The presence of bound cyanide in the naturally inactivated form of nitrate reductase of Chlorella vulgaris.
The presence of bound cyanide in the naturally inactivated form of nitrate reductase of Chlorella vulgaris.
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小球藻自然失活形式的硝酸还原酶中存在结合氰化物。
DOI：
10.1016/s0021-9258(19)42221-7
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发表时间：
1974
期刊：
影响因子：
--
通讯作者：
L. P. Solomonson
中科院分区：
文献类型：
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作者：
G. Lorimer;H. Gewitz;W. Völker;L. P. Solomonson
小球藻（Chlorella vulgarisis）的NADH-硝酸盐氧化还原酶（EC 1.6.6.1）在NADH和氰化物存在下，转化为一种无活性的形式，这种形式很容易被铁氰化物重新激活。用H14 CN进行的实验表明，失活过程与每单位失活酶0.066纳摩尔氰化物与蛋白质的牢固结合有关。这种氰化物结合与灭活量成线性比例。在不存在NADH或等效还原剂的情况下，没有牢固的氰化物结合，也没有发生失活。当酶被铁氰化物重新激活时，结合的14 C被释放。在细胞破碎前用氨处理几个小时后，粗细胞提取物含有主要为无活性形式的硝酸还原酶。对细胞破碎方法的严格检查表明，酶的失活确实发生在体内。经300倍纯化后，该体内失活酶的活化导致每单位酶活化释放0.066 nmole HCN。硝酸还原酶在体内的失活过程涉及到还原酶与氰化物形成牢固的络合物，还原酶与HCN的反应式为：Er+ HCN（ka）/HCN/（kd）Er-HCN测得ka为1.25 × 10 ~（-6）m ~（-1）min ~（-1），kd为4.5 × 10 ~（-4）min ~（-1），解离常数Kd= 3.6 × 10 ~（-10）m ~（-1）。
In the presence of NADH and cyanide, NADH-nitrate oxidoreductase (EC 1.6.6.1) fromChlorella vulgarisis converted to an inactive form which is readily reactivated by ferricyanide. Experiments with H14CN indicated that the inactivation process is associated with the firm binding to the protein of 0.066 nmole of cyanide per unit of enzyme inactivated. This cyanide binding was linearly proportional to the amount of inactivation. No firm cyanide binding and no inactivation occurred in the absence of NADH or an equivalent reductant. The bound14C was released when the enzyme was reactivated by ferricyanide. After the cells had been treated with ammonia for several hours prior to disruption, the crude cell extracts contained the nitrate reductase primarily in the inactive form. Critical examination of the methods of cell disruption suggested that the inactivation of the enzyme had truly occurredin vivo. After 300-fold purification, activation of thisin vivoinactivated enzyme resulted in the release of 0.066 nmole of HCN per unit of enzyme activated. We conclude that the inactivation of nitrate reductasein vivoinvolves the formation of a firmly bound complex of reduced enzyme and cyanide.The reaction between reduced enzyme and HCN may be written:Er+ HCN (ka)/⇄/(kd)Er-HCNThe measured value forkawas 1.25x106m-1min-1, forkd, 4.5x10-4min-1, giving a dissociation constant,Kd= 3.6x10-10m.