Studies on single alkaline phosphatase molecules: Reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation - The death of an enzyme
Studies on single alkaline phosphatase molecules: Reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation - The death of an enzyme
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DOI：
10.1021/ja9540839
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发表时间：
1996-06-05
影响因子：
15
通讯作者：
Dovichi, NJ
中科院分区：
文献类型：
--
作者：
Craig, DB;Arriaga, EA;Dovichi, NJ
单分子碱性磷酸酶在充满荧光底物的毛细管中被捕获。在孵化过程中，每个酶分子都会产生一个荧光产物池。孵化后，产物被扫描通过高灵敏度的激光诱导荧光检测器；峰的面积提供了每个分子活性的精确测量。对捕获的酶分子进行了三项研究。在第一个研究中，在恒温下对同一分子进行复制孵育；产物的量随着孵育时间的延长而线性增加。单一的酶分子表现出一系列的活性；最活跃的分子比最不活跃的分子的活性高10倍以上。在第二项研究中，在连续较高的温度下对同一分子进行复制孵化。高精度地测定了单分子催化反应的活化能。单一的酶分子表现出一定范围的活化能；微观异质性延伸到催化的热力学性质。平均活化能与大块样品分析得到的活化能在实验误差范围内。这些结果与统计热力学的第一个假设是一致的：从单个分子的时间平均得到的热力学性质与由大量分子的集合平均得到的热力学性质是相同的。在第三项研究中，测定了部分热变性后单酶分子的活性。活性分子的数量随着变性程度的增加而成比例减少。然而，在实验上，存活分子的活性与对照酶的活性是无法区分的。碱性磷酸酶的热变性是一个灾难性的过程，在这个过程中，分子经历了不可逆的转化为非活性形式。
Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; microheterogeneity extends to thermodynamic properties of catalysis. The average activation energy is within experimental error of the activation energy obtained from analysis of a bulk sample. These results are consistent with the first postulate of statistical thermodynamics: a thermodynamic property obtained from the time average of an individual molecule is identical to that produced by an ensemble average over a large number of molecules. In the third study, the activity of single enzyme molecules is measured after partial heat denaturation. The number of active molecules decreases in proportion to the extent of denaturation. However, the activity of the surviving molecules is experimentally indistinguishable from the activity of control enzyme. Thermal denaturation of alkaline phosphatase is a catastrophic process, wherein the molecule undergoes irreversible conversion to an inactive form.