Identification of two aspartates and a glutamate essential for the activity of endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus.
Identification of two aspartates and a glutamate essential for the activity of endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus.
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鉴定褶皱链霉菌内切 β-N-乙酰氨基葡萄糖苷酶 H 活性所必需的两种天冬氨酸和一种谷氨酸。
DOI：
10.1006/abbi.1994.1247
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发表时间：
1994
影响因子：
3.9
通讯作者：
P. Lad
中科院分区：
文献类型：
--
作者：
B. Schmidt;E. Ashizawa;A. Jarnagin;S. Lynn;G. Noto;L. Woodhouse;D. Estell;P. Lad
为了鉴定内切-β-N-乙酰氨基葡萄糖苷酶 H (Endo H) 活性所必需的基团，通过寡核苷酸定点诱变，将所有 8 个谷氨酸残基、所有 19 个天冬氨酸和两种色氨酸分别单独替换为谷氨酰胺、天冬酰胺和苯丙氨酸。仅发现变体 D170N、D172N 和 E174Q 的比活性显着低于野生型 Endo H。另一种变体 D173N 未产生可检测量的蛋白质。发现野生型酶具有钟形 pH 活性曲线，该曲线保留在必需天冬氨酸突变体中，但 E174Q 失去了曲线的基本 pH 边缘，表明 E174 是催化所需的质子供体基团的良好候选者。无法明确确定活动所需的一般基础；然而，在必需的天冬氨酸中，D172 是唯一在其他相关葡萄糖苷酶中保守的天冬氨酸。
In order to identify groups essential for the activity of endo-beta-N-acetylglucosaminidase H (Endo H), all 8 glutamate residues, all 19 aspartates, and both tryptophans were individually substituted with glutamines, asparagines, and phenylalanines, respectively, by oligonucleotide site-directed mutagenesis. Only variants D170N, D172N, and E174Q were found to have specific activities significantly less than wild-type Endo H. Another variant, D173N, did not produce detectable amounts of protein. Wild-type enzyme was found to have a bell-shaped pH activity profile, which was retained in the essential aspartate mutants, but E174Q lost the basic pH limb of the curve, indicating that E174 is good candidate for the proton donating group necessary for catalysis. The general base needed for activity could not be unambiguously identified; although, of the essential aspartates, D172 is the only one conserved in other related glucosidases.