研究生: |
陳珈丰 Chen, Jia-Fong. |
---|---|
論文名稱: |
中華眼鏡蛇毒L型胺基酸氧化酶受質專一性探討 The amino acid substrate specificity of L-amino acid oxidase (LAAO) from Taiwan cobra (Naja atra) |
指導教授: |
吳文桂
Wu, Wen-Guey |
口試委員: |
簡昆鎰
Chien, Kun-Yi 李紹禎 Lee, Shao-Chen |
學位類別: |
碩士 Master |
系所名稱: |
生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology |
論文出版年: | 2019 |
畢業學年度: | 107 |
語文別: | 中文 |
論文頁數: | 84 |
中文關鍵詞: | L型胺基酸氧化酶 、中華眼鏡蛇L型胺基酸氧化酶 |
外文關鍵詞: | L-amino acid oxidase, LAAO, Naja atra L-amino acid oxidase, NA-LAAO, Taiwan cobra ( Naja atra) |
相關次數: | 點閱:2 下載:0 |
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本篇論文主要探討粗蛇毒的L型胺基酸氧化酶相對活性測試與中華眼鏡蛇L型胺基酸氧化酶(NA-LAAO)的受質專一性。在粗蛇毒中,龜殼花的LAAO含量較蝙蝠蛇科的LAAO多,但活性卻沒有較強,而在受質為Ile時,龜殼花LAAO相對活性較蝙蝠蛇科的LAAO高。利用 FPLC將中華眼鏡蛇粗蛇毒使用不同的管柱分離純化出NA-LAAO,由米氏曲線得到NA-LAAO在11種胺基酸下的Km、Vmax、kcat及受質專一性,NA-LAAO與其他蛇毒LAAO相同,在疏水性胺基酸有較高的受質專一性,於binding pocket中,NA-LAAO胺基酸序列第242號胺基酸與紅口蝮LAAO(CR-LAAO)及山蝰LAAO(DR-LAAO)不同,DR-LAAO及CR-LAAO在該位置胺基酸為His,NA-LAAO為Ser,在Arg受質專一性結果NA-LAAO為DR-LAAO的兩倍,中華眼鏡蛇與孟加拉眼鏡蛇於演化及地理位置上較相近,但於中華眼鏡蛇L型胺基酸氧化酶(NA-LAAO)與孟加拉眼鏡蛇L型胺基酸氧化酶(NK-LAAO)的受質專一性結果卻不盡相同,因此利用蛋白質結構、胺基酸序列比對與受質專一性去探討NA-LAAO與NK-LAAO的差異,並且提出中華眼鏡蛇L型胺基酸氧化酶會影響mTORC1及IDO-1進而使人體免疫系統受到抑制的假說。
SV-LAAO, also known as snake venom L-amino acid oxidase, is a high molecular weight flavoenzyme which catalyzes L-amino acids and produce α-keto acids, ammonia and H2O2. In this study, we study the relative activity of snake venom L-amino acid oxidase (SV-LAAO) from various snake venom and the substrate specificity of Naja atra L-amino acid oxidase (NA-LAAO). Although Trimeresurus mucrosquamatus venom contains more LAAO than elapidae venom: Naja atra (NA), Naja kaouthia (NK), Naja nivea (NN), the activity of TM-LAAO is lower than NA-LAAO, NK-LAAO, NN-LAAO. We purified NA-LAAO and utilized the Michealis-Menten curve to calculate Km, Vmax, kcat and substrate specificity with 11 kinds of amino acids. Compared with other SV-LAAO, NA-LAAO has stronger activity toward hydrophobic amino acids, such as Leu, Phe and Met. The binding pocket of NA-LAAO is different from Calloselasma rhodostoma LAAO (CR-LAAO) and Daboia russelii LAAO (DR-LAAO) by residue 242. DR-LAAO and CR-LAAO has His but NA-LAAO has Ser at residue 242. In addition, NA-LAAO has stronger activity toward Arg than DR-LAAO and CR-LAAO. Naja atra is closely related to Naja kaouthia in phylogenetic and geographic location so we compare the substrate specificity and 3D-structure of NA-LAAO with Naja kaouthia L-amino acid oxidase (NK-LAAO). According to the sequence alignment and structural comparison, NA-LAAO is different from NK-LAAO by 12 residues in sequence and the Y-shape channel of NA-LAAO adopts more hydrophobicity than NK-LAAO. Given that Leu and Arg are the upstream signals of mTORC1 and mTORC1 which can affect T-cell proliferation and activation, IDO-1(Indoleamine 2,3-dioxygenase 1) can also catalyzis Trp and its product can make vasodilation. Consequently, our group proposed that NA-LAAO catalyzes Leu, Arg, Trp to affect mTORC1 and together with IDO-1 inhibit the human immune system and influence the blood pressure.
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