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

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 mu M 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 degrees 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 10(7) 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.