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J. Biol. Chem., Vol. 282, Issue 16, 12022-12029, April 20, 2007

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The Generation of the Oxidized Form of Creatine Kinase Is a Negative Regulation on Muscle Creatine Kinase^*

Tong-Jin Zhao, Yong-Bin Yan, Yang Liu, and Hai-Meng Zhou^¶||1

From the Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China, the State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China, the ^¶Protein Science Laboratory of the Ministry of Education, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China, and the ^||Yangtze Delta Region Institute of Tsinghua University, Jiaxing, 314050 Zhejiang, China

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 Cys⁷⁴ and Cys¹⁴⁶ 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.

Received for publication, November 7, 2006 , and in revised form, December 12, 2006.

^* This work was supported by funds from the National Natural Science Foundation (30670460, 30500084, 30429001, and 30221003) and by funds from Jiaxing, Zhejiang. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 86-10-6278-4700; Fax: 86-10-6277-2245; E-mail: zhm-dbs{at}mail.tsinghua.edu.cn.

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