LOCALIZATION OF THE ACTIVE-SITE OF PYRUVATE-CARBOXYLASE BY ELECTRON-MICROSCOPIC EXAMINATION OF AVIDIN-ENZYME COMPLEXES
LOCALIZATION OF THE ACTIVE-SITE OF PYRUVATE-CARBOXYLASE BY ELECTRON-MICROSCOPIC EXAMINATION OF AVIDIN-ENZYME COMPLEXES
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DOI：
10.1111/j.1432-1033.1983.tb07448.x
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
KEECH, DB
中科院分区：
文献类型：
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作者：
JOHANNSSEN, W;ATTWOOD, PV;KEECH, DB
用电镜研究了用鸡肝丙酮酸羧化酶滴定亲和素上生物素结合位点不同阶段的负染色酶-亲和素复合物。当亲和素与酶的比例在2:1和1:2之间时，酶-亲和素复合物形成线性的、无支链的聚合物；超过这些限制，只能看到单酶四聚体。观察到的单个亲和素分子似乎具有长方体结构。聚合物中酶四聚体的取向和尺寸表明，在单分子中观察到的四面体结构被保留了下来。从聚合物的结构和对单个酶-亲和素复合物的观察来看，酶上的生物素基团可能位于每个亚基的外表面，靠近亚基间连接处，可能在3纳米范围内。
Negatively stained enzyme-avidin complexes, seen at different stages of the titration of the biotin-binding sites on avidin with chicken liver pyruvate carboxylase, were studied using EM. Formation of linear, unbranched polymers of the enzyme-avidin complex occurs when the ratio of avidin to enzyme is between 2:1 and 1:2; beyond these limits only single enzyme tetramers are visible. The single avidin molecules observed seem to have a cuboid structure. The orientation and dimensions of the enzyme tetramers within the polymers indicate that the tetrahedron-like structure, observed in the single molecules, has been preserved. From the structure of the polymers and the observation of single enzyme-avidin complexes, the biotin groups on the enzyme are probably located on the external faces of each subunit close to and probably within 3 nm of the intersubunit junction.