Substrate-binding Site of Ubiquitous Mitochondrial Creatine Kinase from <i>Homo sapiens</i>

doi:10.13523/j.cb.20180504

China Biotechnology

2018, Vol. 38

Issue (5): 24-32 DOI: 10.13523/j.cb.20180504

Substrate-binding Site of Ubiquitous Mitochondrial Creatine Kinase from Homo sapiens

Hao-yi MENG¹,Dan-yang LI³,Zheng-yang SUN¹,Zhao-yong YANG³,Zhi-fei ZHANG^2,^***(

),Li-jie YUAN^1,^***(

)

1 Hebei Key Laboratory for Chronic Diseases, Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases,School of Basic Medical Science, North China University of Science and Technology, Tangshan 063210,China
2 School of Pharmacy, North China University of Science and Technology, Tangshan 063210,China
3 Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China

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Abstract

To investigate the function of the key residues in the binding site of human uMtCK onto substrate creatine (Cr) and ATP, nine mutants of human uMtCK were constructed by using site-directed mutagenesis and analyzed by enzyme kinetics, circular dichroism spectra and protein structural simulation. The $K m Cr$ value of Q313A and R336A and $K m ATP$ values of R125A and R287A were 2.6, 2.9, 3.2 and 4.2 folds higher than that of the wild-type (WT), and the k_cat values of Q313A, R125A, R287A and R336A 19%, 55%, 72% and 38% lower than that of WT, respectively. Meanwhile, no significant changes of circular dichroism spectra of the mutants were observed compared with WT. The structure model analysis indicated that the mutations altered the hydrogen bonds between the key amino acid residues and substrates, thus creating an unfavorable local environment for substrate binding and catalysis, as reflected by the increased K_m and decreased k_cat of mutants, consequently inactivation of human uMtCK.

Key words： Creatine kinase Site-directed mutagenesis Enzyme kinetics Structural simulation

Received: 17 December 2017 Published: 05 June 2018

ZTFLH:

Q55

Corresponding Authors: Zhi-fei ZHANG,Li-jie YUAN E-mail: zhifeiz@outlook.com;yuanlijie1970@163.com

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	Hao-yi MENG
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Cite this article:

Hao-yi MENG,Dan-yang LI,Zheng-yang SUN,Zhao-yong YANG,Zhi-fei ZHANG,Li-jie YUAN. Substrate-binding Site of Ubiquitous Mitochondrial Creatine Kinase from Homo sapiens. China Biotechnology, 2018, 38(5): 24-32.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180504 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I5/24

Fig.1 The overall structure of uMtCK a) The crystal structure of uMtCK is formed by an octameric fashion (b)N-terminal domain (residues 1-95, red), C-terminal domain (residues 120-379, green), linker region (residues 96-119, yellow)

Table 1 Primer used for construction of site-directed mutations

Fig.2 Amino acid sequence alignment of CKs The residues of uMtCK interacting with the binding site of the creatine and ATP are marked with stars

Fig.3 SDS-PAGE analysis of purified recombinant uMtCK and mutants M: Protein marker; 1-11: uMtCK, uMtCKR125A, uMtCKR127A, uMtCKE227A, uMtCKR231A, uMtCKR287A, uMtCKQ313A, uMtCKV320A, uMtCKR315A, uMtCKD330A, uMtCKR336A

Fig.4 Effect of optimum temperature and pH on the acitivity of uMtCK and mutants

	$K m Cr$ (mmol/L)	$K m ATP$ (mmol/L)	k_cat(/s)
uMtCK	11.2±0.5	1.4±0.2	47±1.7
R125A	16.2±0.9	4.5±0.4	13±0.9
R287A	13.7±1.5	5.9±1.2	29±2.2
Q313A	29.4±0.7	1.5±0.5	38±3.1
R336A	32.4±2.1	3.7±0.2	21±1.8

Table 2 Kinetic parameters obtained for uMtCK and mutants

Table 3 The content of secondary structure in uMtCK and mutants

Fig.5 Circular dichroism of uMtCK and mutants

Fig.6 A close-up view of creatine binding pocket and ATP binding site Creatine (blue), ATP (red), the structural figure were generated with PyMOL (http://www.pymol.org)

Fig.7 Structural analysis of the uMtCK and mutants (a),(b) The models of the creatine and its surroundings (c), (d) The models of the ATP and its surroundings The entire protein is shown in cartoon. ATP, creatine, and residues interacting with them are displayed in stick and labeled. The dotted lines indicate hydrogen-bonding network around the residue at creatine or ATP. The structural figures were generated with PyMOL (http://www.pymol.org)


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