The generation of the oxidized form of creatine kinase is a negative regulation on muscle creatine kinase.
Muscle creatine kinase (CK) is a crucial enzyme in energy metabolism, and it exists in two forms, the reduced form (R-CK) and the oxidized form (O-CK). In contrast with R-CK, O-CK contained an intrachain disulfide bond in each subunit. Here we explored the properties of O-CK and its regulatory role on muscle CK. The intrachain disulfide bond in O-CK was demonstrated to be formed between Cys(74) and Cys(146) by site-directed mutagenesis. Biophysical analysis indicated that O-CK showed decreased catalytic activity and that it might be structurally unstable. Further assays through guanidine hydrochloride denaturation and proteolysis by trypsin and protease K revealed that the tertiary structure of O-CK was more easily disturbed than that of R-CK. Surprisingly, O-CK, unlike R-CK, cannot interact with the M-line protein myomesin through biosensor assay, indicating that O-CK might have no role in muscle contraction. Through in vitro ubiquitination assay, CK was demonstrated to be a specific substrate of muscle ring finger protein 1 (MURF-1). O-CK can be rapidly ubiquitinated by MURF-1, while R-CK can hardly be ubiquitinated, implying that CK might be degraded by the ATP-ubiquitin-proteasome pathway through the generation of O-CK. The results above were further confirmed by molecular modeling of the structure of O-CK. Therefore, it can be concluded that the generation of O-CK was a negative regulation of R-CK and that O-CK might play essential roles in the molecular turnover of MM-CK.
Zhao
Tong-Jin
TJ
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Yan
Yong-Bin
YB
Liu
Yang
Y
Zhou
Hai-Meng
HM
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry*; D004220:Disulfides; D019286:Endopeptidase K / Q000737:chemistry; D006147:Guanine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014357:Trypsin / Q000494:pharmacology; D025801:Ubiquitin / Q000737:chemistry
D004220:Disulfides; D025801:Ubiquitin; D006147:Guanine; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D014357:Trypsin; D019286:Endopeptidase K; D010100:Oxygen