STUDIES ON ATP CITRATE LYASE OF RAT LIVER .3. REACTION MECHANISM
STUDIES ON ATP CITRATE LYASE OF RAT LIVER .3. REACTION MECHANISM
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DOI：
10.1093/oxfordjournals.jbchem.a128753
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发表时间：
1968-01-01
影响因子：
2.7
通讯作者：
TAKEDA, Y
中科院分区：
文献类型：
--
作者：
INOUE, H;SUZUKI, F;TAKEDA, Y
用大鼠肝脏单一均一制剂研究了ATP柠檬酸裂解酶[EC 4.1.3.8]的作用方式。酶与标记的柠檬酸盐孵育，孵育后，通过Sephadex G-50柱层析分离蛋白部分。在三磷酸腺苷和镁离子同时存在的情况下，酶-柠檬酸形成了复合体。当这种酶-标准复合体被分离出来，然后与辅酶A孵育时，在没有添加ATP的情况下证明了乙酰辅酶A的形成。当8-14C-ATP和[Gamma]-32P-ATP分别与该酶孵育时，只有[Gamma]-32P-ATP才能获得与蛋白质相关的放射性，而8-14C-ATP则不能。与此相一致的是，观察到了快速的ATP-ADP交换，而不是ATP-PI[无机磷]交换。这两个反应完全依赖于镁离子的存在。通过Sephadex G-50柱层析分离~(32)P标记的酶-磷酸络合物，然后在辅酶A[辅酶A]存在下与标记的柠檬酸盐孵育，在没有添加ATP的情况下，根据结合磷酸盐定量地生成草酰乙酸酯。在这种转化中，不需要镁++。加入柠檬酸后，结合磷酸盐几乎完全从酶中释放出来。ATP柠檬酸裂解酶的反应总体上是可逆的，但在切割方向上反应较强。ATP柠檬酸裂解酶的反应机理可概括为：酶+AT一磷酸+ADP；酶-磷酸盐+柠檬酸酶+PI；酶-柠檬酸+辅酶A；面基辅酶A+草酰乙酸酯+酶。并对反应中间体的性质进行了描述。
The mode of action of ATP citrate lyase [EC 4.1.3.8] was investigated using a single homogeneous preparation from rat liver. The enzyme was incubated with labeled citrate and after incubation the protein portion was isolated through a column of Sephadex G-50. An enzyme-citrate complex was formed in the presence of both ATP and Mg++. When this enzyme-critrate complex was isolated and then incubated with CoA, the formation of acetyl-CoA was demonstrated in the absence of added ATP. When 8-14C-ATP and [gamma]-32p-ATp were separately incubated with the enzyme, radioactivity associated with protein was obtained only with [gamma]-32p-ATP, but not with 8-14C-ATP. In accord with this, a rapid ATP-ADP exchange, but not an ATP-Pi [inorganic phosphate] exchange, was observed. These 2 reactions were solely dependent on the presence of Mg++. Therefore, the reaction involves the formation of an enzyme-phosphate complex.Whenthe enzyme-phosphate complex, labeled with 32P, was isolated through a Sephadex G-50 column and then incubated with labeled citrate in the presence of CoA [coenzyme A], oxaloacetate was formed quantitatively on the basis of bound phosphate in the absence of added ATP. Mg++ was unnecessary in this conversion. Bound phosphate was almost completely released from the enzyme on addition of citrate. The over-all reaction of ATP citrate lyase was reversible, though it was stronger in the cleavage direction. The reaction mechanism of ATP citrate lyase can be summarized as follows: enzyme + ATPrienzyme-phosphate + ADP; enzyme-phosphate + citrateienzyme-citrate + Pi; enzyme-citrate + CoA^facetyl-CoA + oxaloacetate + enzyme. The properties of the reactive intermediates were also described.