Effect of cysteine modification on creatine kinase aggregation.
We studied the effect of cysteine modification on creatine kinase (CK) aggregation as well as the kinetics of the process. We found that CK aggregation was modulated by different pH conditions in the presence of Zn2+, which is a CK aggregation trigger. The CK aggregation followed first-order kinetics, and this was effectively suppressed in acidic conditions. Even under the acidic condition, cysteine modification at the active site with using 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) induced conspicuous aggregation in a dose-dependent manner. This aggregation process is directly related with decreasing the change of transition free-energy (DeltaDeltaG(AG)). When dithiothreitol (DTT) was applied to the reaction system, the aggregates were significantly reduced: DTT treatment can fully reactivate (higher than 80%) the inactive CK that was separated from CK aggregates, whereas CK was completely inactivated by Zn2+ and DTNB. Some added osmolytes such as glycine and proline were able to successfully block CK aggregation by increasing the DeltaDeltaG(AG) as well as by suppressing the hydrophobic CK surface. Our study suggests the effect of cysteine modification on the unfavorable aggregation of CK and on the aggregation process that followed first-order kinetics with the accompanying changes of transitional free energy and disruptions of the hydrophobic surface. We also demonstrate the successful protocol to block the aggregation.
Zou
He-Chang
HC
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China.
Lü
Zhi-Rong
ZR
Wang
Ye-Jing
YJ
Zhang
Ying-Mei
YM
Zou
Fei
F
Park
Yong-Doo
YD
D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D003634:DDT / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D005998:Glycine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D011392:Proline / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects*; D020836:Protein Structure, Quaternary; D011817:Rabbits; D013816:Thermodynamics; D015032:Zinc / Q000494:pharmacology
D004228:Dithionitrobenzoic Acid; D011392:Proline; D003634:DDT; D003402:Creatine Kinase; D015032:Zinc; D003545:Cysteine; D005998:Glycine