厭氧菌Desulfovibrio gigas的三磷酸腺苷硫酸化酶的純化,特性以及初步的結晶學分析

Purification, Characterization, Preliminary x-ray diffraction analysis of ATP Sulfurylase from Desulfovibrio gigas.

李泱寰(Yang-Huan Lee)

指導教授 ： 陳俊榮

國立清華大學/理學院/物理學系/碩士(2013年)

摘要

三磷酸腺苷硫酸化酶(ATP Sulfurylase , EC 2.7.7.4)在細胞中將三磷酸腺苷(Adenosine-5'-triphosphate)以及硫酸催化為焦磷酸根(PPi)以及adenosine 5'-phosphosulfate (APS)，這個過程在細胞內硫酸還原作用中的同化(assimilatory)路徑以及異化(dissimilatory)路徑皆佔有重要的角色∘ 我們在厭氧菌Desulfovibrio gigas的週質中得到三磷酸腺苷硫酸化酶，以蛋白質N端定序以及液態層析質譜儀(LC/MS/MS)鑑定蛋白質，接著進行紫外線吸收光譜，同步輻射光源圓二色光譜(SRCD)分析三磷酸腺苷硫酸化酶的特性。並藉由懸滴蒸氣擴散法(hanging drop vapor diffusion method)得到三磷酸腺苷硫酸化酶的晶體，利用同步輻射進行X光繞射分析，解析度為2.8Å，Mattew’s coefficient為2.33(Å^3/Dalton)，solvent content為47.21%，每單位asymmetric unit的分子數為1， initial linear R-factor為0.064。 使用Molecular replacment和Heavy atom method進行蛋白質的結構鑑定的實驗正在進行中，並希望藉由進一步實驗得知酵素的催化機制。

關鍵字

三磷酸腺苷硫酸化酶 ； 異化硫酸鹽還原 ； 三磷酸腺苷硫酸化酶

並列摘要

ATP Sulfurylase (ATPS) catalyzes ATP (Adenosine-5'-triphosphate) and sulfate into adenosine 5'-phosphosulfate (APS) and pyrophosphate (PPi), which plays an important role in both assimmilatory and dissimlatory sulfate reduction. ATPS was purifued from Desulfovibrio gigas periplasmic part. SDS-page, UV wavescan, N-terminal sequencing, Synchrotron Radiation Circular Dichroism (SRCD) are used to identify and characterized ATP Sulfurylase. The crystallization condition was found and optimized to produce the crystal of ATP Sulfurylase. Xray diffraction of ATPS crystal allows structural determination that provides a better understanding of the zine containing enzyme. By the unit-cell parameter, the calculated Mattew’s coeffcient and solvent content are 2.33 (Å^3/Dalton) and 47.21%, respectively. Furthermore, Mattew’s coeffcient suggested that the presense of a monomer in the asymmetric unit. The initial linear R-factor is 0.064. The structure determination using MR and heavy atom method are in progress to elucidate its function.

並列關鍵字

ATP Sulfurylase ； dissimilatory sulfate reduction ； sulfate adenylyltransferase

參考文獻

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