title: Hydrogen peroxide targets the cysteine at the active site and irreversibly inactivates creatine kinase.
abstract: In our study, we showed that at a relatively low concentration, H(2)O(2) can irreversibly inactivate the human brain type of creatine kinase (HBCK) and that HBCK is inactivated in an H(2)O(2) concentration-dependent manner. HBCK is completely inactivated when incubated with 2mM H(2)O(2) for 1h (pH 8.0, 25°C). Inactivation of HBCK is a two-stage process with a fast stage (k(1)=0.050 ± 0.002 min(-1)) and a slow (k(2)=0.022 ± 0.003 min(-1)) stage. HBCK inactivation by H(2)O(2) was affected by pH and therefore we determined the pH profile of HBCK inactivation by H(2)O(2). H(2)O(2)-induced inactivation could not be recovered by reducing agents such as dl-dithiothreitol, N-acetyl-L-cysteine, and l-glutathione reduced. When HBCK was treated with DTNB, an enzyme substrate that reacts specifically with active site cysteines, the enzyme became resistant to H(2)O(2). HBCK binding to Mg(2+)ATP and creatine can also prevent H(2)O(2) inactivation. Intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence data showed no tertiary structure changes after H(2)O(2) treatment. The thiol group content of H(2)O(2)-treated HBCK was reduced by 13% (approximately 1 thiol group per HBCK dimer, theoretically). For further insight, we performed a simulation of HBCK and H(2)O(2) docking that suggested the CYS283 residue could interact with H(2)O(2). Considering these results and the asymmetrical structure of HBCK, we propose that H(2)O(2) specifically targets the active site cysteine of HBCK to inactivate HBCK, but that substrate-bound HBCK is resistant to H(2)O(2). Our findings suggest the existence of a previously unknown negative form of regulation of HBCK via reactive oxygen species.
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, PR China.
authors.forename: Chang
authors.initials: C
authors.lastname: Li
authors.forename: Shiqing
authors.initials: S
authors.lastname: Sun
authors.forename: Daeui
authors.initials: D
authors.lastname: Park
authors.forename: Hyoung Oh
authors.initials: HO
authors.lastname: Jeong
authors.forename: Hae Young
authors.initials: HY
authors.lastname: Chung
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Liu
authors.forename: Hai-Meng
authors.initials: HM
authors.lastname: Zhou
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000032:analysis; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D020134:Catalytic Domain; D052276:Creatine Kinase, BB Form / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004229:Dithiothreitol / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000009:adverse effects*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000009:adverse effects; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D000817:Anilino Naphthalenesulfonates; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; C027132:1-anilino-8-naphthalenesulfonate; D004228:Dithionitrobenzoic Acid; D006861:Hydrogen Peroxide; D052276:Creatine Kinase, BB Form; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol; D000111:Acetylcysteine