人类线粒体肌酸激酶uMtCK的底物结合位点分析 <sup>*</sup>

doi:10.13523/j.cb.20180504

中国生物工程杂志

2018, Vol. 38

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

研究报告

人类线粒体肌酸激酶uMtCK的底物结合位点分析 ^*

孟浩毅¹,李丹阳³,孙正阳¹,杨兆勇³,张志斐^2,^***(

),袁丽杰^1,^***(

)

1 华北理工大学基础医学院河北省慢性疾病重点实验室唐山市慢性病临床基础研究重点实验室唐山 063000
2 华北理工大学药学院唐山 063000
3 中国医学科学院医药生物技术研究所北京 100050

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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摘要：

研究人类线粒体肌酸激酶uMtCK的结合位点,将其与底物肌酸和ATP结合有关的关键氨基酸进行突变,并对突变体进行酶动力学和圆二色谱数据分析,探讨这些关键氨基酸在底物识别和催化过程中的作用。结果显示,与野生酶相比,突变体Q313A和R336A的 $K m Cr$ 分别提高了2.6和2.9倍,k_cat下降了19%和55%;同样地,与ATP结合相关的突变体R125A和R287A分别使得 $K m ATP$ 升高了3.2和4.2,k_cat下降了72%和38%。以上结果表明突变体R125A、R287A、Q313A和R336A影响对底物的结合,同时也降低了酶促反应的速度。利用圆二色谱比较野生酶与不同突变体的二级结构并无明显变化,但进一步的结构模拟表明底物结合位点氨基酸在与底物之间的氢键对底物的识别和酶催化过程中发挥着重要作用。

关键词： 肌酸激酶; 定点突变; 酶动力学; 结构模拟

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

收稿日期: 2017-12-17 出版日期: 2018-06-05

ZTFLH:

Q55

基金资助: * 河北省自然科学基金(C2014209137);中国医学科学院医学与健康科技创新工程资助项目(2016-I2M-3-022);中国医学科学院医学与健康科技创新工程资助项目(2016-I2M-3-012)

通讯作者: 张志斐,袁丽杰 E-mail: zhifeiz@outlook.com;yuanlijie1970@163.com

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引用本文:

孟浩毅,李丹阳,孙正阳,杨兆勇,张志斐,袁丽杰. 人类线粒体肌酸激酶uMtCK的底物结合位点分析 ^*[J]. 中国生物工程杂志, 2018, 38(5): 24-32.

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.

链接本文:

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

图1 uMtCK三维结构

表1 定点突变设计的引物

图2 CK的同源序列比对

图3 uMtCK及其突变体的表达与纯化

图4 uMtCK及其突变体的最适温度和pH

	$K m Cr$ (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

表2 uMtCK及突变体催化动力学常数

表3 uMtCK及其突变体二级结构的含量

图5 uMtCK及其突变体的CD光谱

图6 uMtCK的肌酸和ATP底物结合腔

图7 uMtCK及其突变体的三维结构分析

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