoyl adenosine, Autoinhibition, Calmodulin, Myosin light chain kinase, Pseudosubstrate",

author = "Kennelly, {Peter J.} and Colburn, {Joel C.} and James Lorenzen and Edelman, {Arthur M.} and Stull, {James T.} and Krebs, {Edwin G.}",

note = "Funding Information: Acknowledgements: We would like toacknowledget he help of Floyd Kennedy in the preparation of proteins and Maria Harrylock for assistancein the purification and analysisa nd Roger Wade for amino acid analyseso f the syntheticp eptidesu sedi n thesee xperimentsT. his work was supported in part by a postdoctoral fellowship from the Muscular Dystrophy Association awardedt o P.J.K.",

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doi = "10.1016/0014-5793(91)80977-B",

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AU - Lorenzen, James

AU - Edelman, Arthur M.

AU - Stull, James T.

AU - Krebs, Edwin G.

N1 - Funding Information: Acknowledgements: We would like toacknowledget he help of Floyd Kennedy in the preparation of proteins and Maria Harrylock for assistancein the purification and analysisa nd Roger Wade for amino acid analyseso f the syntheticp eptidesu sedi n thesee xperimentsT. his work was supported in part by a postdoctoral fellowship from the Muscular Dystrophy Association awardedt o P.J.K.

PY - 1991/7/29

Y1 - 1991/7/29

N2 - 5′-p-Fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3= -0.040 min-1 and -0.038 mint-1, and Ki=0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.

AB - 5′-p-Fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3= -0.040 min-1 and -0.038 mint-1, and Ki=0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.

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KW - Myosin light chain kinase

KW - Pseudosubstrate

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Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme

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