THE N-TERMINAL HEPTAPEPTIDE OF MITOCHONDRIAL CREATINE-KINASE IS IMPORTANT FOR OCTAMERIZATION

Mitochondrial creatine kinase (Mi-CK) isoenzymes, in contrast to cytos olic CKs, form octameric molecules composed of four stable dimers. Oct amers and dimers are interconvertible. Removal of the N-terminal penta peptide of chicken cardiac Mi-CK (Mi(b)-CK) by limited proteolysis dra stically destabilized the octamer. The role of the charged amino acids within the N-terminal heptapeptide was studied in detail by progressi vely substituting the four charged residues by uncharged ones. In thes e altered proteins, the octamer/dimer ratio at equilibrium conditions was shifted toward the dimer. Also, the in vitro dissociation rate of octamers into dimers was increased in correlation to the number of cha rged residues eliminated. Point mutant E4Q, with only one positive cha rged amino acid removed, already displayed a 50-fold higher equilibriu m constant and a 13-fold increased dissociation rate compared to wild- type Mi(b)-CK. Mutant 4-7, having all four charged residues in the N-t erminal heptapeptide substituted, showed a 100-fold higher equilibrium constant and a 146-fold increased dissociation rate. The correspondin g values for double mutant E4Q/K5L were intermediate between the singl e and quadruple mutants. This strongly suggests that the charged amino acids in the N-terminal heptapeptide of Mi(b)-CK, and therefore ionic interactions mediated by the N-terminal moiety, play an important rol e in forming and stabilizing the octameric molecule. The role of dimer -octamer interconversion in vivo as a possible regulator of contact si te formation and of mitochondrial oxidative phosphorylation is discuss ed.