Kinetic studies of calcium and magnesium binding to troponin C.
The kinetic mechanism of calcium binding was investigated for the high-affinity calcium-magnesium sites of troponin C (TN-C), for the C-terminal fragment containing only the high-affinity sites (TR2) and for the TN-C:TN-I (where TN-I represents the inhibitory subunit of troponin) complex. Rate constants were measured by the change in fluorescence of the proteins labeled with 4-(N-iodoacetoxyethyl-N-methyl-7-nitrobenz-2-oxa-1,3-diazole at Cys 98. Rate constants for calcium dissociation were also measured using the fluorescent calcium chelating agent quin 2. Calcium binding to TR2 at 4 degrees C is a two-step process at each binding site. (formula; see text) A first order transition (k1 = 700 s-1) follows the formation of a weakly bound collision complex (K0 = 2.5 X 10(3) M-1). The two sits of the labeled protein are distinguishable because of a 2-4-fold difference in rate constants of calcium dissociation. The kinetic evidence is consistent with additive changes in structure induced by calcium binding to two identical or nearly identical high-affinity sites. The mechanism for TN-C:TN-I is similar to TR2. TN-C gave complex kinetic behavior for calcium binding but calcium dissociation occurred with the same rate constants found for TR2. Calcium binding to the high-affinity sites of TnC can be interpreted by the same mechanism as for TR2 but an additional reaction possibly arriving from calcium binding to the low-affinity sites leads to a high-fluorescence intermediate state which is detected by the fluorophore. The interactions between the two classes of sites are interpreted by a model in which calcium binding at the high-affinity sites reverses the fluorescence change induced by calcium binding at the low-affinity sites. Magnesium binding to the calcium-magnesium sites of TR2 and TN-C occurs by the same two-step binding mechanism with a smaller value for K0 and a 5-fold larger rate constant of dissociation.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008433:Mathematics; D010069:Oxadiazoles; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D014336:Troponin; D019209:Troponin C; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; D008274:Magnesium; D002118:Calcium