Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33→Ala33, Asp60→Ala60, Ser62→Ala62, and Thr220→Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (ΔΔGT). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the Km values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations.
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Research Article|
April 11 2006
Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation
Zhong-liang Zheng;
Zhong-liang Zheng
*National Key Laboratory of Virology, Life Sciences College, Wuhan University, Wuhan 430072, China
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Mao-qing Ye;
Mao-qing Ye
*National Key Laboratory of Virology, Life Sciences College, Wuhan University, Wuhan 430072, China
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Zhen-yu Zuo;
Zhen-yu Zuo
*National Key Laboratory of Virology, Life Sciences College, Wuhan University, Wuhan 430072, China
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Zhi-gang Liu;
Zhi-gang Liu
*National Key Laboratory of Virology, Life Sciences College, Wuhan University, Wuhan 430072, China
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Keng-chang Tai;
Keng-chang Tai
†Department of Life Sciences, National Tsing Hua University, Hsinchu 30043, Taiwan
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Guo-lin Zou
Guo-lin Zou
1
*National Key Laboratory of Virology, Life Sciences College, Wuhan University, Wuhan 430072, China
1To whom correspondence should be addressed (email zouguolin@whu.edu.cn).
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Publisher: Portland Press Ltd
Received:
May 11 2005
Revision Received:
January 13 2006
Accepted:
January 17 2006
Accepted Manuscript online:
January 17 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 395 (3): 509–515.
Article history
Received:
May 11 2005
Revision Received:
January 13 2006
Accepted:
January 17 2006
Accepted Manuscript online:
January 17 2006
Citation
Zhong-liang Zheng, Mao-qing Ye, Zhen-yu Zuo, Zhi-gang Liu, Keng-chang Tai, Guo-lin Zou; Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation. Biochem J 1 May 2006; 395 (3): 509–515. doi: https://doi.org/10.1042/BJ20050772
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