Kinetics of assembly and dissociation of the mitochondrial creatine kinase octamer. A fluorescence study

The dissociation of octameric mitochondrial creatine kinase (Mi-CK) into dimers induced by the transition-state analogue complex (TSAC) mixture (creatine+Mg(2+)+ADP+NO3-) is accompanied by a large (25.2%) decrease in Trp fluorescence. This effect is caused by a Trp residue situated at the dimer-dimer interface within the octamer, which becomes susceptible to solvent quenching upon octamer dissociation. Octamer formation, induced by adding excess EDTA to TSAC-dissociated Mi-CK, involves a transient tetrameric species, whereas the dissociation reaction proceeds in a one-step, all-or-none fashion. From fluorescence spectroscopic investigations of the octamer formation and dissociation reactions, a first-order dissociation rate constant of 0.19 min-1 and a bimolecular association rate constant of 318 M-1 s-1 at 30 degrees C were obtained. The octamers formed after EDTA addition can be dissociated again by lowering the temperature to 4 degrees C, indicating a substantial hydrophobic contribution to the interactions stabilizing the octamer.