TY - JOUR
T1 - Exploration of the polar microenvironment around the reactive cysteine in rabbit muscle creatine kinase
AU - He, Hua Wei
AU - Li, Jie
AU - Zhao, Tong Jin
AU - Ma, Yong
AU - Shi, Feng
AU - Zhou, Hai Meng
N1 - Funding Information:
This investigation was supported by funds from the National Natural Science Foundation of China (nos. 30500084 and 30221003), and funds from Jiaxing, Zhejiang.
PY - 2007/10/1
Y1 - 2007/10/1
N2 - The polar microenvironment around the reactive Cys283 of rabbit muscle creatine kinase was explored using kinetic analysis of substrates reaction in the presence of modifiers. In the present study, three specific sulphydryl reagents, 5,5′-dithiobis(2-nitrobenzoic acid), 6,6′-dithiodinicotinic acid and 2,2′-dithiodipyridine, were applied as modifiers to react with Cys283 of creatine kinase. The inactivation kinetics of creatine kinase by the modifiers was analyzed. The microscopic rate constants for reactions of the modifiers with free enzyme and enzyme-substrate complexes were also determined. The results suggested that the inactivation rate of creatine kinase by 5,5′-dithiobis(2-nitrobenzoic acid) was the fastest, followed by 6,6′-dithiodinicotinic acid and then 2,2′-dithiodipyridine. Interestingly, 5,5′-dithiobis(2-nitrobenzoic acid) and 6,6′-dithiodinicotinic acid functioned as non-complexing modifiers, while 2,2′-dithiodipyridine did a complexing modifier. The results here indicated that the electrophilic group was predominant around Cys283, and that the presence of substrates seemed to have different effects on the inactivation reactions of creatine kinase by the three modifiers. Furthermore, the findings in this study may provide a novel explanation for the low pKa value of Cys283.
AB - The polar microenvironment around the reactive Cys283 of rabbit muscle creatine kinase was explored using kinetic analysis of substrates reaction in the presence of modifiers. In the present study, three specific sulphydryl reagents, 5,5′-dithiobis(2-nitrobenzoic acid), 6,6′-dithiodinicotinic acid and 2,2′-dithiodipyridine, were applied as modifiers to react with Cys283 of creatine kinase. The inactivation kinetics of creatine kinase by the modifiers was analyzed. The microscopic rate constants for reactions of the modifiers with free enzyme and enzyme-substrate complexes were also determined. The results suggested that the inactivation rate of creatine kinase by 5,5′-dithiobis(2-nitrobenzoic acid) was the fastest, followed by 6,6′-dithiodinicotinic acid and then 2,2′-dithiodipyridine. Interestingly, 5,5′-dithiobis(2-nitrobenzoic acid) and 6,6′-dithiodinicotinic acid functioned as non-complexing modifiers, while 2,2′-dithiodipyridine did a complexing modifier. The results here indicated that the electrophilic group was predominant around Cys283, and that the presence of substrates seemed to have different effects on the inactivation reactions of creatine kinase by the three modifiers. Furthermore, the findings in this study may provide a novel explanation for the low pKa value of Cys283.
KW - Chemical modification
KW - Creatine kinase
KW - Cysteine
KW - Inactivation
KW - Kinetics
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U2 - 10.1016/j.ijbiomac.2007.05.004
DO - 10.1016/j.ijbiomac.2007.05.004
M3 - Article
C2 - 17592740
AN - SCOPUS:34548222651
SN - 0141-8130
VL - 41
SP - 361
EP - 368
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
IS - 4
ER -