利用光谱技术解析绿豆两种谷氨酸脱羧酶结构的研究

doi:10.3964/j.issn.1000-0593(2020)12-3953-10

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摘要：谷氨酸脱羧酶（GAD）可催化谷氨酸的α-脱羧反应以产生γ-氨基丁酸（GABA）。首先提取并纯化了两种绿豆[Vigna radiate（L.）] GAD并分析了它们的酶学性质。研究发现绿豆中的两种GAD均为二聚体，分子量分别为155 kDa（GAD1）和75 kDa（GAD2）。GAD2含有更多的α-螺旋和β-折叠结构，因而表现出更高稳定性。GAD1和GAD2的三级结构趋于“暴露态”，GAD1具有更强的分子柔性且更趋于暴露态，而GAD2的分子构象更致密保守。研究发现pH和温度引起的结构变化是GAD酶学性质降低的主要原因。Ca²⁺参与结合GAD的钙调蛋白结合结构域，并诱导形成了“埋入式”紧凑的分子结构。SDS可诱导的GAD分子结构解折叠降低了酶的活性。KI，MgSO₄，AgNO3和SDS显著抑制了GAD1和GAD2酶活性。吐温80，Ca²⁺和Cu²⁺可以显著激活GAD1和GAD2的酶学活性，而Fe²⁺仅增加GAD2的酶学活性。

关键词：谷氨酸脱羧酶；绿豆；结构；光谱

Abstract：Glutamate decarboxylase (GAD) catalyzes an α-decarboxylation reaction of glutamate to produce γ-aminobutyric acid (GABA). Interestingly, two kinds of mung bean [Vigna radiate (L.)] GADs were firstly extracted and purified in this research. Two GADs in mung bean were both dimers with a molecular mass of 155 kDa (GAD1) and 75 kDa (GAD2). The enzymatic properties of GAD1 and GAD2 were detected in this research. Infrared spectroscopy analysis revealed that more higher-ordered structure contents, α-helix and β-sheet structures, was found in GAD2, which determined the higher stability of GAD2. It was analyzed by Raman that the molecular structures of GAD1 and GAD2 are generally “exposed”. Fluorescence analysis revealed that GAD1 had a more flexible and exposed molecular structure, while the conformation of GAD2 was more compact and conservative. It was found that pH and temperature-induced structural change decreased the enzyme activity. Ca²⁺ was involved in binding the calmodulin-binding domain of GADs and induced a “buried” and compact structure. The unfolding of GAD induced by SDS, impaired the enzyme activity. KI, MgSO₄, AgNO₃, and SDS significantly inhibited GAD1 and GAD2 activities. Tween 80, Ca²⁺ and Cu²⁺ could significantly activate GAD1 and GAD2, and Fe²⁺ only increased GAD2 activity.

Key words：Glutamate decarboxylase; Mung bean; Structure; Spectroscopy

收稿日期: 2019-10-23 修订日期: 2020-09-25

中图分类号:

TS201.2

基金资助: NNSF of China (31301501) and National Key Research and Development Program of China (2017YFD0401203)

通讯作者: 王宪青，杨勇 E-mail: yangyong7904@163.com; xqwang1977@126.com

作者简介: WANG Xian-qing, (1977—), associate professor, College of Food Science，Heilongjiang Bayi Agricultural University e-mail: xqwang1977@126.com

引用本文:

王宪青，魏彤，杨勇，石彦国. 利用光谱技术解析绿豆两种谷氨酸脱羧酶结构的研究[J]. 光谱学与光谱分析, 2020, 40(12): 3953-3962.
WANG Xian-qing, WEI Tong, YANG Yong, SHI Yan-guo. Molecular Structure of Two Glutamate Decarboxylases From Mung Bean [Vigna Radiate (L.)] Analyzed by Spectroscopy. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2020, 40(12): 3953-3962.

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