小分子量腺苷酸基琥珀酸合成酶的结构与功能及其产物的药理学研究
发布时间：2018-08-28 06:38
【摘要】：腺苷酸基琥珀酸合成酶(Adenylosuccinate synthase,简称AdSS)存在于所有生物体内,它可以在GTP去磷酸生成GDP时用IMP和L-Asp合成s-AMP。基因组研究结果证明几乎所有的常温环境中生存的生物来源AdSS含有一条430-460个氨基酸残基的肽链,而部分嗜热古细菌和病毒来源AdSS比常温生物来源AdSS缺失了90-120个氨基酸。这些小分子量AdSS可以归为的一类。迄今为止,对常温生物来源AdSS的结构和功能的研究成果比较丰富,而对小分子量AdSS的结构和功能还未知。 本人的导师谢勇副研究员在前期研究中成功测定了嗜热生物来源AdSS：PhAdSS的2.5A分辨率晶体结构,这是嗜热生物来源AdSS晶体结构的首例,为研究小分子量AdSS的结构和功能的特性奠定基础。在导师的指导下,本人承担了PhAdSS的空间结构和功能特殊性的相互关系的研究。通过开展的酶促动力学分析和共结晶结构分析,成功测定了PhAdSS的米氏常数和PhAdSS与GTP和IMP共结晶后的2.7A分辨率的晶体结构。发现了PhAdSS具有很好的热稳定性,在323K环境中显示几乎等同于常温生物来源AdSS的活性,依据酶促动力学参数以及共结晶结构分析的结果论述了PhAdSS独特的空间结构特征和底物结合机制。 s-AMP作为AdSS的产物,是AMP的结构类似物之一,可能具有激活AMPK改善糖脂代谢的药理学活性,但未发现相关的研究报道。为了证实这一猜想,本人开展了S-AMP的药理学功能初探。研究结果证实s-AMP具有较好的降脂活性且细胞毒性低,可以作为降血脂新药的先导化合物开展作用机制和代谢机制的研究。PhAdSS可以作为新型生物催化剂实现s-AMP的大规模合成,为s-AMP的提供原料保障。 在开展PhAdSS的结构与功能研究的同时,还承担了人体来源Tks4的C-端SH3结构域的晶体学研究,成功实现了Tks4的C-端SH3结构域大肠杆菌表达、纯化和结晶化。利用上海同步辐射光源生物大分子光束线站测定了2.3A分辨率的X射线衍射数据,计算了Tks4的C-端SH3结构域晶体的晶体学参数。从晶体内分子的排列方式预测了Tks4的C-端SH3结构域具有独特的相互作用模式。
[Abstract]:Adenylate succinic acid synthase (Adenylosuccinate synthase,) is present in all organisms. It can synthesize s-AMPs by IMP and L-Asp when GTP dephosphoric acid produces GDP. The results of genomic studies showed that almost all living organisms in normal temperature environment had a peptide chain of 430-460 amino acid residues, while some thermophilic paleobacteria and virus AdSS lacked 90-120 amino acids compared with normal temperature biological source AdSS. These small molecular weight AdSS can be grouped into a class. Up to now, researches on the structure and function of AdSS at room temperature are abundant, but the structure and function of AdSS with small molecular weight are unknown. In previous studies, my mentor, Xie Yong, has successfully determined the 2.5A resolution crystal structure of thermophilic biological source AdSS:PhAdSS, which is the first example of thermophilic biological source AdSS crystal structure. It lays a foundation for the study of the structure and function of small molecular weight AdSS. Under the guidance of the tutor, I undertook the research on the relationship between spatial structure and functional particularity of PhAdSS. The Michlet constant of PhAdSS and the crystal structure of PhAdSS cocrystallized with GTP and IMP were determined successfully by enzymatic kinetic analysis and eutectic structure analysis. It was found that PhAdSS had good thermal stability and showed almost the same activity as AdSS at 323K. Based on the results of enzymatic kinetic parameters and eutectic structure analysis, the unique spatial structure and substrate binding mechanism of PhAdSS were discussed. As a product of AdSS, s-AMP is one of the structural analogues of AMP. It may have pharmacological activity to activate AMPK to improve glycolipid metabolism, but no related studies have been reported. In order to confirm this conjecture, the pharmacological function of S-AMP was studied. The results show that s-AMP has good lipid-lowering activity and low cytotoxicity. It can be used as a leading compound to develop the mechanism of action and metabolism of new lipid-lowering drugs. PhAdSS can be used as a new biocatalyst to realize the large-scale synthesis of s-AMP. Provide raw material guarantee for s-AMP. While the structure and function of PhAdSS were studied, the crystallographic study of the C-terminal SH3 domain of human Tks4 was carried out. The expression, purification and crystallization of the C-terminal SH3 domain of Tks4 were successfully realized. The X-ray diffraction data of 2.3A resolution were measured by using the Shanghai Synchrotron radiation Source Biomacromolecule Beam Line Station, and the crystallographic parameters of the SH3 domain crystal at the C-terminal of Tks4 were calculated. It is predicted that the C-terminal SH3 domain of Tks4 has a unique interaction mode from the arrangement of molecules in the crystal.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
