A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle

Wen Ji Dong, Chien Kao Wang, Albert M. Gordon, Steven Rosenfeld, Herbert C. Cheung

Research output: Contribution to journalArticle

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Abstract

The kinetics of the binding of Ca2+ to the single regulatory site of cardiac muscle troponin was investigated by using troponin reconstituted from the three subunits, using a monocysteine mutant of troponin C (cTnC) labeled with the fluorescent probe 2-[(4'-(iodoacetamido)anilino]naphthalene-6- sulfonic acid (IAANS) at Cys-35. The kinetic tracings of binding experiments for troponin determined at free [Ca2+] > 1 μM were resolved into two phases. The rate of the fast phase increased with increasing [Ca2+], reaching a maximum of about 35 s-1 at 4 °C, and the rate of the slow phase was approximately 5 s-1 and did not depend on [Ca2+]. Dissociation of bound Ca2+ occurred in two phases, with rates of about 23 and 4 s-1. The binding and dissociation results obtained with the binary complex formed between cardiac troponin I and the IAANS-labeled cTnC mutant were very similar to those obtained from reconstituted troponin. The kinetic data are consistent with a three-step sequential model similar to the previously reported mechanism for the binding of Ca2+ to a cTnC mutant labeled with the same probe at Cys-84 (Dong et al. (1996) J. Biol. Chem. 271, 688-694). In this model, the initial binding in the bimolecular step to form the Ca2+- troponin complex is assumed to be a rapid equilibrium, followed by two sequential first-order transitions. The apparent bimolecular rate constant is 5.1 x 107 M-1 s-1, a factor of 3 smaller than that for cTnC. The rates of the first-order transitions are an order of magnitude smaller for troponin than for cTnC. These kinetic differences form a basis for the enhanced Ca2+ affinity of troponin relative to the Ca2+ affinity of isolated cTnC. Phosphorylation of the monocysteine mutant of troponin I by protein kinase A resulted in a 3-fold decrease in the bimolecular rate constant but a 2-fold increase in the two observed Ca2+ dissociation rates. These changes in the kinetic parameters are responsible for a 5-fold reduction in Ca2+ affinity of phosphorylated troponin for the specific site.

Original languageEnglish (US)
Pages (from-to)19229-19235
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number31
DOIs
StatePublished - Aug 1 1997
Externally publishedYes

Fingerprint

Troponin
Muscle
Myocardium
Kinetics
Troponin I
Sulfonic Acids
Rate constants
Troponin C
Phosphorylation
Cyclic AMP-Dependent Protein Kinases
Fluorescent Dyes
Kinetic parameters

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle. / Dong, Wen Ji; Wang, Chien Kao; Gordon, Albert M.; Rosenfeld, Steven; Cheung, Herbert C.

In: Journal of Biological Chemistry, Vol. 272, No. 31, 01.08.1997, p. 19229-19235.

Research output: Contribution to journalArticle

Dong, Wen Ji ; Wang, Chien Kao ; Gordon, Albert M. ; Rosenfeld, Steven ; Cheung, Herbert C. / A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 31. pp. 19229-19235.
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AU - Cheung, Herbert C.

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N2 - The kinetics of the binding of Ca2+ to the single regulatory site of cardiac muscle troponin was investigated by using troponin reconstituted from the three subunits, using a monocysteine mutant of troponin C (cTnC) labeled with the fluorescent probe 2-[(4'-(iodoacetamido)anilino]naphthalene-6- sulfonic acid (IAANS) at Cys-35. The kinetic tracings of binding experiments for troponin determined at free [Ca2+] > 1 μM were resolved into two phases. The rate of the fast phase increased with increasing [Ca2+], reaching a maximum of about 35 s-1 at 4 °C, and the rate of the slow phase was approximately 5 s-1 and did not depend on [Ca2+]. Dissociation of bound Ca2+ occurred in two phases, with rates of about 23 and 4 s-1. The binding and dissociation results obtained with the binary complex formed between cardiac troponin I and the IAANS-labeled cTnC mutant were very similar to those obtained from reconstituted troponin. The kinetic data are consistent with a three-step sequential model similar to the previously reported mechanism for the binding of Ca2+ to a cTnC mutant labeled with the same probe at Cys-84 (Dong et al. (1996) J. Biol. Chem. 271, 688-694). In this model, the initial binding in the bimolecular step to form the Ca2+- troponin complex is assumed to be a rapid equilibrium, followed by two sequential first-order transitions. The apparent bimolecular rate constant is 5.1 x 107 M-1 s-1, a factor of 3 smaller than that for cTnC. The rates of the first-order transitions are an order of magnitude smaller for troponin than for cTnC. These kinetic differences form a basis for the enhanced Ca2+ affinity of troponin relative to the Ca2+ affinity of isolated cTnC. Phosphorylation of the monocysteine mutant of troponin I by protein kinase A resulted in a 3-fold decrease in the bimolecular rate constant but a 2-fold increase in the two observed Ca2+ dissociation rates. These changes in the kinetic parameters are responsible for a 5-fold reduction in Ca2+ affinity of phosphorylated troponin for the specific site.

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