KINETIC-STUDIES OF CALCIUM-BINDING TO THE REGULATORY SITE OF TROPONIN-C FROM CARDIAC-MUSCLE

We have studied the kinetics of the structural transitions induced by calcium binding to the single, regulatory site of cardiac troponin C b y measuring the rates of calcium-mediated fluorescence changes with a monocysteine mutant of the protein (C35S) specifically labeled at Cys- 84 with the fluorescent probe -[4'-(iodoacetamido)anilino]naphthalene- 6-sulfonic acid. At 4 degrees C, the binding kinetics determined in th e presence of Mg2+ was resolved into two phases with positive amplitud e, which were completed in less than 100 ms, The rate of the fast phas e increased linearly with [Ca2+] reaching a maximum of similar to 590 s(-1), and that of the slow phase was approximately 100 s(-1) and did not depend on Ca2+ concentration, Dissociation of bound Ca2+ from the regulatory site occurred with a rate of 102 s(-1), whereas the dissoci ation from the two high affinity sites was about two orders of magnitu de slower, These results are consistent with the following scheme for the binding of Ca2+ to the regulatory site: [GRAPHICS] where the aster isks denote states with enhanced fluorescence, The apparent second-ord er rate constant for calcium binding is K(o)k(1) = 1.4 x 10(8) M(-1) s (-1). The two first-order transitions occur with observed rates of k(1 ) + k(-1) approximate to 590 s(-1) and k(2) + k(-2) approximate to 100 s(-1), and the binding of Ca2+ to the regulatory site is not a simple diffusion-controlled reaction. These transitions provide the first in formation on the rates of Ca2+-induced conformational changes involvin g helix movements in the regulatory domain.