TY - JOUR
T1 - A kinetic model for the binding of Ca2+ to the regulatory site of troponin from cardiac muscle
AU - Dong, Wen Ji
AU - Wang, Chien Kao
AU - Gordon, Albert M.
AU - Rosenfeld, Steven S.
AU - Cheung, Herbert C.
PY - 1997/8/1
Y1 - 1997/8/1
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.
AB - 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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U2 - 10.1074/jbc.272.31.19229
DO - 10.1074/jbc.272.31.19229
M3 - Article
C2 - 9235915
AN - SCOPUS:0030790186
SN - 0021-9258
VL - 272
SP - 19229
EP - 19235
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 31
ER -