What is the clinical evidence from this study?

Intervention: Phosphorylation by protein kinase A vs. Unphosphorylated troponin. Primary outcome: Ca2+ binding kinetics and affinity.

What question did this study set out to answer?

To determine the kinetic mechanism of Ca2+ binding to and dissociation from the single regulatory site of reconstituted cardiac troponin, and to assess how troponin I phosphorylation alters these kinetics.

August 1, 1997Journal of Biological ChemistryOpen Access

A Kinetic Model for the Binding of Ca2+ to the Regulatory Site of Troponin from Cardiac Muscle

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Key result

Phosphorylation of the monocysteine mutant of troponin I by protein kinase A resulted in a 3-fold decrease in the bimolecular rate constant and a 5-fold reduction in Ca2+ affinity.

Key Points

To determine the kinetic mechanism of Ca2+ binding to and dissociation from the single regulatory site of reconstituted cardiac troponin, and to assess how troponin I phosphorylation alters these kinetics.
Reconstituted cardiac troponin from its three subunits using a monocysteine troponin C (cTnC) mutant labeled with the fluorescent reporter IAANS at Cys-35.
Measured Ca2+ association and dissociation rates at 4 °C across free Ca2+ concentrations above 1 µM using stopped-flow fluorescence.
Assessed changes in binding constants and dissociation rates following phosphorylation of the troponin I subunit by protein kinase A.
Ca2+ binding followed biphasic kinetics fitting a three-step sequential model with an apparent bimolecular rate constant of 5.1 × 10^7 M^-1 s^-1 and dissociation rates of ~23 s^-1 and 4 s^-1.
First-order conformational transition rates were an order of magnitude slower in full troponin than in isolated cTnC, underlying the higher Ca2+ affinity of the complete complex.
Protein kinase A phosphorylation of troponin I caused a 3-fold decrease in the bimolecular rate constant and a 2-fold increase in dissociation rates, producing a 5-fold reduction in overall regulatory Ca2+ affinity.

Structured PICO

Population

Reconstituted cardiac muscle troponin using a monocysteine mutant of troponin C (cTnC) labeled with the fluorescent probe IAANS at Cys-35

Exposure

Ca2+ binding and phosphorylation by protein kinase A

Comparator

Isolated cTnC and unphosphorylated troponin

Outcome

Kinetics of Ca2+ binding and dissociationsurrogate

Phosphorylation of cardiac troponin I by protein kinase A reduces the Ca2+ affinity of the regulatory site by altering both binding and dissociation kinetics.

Discussion

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Implication

Refines PKA-troponin I kinetics in mutants; leaves open extension to native protein or human myocardium.

Cite This Study

Dong et al. (1997) studied this question. Phosphorylation by protein kinase A vs. Unphosphorylated troponin was evaluated on Ca2+ binding kinetics and affinity. Phosphorylation of the monocysteine mutant of troponin I by protein kinase A resulted in a 3-fold decrease in the bimolecular rate constant and a 5-fold reduction in Ca2+ affinity.

synapsesocial.com/papers/6a9b7727d6bcd4d1ccf4f8ea https://doi.org/10.1074/jbc.272.31.19229

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A Kinetic Model for the Binding of Ca2+ to the Regulatory Site of Troponin from Cardiac Muscle

Key Points

Structured PICO

Discussion

Implication

Cite This Study

A Kinetic Model for the Binding of Ca2+ to the Regulatory Site of Troponin from Cardiac Muscle

Key Points

Structured PICO

Discussion

Implication

Cite This Study

Also Consider

Also Consider