Divergent enzyme kinetics and structural properties of the two human mitochondrial creatine kinase isoenzymes

The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK an d sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is kno wn about uMtCK in general, or about sMtCK of human origin. Here we have het erologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occur red as octamers, which can dissociate into dimers. Distinct Stokes' radii o f uMtCK and sMtCK in solution were indicative for conformational difference s between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octame r structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 Angstrom, indicating a 'flattening' of the octam er on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (K-d > K-m) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower K-m for creatine (1.01 mM) and ATP (0.11 mM) as compare d to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).