A model of mitochondrial creatine kinase binding to membranes: adsorption constants, essential amino acids and the effect of ionic strength.
The quantitative aspects of mitochondrial creatine kinase (mitCK) binding to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the influence of protein concentration, pH, ionic strength and substrate concentration on the enzyme adsorption. An analysis of our own data as well as of the data from the literature is consistent with the adsorption site of the octameric mitCK being composed of 4 amino acid residues with pK = 8.8 in the free enzyme. The pK value changes to 9.8 upon binding of the protein to the membrane. Lysine is suggested as the main candidate to form the adsorption site of mitCK. Deprotonated octameric mitCK easily dissociated from the membrane (Ka = 0.39 mM at ionic strength I = 7.5 mM and 5 degrees C); after protonation its affinity increased many times (Kah = 39 nM). Determination of mitCK adsorption capacity by another method at pH 7.4, when the enzyme is almost protonated, gave Kah = 15 nM. The effect of ionic strength on mitCK adsorption may be described in terms of Debye-Hückel's theory for activity coefficients assuming the charges of the interacting species to be +4 and -4. The dissociation constant for the mitCK-membrane complex at pH 7.4 and I = 0 was evaluated by different approaches as approx. 1 nM. Extramitochondrial ATP (or ADP) shifted greatly the equilibrium between the adsorbed and the free mitCK towards the solubilized state, since in the adsorbed protein the external ligands had access to four binding sites and in the free protein to eight sites.
Fedosov
S N
SN
Department of Chemistry, Aarhus University, Denmark.
Belousova
L V
LV
Plesner
I W
IW
D000327:Adsorption; D000818:Animals; D002417:Cattle; D003402:Creatine Kinase / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007038:Hypotonic Solutions; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008961:Models, Structural; D008962:Models, Theoretical
D007038:Hypotonic Solutions; D046911:Macromolecular Substances; D003402:Creatine Kinase