本文编号：2208587
[Abstract]:Adenylate succinic acid synthase (Adenylosuccinate synthase,) is present in all organisms. It can synthesize s-AMPs by IMP and L-Asp when GTP dephosphoric acid produces GDP. The results of genomic studies showed that almost all living organisms in normal temperature environment had a peptide chain of 430-460 amino acid residues, while some thermophilic paleobacteria and virus AdSS lacked 90-120 amino acids compared with normal temperature biological source AdSS. These small molecular weight AdSS can be grouped into a class. Up to now, researches on the structure and function of AdSS at room temperature are abundant, but the structure and function of AdSS with small molecular weight are unknown. In previous studies, my mentor, Xie Yong, has successfully determined the 2.5A resolution crystal structure of thermophilic biological source AdSS:PhAdSS, which is the first example of thermophilic biological source AdSS crystal structure. It lays a foundation for the study of the structure and function of small molecular weight AdSS. Under the guidance of the tutor, I undertook the research on the relationship between spatial structure and functional particularity of PhAdSS. The Michlet constant of PhAdSS and the crystal structure of PhAdSS cocrystallized with GTP and IMP were determined successfully by enzymatic kinetic analysis and eutectic structure analysis. It was found that PhAdSS had good thermal stability and showed almost the same activity as AdSS at 323K. Based on the results of enzymatic kinetic parameters and eutectic structure analysis, the unique spatial structure and substrate binding mechanism of PhAdSS were discussed. As a product of AdSS, s-AMP is one of the structural analogues of AMP. It may have pharmacological activity to activate AMPK to improve glycolipid metabolism, but no related studies have been reported. In order to confirm this conjecture, the pharmacological function of S-AMP was studied. The results show that s-AMP has good lipid-lowering activity and low cytotoxicity. It can be used as a leading compound to develop the mechanism of action and metabolism of new lipid-lowering drugs. PhAdSS can be used as a new biocatalyst to realize the large-scale synthesis of s-AMP. Provide raw material guarantee for s-AMP. While the structure and function of PhAdSS were studied, the crystallographic study of the C-terminal SH3 domain of human Tks4 was carried out. The expression, purification and crystallization of the C-terminal SH3 domain of Tks4 were successfully realized. The X-ray diffraction data of 2.3A resolution were measured by using the Shanghai Synchrotron radiation Source Biomacromolecule Beam Line Station, and the crystallographic parameters of the SH3 domain crystal at the C-terminal of Tks4 were calculated. It is predicted that the C-terminal SH3 domain of Tks4 has a unique interaction mode from the arrangement of molecules in the crystal.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
【参考文献】
相关期刊论文 前1条
1 苏岩;郭鹏;季宇彬;;AMPKγ基因沉默对AMPK活化及调血脂作用的影响[J];药学学报;2013年06期
，本文编号：2208587
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