Allosteric interactions in glycerol dehydratase. Purification of enzyme and effects of positive and negative cooperativity for glycerol.
Allosteric interactions in glycerol dehydratase. Purification of enzyme and effects of positive and negative cooperativity for glycerol.
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甘油脱水酶的变构相互作用。
DOI：
10.1016/0003-9861(74)90189-1
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发表时间：
1974
影响因子：
3.9
通讯作者：
B. Johnson
中科院分区：
文献类型：
--
作者：
A. Stroinski;J. Pawelkiewicz;B. Johnson
提出了一种从产气杆菌中纯化甘油脱水酶脱辅基酶（AB 复合物）的改进方法。达到了百倍净化。由于甘油对 AB 复合物的稳定作用，这种纯化成为可能。使用 Sephadex G-200 色谱法，在甘油存在的甘氨酸缓冲液中发现 AB 复合物的最高缔合度。与甘油相反，钾离子似乎削弱了将亚基 A 和 B 结合在一起的力。这可能是发挥甘油脱水酶活性所必需的钾的作用，因为全酶活性需要钾。从动力学研究看来，该酶在甘油结合位点方面表现出同养效应，可以从正协同性（在存在甘氨酸缓冲液的情况下）扩展到负协同性（在存在乙醇胺缓冲液的情况下）。在甘氨酸缓冲液中，酶上甘油结合位点之间的高协同性可以通过添加磷酸盐来消除。通过降低希尔系数的值和增加酶促反应的 V，磷酸盐似乎充当变构激活剂。
An improved method for the purification of the apoenzyme (AB complex) of glycerol dehydratase fromAerobacter aerogenesis presented. One hundredfold purification Was achieved. This purification was possible due to stabilization of the AB complex by glycerol. Using chromatography on Sephadex G-200, the highest degree of association of AB complex was found in glycine buffer in the presence of glycerol.Potassium ions, in contrast to glycerol, seem to weaken the forces which bind subunits A and B together. This may be the action of potassium necessary for the performance of glycerol dehydratase activity, since potassium is required for holoenzyme activity.From kinetic studies it appears that the enzyme exhibits homotrophic effects with regard to glycerol binding sites, which can he extended from positive (in the presence of glycine buffer) to negative (in the presence of ethanolamine buffer) cooperativity. The high cooperativity between glycerol binding sites on the enzyme, in glycine buffer, can be abolished by the addition of phosphate. By decreasing the value of the Hill coefficient and increasing theVof the enzymatic reaction, phosphate seems to act as an allosteric activator.