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.
Gross
M
M
Swiss Federal Institute of Technology, Institute for Cell Biology, ETH-Hönggerberg, Zürich.
Wallimann
T
T
D000818:Animals; D002645:Chickens; D003402:Creatine Kinase / Q000737:chemistry*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D011108:Polymers; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D011108:Polymers; D014364:Tryptophan; D003402:Creatine Kinase