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Three-dimensional structural analysis of non-pepsin-type acid proteinase using NMR and x-ray crystaliography

使用 NMR 和 X 射线晶体学分析非胃蛋白酶型酸性蛋白酶的三维结构

基本信息

批准号：
05680571
负责人：
TANOKURA Masaru
金额：
$ 1.34万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Proteinase A [EC3.4.23.6] secreted by the fungus Aspergillus niger var.macrosporus is a non-pepsin-type acid proteinase. It is distinctly different from ordinary pepsin-type aspartic proteinases in primary structure, substrate specificity and reactivity to pepstatin. It remains to be elucidated which residues participate in the catalysis of the enzyme and how the mechanism operates. To make clear these queries, the three-dimensional structure of proteinase A was studied by nuclear magnetic resonance (NMR) and X-ray crystallography. The pH dependence of NMR spectra indicated that a conformational change occurs around pH 4.5 and the protein denatures irreversibly at the pH higher than 6. Distribution of cross peaks in the fingerprint region indicated that the enzyme is rich in beta-sheet and contains a tightly packed core in the molecule. Homonuclear and heteronuclear 3D-NMR spectra were measured using isotope-labeled samples. During the experiment, neither autolysis nor denaturation occurred. The spectral analysis and resonance assignments are in progress. On the other hand, the crystals of proteinase A grown at the pH around 2 gave the intensity data up to about 1.5 * resolution with the Weissenberg camera at the station BL6A2 at the Photon Factory, National Laboratory for High Energy Physics. The electron density map was obtained using the data of three isomorphous heavy atom derivatives. It showed that the protein has the molecular shape like croissant.

蛋白酶A [EC3.4.23.6]是由尼日尔黑曲霉大孢变种分泌的一种非胃蛋白酶型酸性蛋白酶。它在一级结构、底物特异性和对胃蛋白酶抑制剂的反应性方面与普通胃蛋白酶型天冬氨酸蛋白酶有明显的不同。哪些残基参与了酶的催化作用以及该机制如何运作仍有待阐明。为了澄清这些疑问，蛋白酶A的三维结构的核磁共振（NMR）和X射线晶体学研究。NMR谱的pH依赖性表明，在pH 4.5附近发生构象变化，并且在pH高于6时蛋白质不可逆地变性。交叉峰在指纹区域的分布表明，该酶富含β-折叠，并在分子中含有紧密堆积的核心。使用同位素标记的样品测量均方和异方3D-NMR光谱。在实验过程中，既没有发生自溶也没有发生变性。频谱分析和共振分配正在进行中。另一方面，在约2的pH下生长的蛋白酶A的晶体用位于光子工厂（Photon Factory），国家高能物理实验室（National Laboratory for High Energy Physics）的BL 6A 2站的Weissenberg照相机给出了高达约1.5倍分辨率的强度数据。利用三种同晶重原子衍生物的数据得到了电子密度图。结果表明，该蛋白具有羊角面包的分子形态。