Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin

Donald K. Blumenthal, James T. Stull

Research output: Contribution to journalArticle

199 Citations (Scopus)

Abstract

Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin to form the complex, Ca4 2+·calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca4 2+·calmodulin·enzyme. Formation of the holoenzyme follows simple hyperbolic kinetics, indicating a 1:1 stoichiometry of Ca4 2+·calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa 2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa 2+ and KCM were found to have values of 10 μM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.

Original languageEnglish (US)
Pages (from-to)5608-5614
Number of pages7
JournalBiochemistry
Volume19
Issue number24
StatePublished - 1980

Fingerprint

Skeletal Muscle Myosins
Myosin-Light-Chain Kinase
Calmodulin
Chemical activation
Calcium
Catalytic Domain
Holoenzymes
Equilibrium constants
Enzymes
Stoichiometry
Activation Analysis
Biological Phenomena
Kinetics
Enzyme Activation
Metals
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin. / Blumenthal, Donald K.; Stull, James T.

In: Biochemistry, Vol. 19, No. 24, 1980, p. 5608-5614.

Research output: Contribution to journalArticle

@article{2d2709009b1840cb8cf2ea4c97191233,
title = "Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin",
abstract = "Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin to form the complex, Ca4 2+·calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca4 2+·calmodulin·enzyme. Formation of the holoenzyme follows simple hyperbolic kinetics, indicating a 1:1 stoichiometry of Ca4 2+·calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa 2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa 2+ and KCM were found to have values of 10 μM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.",
author = "Blumenthal, {Donald K.} and Stull, {James T.}",
year = "1980",
language = "English (US)",
volume = "19",
pages = "5608--5614",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "24",

}

TY - JOUR

T1 - Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin

AU - Blumenthal, Donald K.

AU - Stull, James T.

PY - 1980

Y1 - 1980

N2 - Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin to form the complex, Ca4 2+·calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca4 2+·calmodulin·enzyme. Formation of the holoenzyme follows simple hyperbolic kinetics, indicating a 1:1 stoichiometry of Ca4 2+·calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa 2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa 2+ and KCM were found to have values of 10 μM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.

AB - Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin to form the complex, Ca4 2+·calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca4 2+·calmodulin·enzyme. Formation of the holoenzyme follows simple hyperbolic kinetics, indicating a 1:1 stoichiometry of Ca4 2+·calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa 2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa 2+ and KCM were found to have values of 10 μM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.

UR - http://www.scopus.com/inward/record.url?scp=0019182299&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0019182299&partnerID=8YFLogxK

M3 - Article

C2 - 6893940

AN - SCOPUS:0019182299

VL - 19

SP - 5608

EP - 5614

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 24

ER -