Creatine kinase isoenzymes specificities: histidine 65 in human CK-BB, a role in protein stability, not in catalysis

Creatine kinases (CK) play a prominent role in cell energy distribution thr ough an energy shuttle between mitochondria and other organelles. Human bra in CK was cloned and overexpressed in COS-7 cells. We then deleted His-65 a nd/or Pro-66 situated near the center of a flexible loop as shown by X-ray crystallography on mitochondrial and cytosolic CK. The Delta H65 mutant had nearly the same affinity for its substrates as wild isoenzyme, but its sta bility was very low. Unlike Delta H65, Delta H65P66 had a eightfold decreas ed affinity for creatine phosphate and was unable to dephosphorylate cycloc reatine phosphate. Our results demonstrate that, despite an overall similar shape of the proteins, this loop accounts for some subtle differences in i soenzyme functions. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.