The N-terminal heptapeptide of mitochondrial creatine kinase is important for octamerization.
Mitochondrial creatine kinase (Mi-CK) isoenzymes, in contrast to cytosolic CKs, form octameric molecules composed of four stable dimers. Octamers and dimers are interconvertible. Removal of the N-terminal pentapeptide of chicken cardiac Mi-CK (Mib-CK) by limited proteolysis drastically destabilized the octamer. The role of the charged amino acids within the N-terminal heptapeptide was studied in detail by progressively substituting the four charged residues by uncharged ones. In these 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 charged residues eliminated. Point mutant E4Q, with only one positive charged amino acid removed, already displayed a 50-fold higher equilibrium constant and a 13-fold increased dissociation rate compared to wild-type Mib-CK. Mutant 4-7, having all four charged residues in the N-terminal heptapeptide substituted, showed a 100-fold higher equilibrium constant and a 146-fold increased dissociation rate. The corresponding values for double mutant E4Q/K5L were intermediate between the single and quadruple mutants. This strongly suggests that the charged amino acids in the N-terminal heptapeptide of Mib-CK, and therefore ionic interactions mediated by the N-terminal moiety, play an important role in forming and stabilizing the octameric molecule. The role of dimer-octamer interconversion in vivo as a possible regulator of contact site formation and of mitochondrial oxidative phosphorylation is discussed.
Kaldis
P
P
Swiss Federal Institute of Technology, Institute for Cell Biology, ETH-Hönggerberg, Zürich.
Furter
R
R
Wallimann
T
T
D000595:Amino Acid Sequence; D001704:Biopolymers / Q000737:chemistry; D003402:Creatine Kinase / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D013379:Substrate Specificity
D001704:Biopolymers; D010446:Peptide Fragments; D003402:Creatine Kinase