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) bind ing to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the i nfluence of protein concentration, pH, ionic strength and substrate co ncentration on the enzyme adsorption. An analysis of our own data as w ell as of the data from the literature is consistent with the adsorpti on 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 bi nding 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 (K-a = 0.39 mM at ionic st rength I = 7.5 mM and 5 degrees C); after protonation its affinity inc reased many times (K-ah = 39 nM). Determination of mitCK adsorption ca pacity by another method at pH 7.4, when the enzyme is almost protonat ed, gave K-ah = 15 nM. The effect of ionic strength on mitCK adsorptio n may be described in terms of Debye-Huckel's theory for activity coef ficients 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. Ex tramitochondrial ATP (or ADP) shifted greatly the equilibrium between the adsorbed and the free mitCK towards the solubilized state, since i n the adsorbed protein the external ligands had access to four binding sites and in the free protein to eight sites.