阿拉伯芥 AtMAPR2 重組蛋白質之過氧化氫酶活性及形成寡聚體條件之探討

Peroxidase activity and oligomerization analysis of Arabidopsis AtMAPR2 recombinant protein

江旻壕(Min-Hao Chiang)

指導教授 ： 楊健志

國立臺灣大學/生命科學院/生化科技學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10733

摘要

阿拉伯芥中的 AtMAPR2 蛋白質屬於功能多元的 MAPR 家族, 是阿拉伯芥 MSBP1 的同源蛋白質。在過往研究中，以紫外線-可見光光譜及電子自旋共振光譜分析利用大腸桿菌表現之 AMAPR2 重組蛋白質，發現其具有與血基質結合的特性。本研究是探討 AtMAPR2 重組蛋白質之酵素活性及形成寡聚體之特性。使用 ABTS 作為過氧化氫反應之基質，發現 AtMAPR2 具有過氧化氫酶活性 (kcat = 0.960 s-1, Km = 0.168 mM)。將 AtMAPR2 之三個酪胺酸基團 (Y38, Y44, Y92) 進行點突變，探討其對血基質結合能力與酵素活性之影響。發現 Y38F、 Y44F、Y38F/Y44F 等點突變組別會使血基質結合能力輕微下降，而 Y92F、Y44F/Y92F 組別則會使其血基質結合能力劇烈下降。上述點突變影響酵素活性的趨勢與其降低蛋白質與血基質結合能力有一致趨勢。利用粒徑篩析層析法分析蛋白質組成，可觀察到當存在過量血基質時，AtMAPR2 可形成單體、二聚體、三聚體、四聚體及高聚合體。利用沉降速度分析式超高速離心分析蛋白質的分子量及聚合度也發現，添加血基質會造成部分 AtMAPR2 往高分子量聚合。在穿透式電子顯微鏡下，可看到呈現球體及纖維狀的聚合體，可能由數十至數百個單體所組成。根據圓二色光譜分析的結果顯示，添加血基質會輕微影響 AtMAPR2 的二級結構。由於蛋白質序列及二級結構與現有的過氧化氫酶不相似，AtMAPR2 有可能是阿拉伯芥中一種新型態的過氧化氫酶。

關鍵字

AtMAPR ； 血基質 ； 血基質結合蛋白 ； 過氧化氫酶 ； 寡聚體 ； 阿拉伯芥

並列摘要

AtMAPR2, a protein homologous to MSBP1 from Arabidopsis, belongs to a functional diversified protein family of MAPR. Recombinant AtMAPR2 produced in E.coli was shown to bind heme using UV-Vis spectroscopy and electron paramagnetic resonance (EPR) spectroscopy in previous study. In this study, the enzymatic and oligomeric properties of recombinant AtMAPR2 were investigated. I found that AtMAPR2 possessed peroxidase activity in vitro when using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as substrate (kcat = 0.960 s-1, Km = 0.168 mM). Role of three Tyr residues (Y38, Y44 and Y92) of AtMAPR2 were studied using site-directed mutagenesis. The heme-binding ability of Y38F, Y44F and Y38F/Y44F were slightly decreased, while that of mutants Y92F and Y44F/Y92F reduced significantly, according to the UV-Vis spectra. The peroxidases activities of AtMAPR2 mutants agreed with their heme-binding ability, with Y38F, Y44F and Y38F/Y44F having smaller effect, and Y92F and Y44F/Y92F were disturbed dramatically. Using size-exclusion chromatography (SEC), oligomeric forms of AtMAPR2 corresponded to dimer, trimer, tetramer and even higher-order oligomer were observed when supplemented with excess hemin. In sedimentation velocity analytical ultracentrifugation (SV-AUC), addition of heme to AtMAPR2 would cause a shift in equilibrium towards the larger species. It is likely that even higher-order oligomers, up to more than hundreds of monomers, were present in either globular or filamentous shape as revealed by TEM. The secondary structure of AtMAPR2 was slightly affected by different hemin concentrations using circular dichroism (CD) spectroscopy. With little sequence and structure similar to present peroxidase, AtMAPR2 could be a novel peroxidase of Arabidopsis.

並列關鍵字

AtMAPR ； heme ； hemoprotein ； peroxidase ； oligomer ； Arabidopsis

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