Creatine kinases (CK) are key enzymes in energy metabolism by catalyzing the reversible transphosphorylation of creatine (Cr) by ATP. It has recently shown that mitochondrial associated creatine kinase (mt-CK) play a central role on preventing mitochondrial ROS generation in rat brain by an ADP re-cycling process. CK isoenzymes are very susceptible to free radical damage and are inactivated by superoxide radicals and peroxynitrite in a concentration-dependent and irreversible manner. In this work we investigated the effects of a pro-oxidant milieu on the specific activity of mt-CK using different concentrations of hydrogen peroxide (H₂O₂). The mitochondria were isolated from mice brain and the specific mt-CK activities were assayed photometrically in the direction of creatine formation in a reaction medium containing H₂O₂ (0-100 μM). Our results shown that the enzyme activity display a dose-dependent decline, without previous incubation, starting with 20 μM of H₂O₂ and reaching about 75% of total inhibition with 100μM of H₂O₂. A more potent inhibition was obtained when the mitochondrial enzyme preparation was pre-incubated for 10 minutes with 100μM H₂O₂. Under these conditions the mt-CK activity was undetectable. These results suggest that the catalytic activity may render the mt-CK less sensible to H₂O₂ inhibition by oxidation of essential residues. In conclusion, this study shows that H₂O₂ has important effects on mt-CK enzymatic activity.

Supported by: FAPERJ, CNPq