Squalene Synthetase III. MECHANISM OF THE REACTION
Squalene Synthetase III. MECHANISM OF THE REACTION
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角鲨烯合成酶 III。
DOI：
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发表时间：
1973
期刊：
影响因子：
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通讯作者：
J. Porter
中科院分区：
文献类型：
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作者：
E. Beytía;A. Qureshi;J. Porter
摘要利用纯化的酵母角鲨烯合成酶对焦磷酸法呢酯转化角鲨烯的反应性质和机理进行了研究。酵母酶的一般性质与从哺乳动物肝脏获得的酶的性质相似。二价金属离子（Mg++或Mn++）对于形成焦磷酸前角鲨烯是必需的，但对于该化合物转化为角鲨烯不是必需的。然而，在Mg++存在下，后一反应的速率增加。酵母角鲨烯合成酶活性被浓度高于50 mM的焦磷酸法呢酯抑制，被N-乙基马来酰亚胺抑制，并且在较小程度上被碘乙酰胺抑制。因此，很明显，巯基或其他亲核试剂是酶活性所必需的。酵母角鲨烯合成酶在pH 7.3至7.5具有最大酶活性。然而，发现酶活性存在很大差异，这取决于所使用的缓冲液。这些差异部分归因于离子强度，并且可能归因于磷酸根离子对酶活性的直接影响。由焦磷酸法呢酯形成角鲨烯和焦磷酸前角鲨烯的初速度研究表明，缩合反应的机理是乒乓式的。对焦磷酸前角鲨烯还原为角鲨烯的初速度动力学和产物抑制的研究表明，该反应是连续有序的。在还原反应中，NADPH是与酶结合的第一个底物，其次是焦磷酸前角鲨烯。第一个释放的产物是焦磷酸盐，其次是角鲨烯。最后离开酶的产物是NADP。提出了角鲨烯合成的化学机理。
Abstract Studies on the properties of the reaction and the mechanism of conversion of farnesyl pyrophosphate to squalene have been carried out with purified yeast squalene synthetase. The general properties of the yeast enzyme are similar to those of the enzyme obtained from mammalian liver. A divalent metal ion (Mg++ or Mn++) is essential for the formation of presqualene pyrophosphate, but not for the conversion of this compound to squalene. However, the rate of the latter reaction is increased in the presence of Mg++. Yeast squalene synthetase activity is inhibited by concentrations above 50 mm of farnesyl pyrophosphate, by N-ethylmaleimide, and to a lesser extent by iodoacetamide. Hence it is evident that a sulfhydryl group or other nucleophile is required for enzyme activity. Yeast squalene synthetase has a maximum enzyme activity at pH 7.3 to 7.5. However, great differences in enzyme activity were found, depending on the buffer used. These differences are due in part to ionic strength, and possibly to a direct effect of phosphate ion on enzymatic activity. Initial velocity studies for the formation of squalene and presqualene pyrophosphate from farnesyl pyrophosphate have shown that the mechanism of the condensing reaction is ping-pong. Initial velocity kinetics and product inhibition studies for the reduction of presqualene pyrophosphate to squalene have shown that this reaction is sequential ordered. In the reduction reaction NADPH is the first substrate to bind to the enzyme, followed by presqualene pyrophosphate. The first product to be released is pyrophosphate, followed by squalene. The last product to leave the enzyme is NADP. A chemical mechanism for squalene synthesis is presented.