Free radical-induced inactivation of creatine kinase: influence on the octameric and dimeric states of the mitochondrial enzyme (Mib-CK).
Free radicals of X-ray-induced water radiolysis, either directly or indirectly via their reaction products, reduce the activity of both dimeric cytoplasmic muscle-type creatine kinase (MM-CK) and octameric mitochondrial creatine kinase (Mi-CK) to virtually zero. Similarly values of the characteristic D(37)-dose of enzyme inactivation (dose required to reduce enzyme activity to 37%) were found for the two isoenzymes of CK under identical conditions. Octamer stability was not significantly affected within the dose range considered. However, both the dissociation of octamers into dimers by a transition-state analogue complex (TSAC), and the reassociation of the dimers into octamers, showed dose-dependent reduction. Binding of the TSAC to the active centre was found to protect the enzyme against inactivation by free radicals. No protection was observed for the radiation-induced decrease of the endogenous tryptophan fluorescence. The experimental results are in line with the following interpretation: (i) the reduction of Mi(b)-CK dimer association is due to free radical-induced modification of Trp-264, situated at the dimer/dimer interface; (ii) the active-site Trp-223 is not a prime target for free radicals and is not involved in the inactivation of the enzyme; (iii) the inhibition of TSAC-induced dissociation of Mi(b)-CK, like enzyme inactivation, is primarily due to a modification of the active-site Cys-278.
Koufen
P
P
Department of Biology, University of Konstanz, Box M638, D-78457 Konstanz, Germany.
Rück
A
A
Brdiczka
D
D
Wendt
S
S
Wallimann
T
T
Stark
G
G
D000818:Animals; D002645:Chickens; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000528:radiation effects; D003600:Cytosol / Q000201:enzymology; D019281:Dimerization; D004307:Dose-Response Relationship, Radiation; D005609:Free Radicals; D007527:Isoenzymes; D008928:Mitochondria / Q000201:enzymology*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014965:X-Rays
D005609:Free Radicals; D007527:Isoenzymes; D017382:Reactive Oxygen Species; D003402:Creatine Kinase