小分子量腺苷酸基琥珀酸合成酶的結(jié)構(gòu)與功能及其產(chǎn)物的藥理學(xué)研究
發(fā)布時(shí)間：2018-08-28 06:38
【摘要】：腺苷酸基琥珀酸合成酶(Adenylosuccinate synthase,簡(jiǎn)稱AdSS)存在于所有生物體內(nèi),它可以在GTP去磷酸生成GDP時(shí)用IMP和L-Asp合成s-AMP�；蚪M研究結(jié)果證明幾乎所有的常溫環(huán)境中生存的生物來(lái)源AdSS含有一條430-460個(gè)氨基酸殘基的肽鏈,而部分嗜熱古細(xì)菌和病毒來(lái)源AdSS比常溫生物來(lái)源AdSS缺失了90-120個(gè)氨基酸。這些小分子量AdSS可以歸為的一類(lèi)。迄今為止,對(duì)常溫生物來(lái)源AdSS的結(jié)構(gòu)和功能的研究成果比較豐富,而對(duì)小分子量AdSS的結(jié)構(gòu)和功能還未知。 本人的導(dǎo)師謝勇副研究員在前期研究中成功測(cè)定了嗜熱生物來(lái)源AdSS：PhAdSS的2.5A分辨率晶體結(jié)構(gòu),這是嗜熱生物來(lái)源AdSS晶體結(jié)構(gòu)的首例,為研究小分子量AdSS的結(jié)構(gòu)和功能的特性奠定基礎(chǔ)。在導(dǎo)師的指導(dǎo)下,本人承擔(dān)了PhAdSS的空間結(jié)構(gòu)和功能特殊性的相互關(guān)系的研究。通過(guò)開(kāi)展的酶促動(dòng)力學(xué)分析和共結(jié)晶結(jié)構(gòu)分析,成功測(cè)定了PhAdSS的米氏常數(shù)和PhAdSS與GTP和IMP共結(jié)晶后的2.7A分辨率的晶體結(jié)構(gòu)。發(fā)現(xiàn)了PhAdSS具有很好的熱穩(wěn)定性,在323K環(huán)境中顯示幾乎等同于常溫生物來(lái)源AdSS的活性,依據(jù)酶促動(dòng)力學(xué)參數(shù)以及共結(jié)晶結(jié)構(gòu)分析的結(jié)果論述了PhAdSS獨(dú)特的空間結(jié)構(gòu)特征和底物結(jié)合機(jī)制。 s-AMP作為AdSS的產(chǎn)物,是AMP的結(jié)構(gòu)類(lèi)似物之一,可能具有激活A(yù)MPK改善糖脂代謝的藥理學(xué)活性,但未發(fā)現(xiàn)相關(guān)的研究報(bào)道。為了證實(shí)這一猜想,本人開(kāi)展了S-AMP的藥理學(xué)功能初探。研究結(jié)果證實(shí)s-AMP具有較好的降脂活性且細(xì)胞毒性低,可以作為降血脂新藥的先導(dǎo)化合物開(kāi)展作用機(jī)制和代謝機(jī)制的研究。PhAdSS可以作為新型生物催化劑實(shí)現(xiàn)s-AMP的大規(guī)模合成,為s-AMP的提供原料保障。 在開(kāi)展PhAdSS的結(jié)構(gòu)與功能研究的同時(shí),還承擔(dān)了人體來(lái)源Tks4的C-端SH3結(jié)構(gòu)域的晶體學(xué)研究,成功實(shí)現(xiàn)了Tks4的C-端SH3結(jié)構(gòu)域大腸桿菌表達(dá)、純化和結(jié)晶化。利用上海同步輻射光源生物大分子光束線站測(cè)定了2.3A分辨率的X射線衍射數(shù)據(jù),計(jì)算了Tks4的C-端SH3結(jié)構(gòu)域晶體的晶體學(xué)參數(shù)。從晶體內(nèi)分子的排列方式預(yù)測(cè)了Tks4的C-端SH3結(jié)構(gòu)域具有獨(dú)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R96
本文編號(hào)：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R96
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 蘇巖;郭鵬;季宇彬;;AMPKγ基因沉默對(duì)AMPK活化及調(diào)血脂作用的影響[J];藥學(xué)學(xué)報(bào);2013年06期
，本文編號(hào)：2208587
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