The dynamics of complex cellular machinery required for methionine synthesis in mammalian cells

人类，像所有生物一样，有专门的分子来执行特定的生物功能。其中一些功能是由专门设计的酶来执行的。尤其是一种叫做蛋氨酸合成酶的酶，它在很多层面上都非常特别。首先，它利用维生素B12发挥作用；它是仅有的两种利用其高活性特性的人类酶之一。然而，氧气会破坏维生素B12，并阻止蛋氨酸合成酶发挥其生物功能。幸运的是，人类有另一种酶，叫做蛋氨酸合成酶还原酶，它可以修复维生素B12，恢复蛋氨酸合成酶的活性。其次，蛋氨酸合酶对人体健康有重要作用，因为它可以减少体内损害细胞和引起心血管疾病的有毒化合物的数量。这种酶还能产生合成DNA和蛋白质所必需的化合物。这种酶的活性受损会阻止细胞分裂和生长的能力。第三，蛋氨酸合酶的结构非常复杂，因为它是由四个单独的刚性单元组成的，这些单元通过柔性连接器连接在一起。其中两个单元与底物分子结合，另一个容纳维生素B12，第四个与修复酶，蛋氨酸合成酶还原酶相互作用。为了使酶发挥作用，所有四个单元必须在特定的时间以特定的顺序移动相对较远的距离。此外，蛋氨酸合酶还原酶也是由移动单元组成的，这些单元必须相互结合和分离，并在关键时刻与蛋氨酸合酶的特定单元结合和分离。与这些单位协调运动相关的复杂性在酶中是罕见的。我们想要测量在特定时间单个单位之间的距离，并确定它们移动的距离和速度。我们还想了解是什么控制着这些单个单位的运动，以及它们的运动是如何同步的。为了解决这些问题，我们将确定这两种酶的分子结构（即酶的每个原子在三维空间中的位置），作为单独的实体以及它们何时相互结合。我们还将尝试解决单个单元的分子结构。从这些信息中，我们将能够检查与运动相关的酶的独特结构特征。为了在关键时刻测量单元之间的距离，我们将对每种酶使用不同的技术。蛋氨酸合成酶还原酶在每个移动单元中都含有一个固有探针，可用于测量单元之间的距离。在一定的物理状态下，这两个探针包含一个电子，其作用就像一个微型磁铁。这些探针被更大的磁铁和微波频率包围，可以报告它们周围的环境（即邻近的原子）以及彼此之间的距离。蛋氨酸合酶不含内禀探针；因此，我们将人工地将它们附着在酶的表面。通过以特定频率照射激光，能量从一个探针转移到下一个探针。然而，传递的能量取决于探针之间的距离。既然我们能够测量随时间传递的能量，我们就可以观察到探测器之间与时间相关的距离。此外，我们将有能力观察单个酶分子，我们将能够确定它们的行为是否相似。这种相对较新的技术可以揭示酶的结构和功能特性的丰富信息。这将是首次将其应用于如此复杂的酶系统。

Humans, like all living things, have specialised molecules for carrying out specific biological functions. Some of these functions are executed by specifically designed enzymes. One enzyme in particular, called methionine synthase, is quite extraordinary on a number of levels. First, it utilizes vitamin B12 to function; it is only one of two human enzymes to exploit its highly reactive properties. Oxygen, however, can damage vitamin B12, and prevent methionine synthase from carring out its biological function. Fortunately, humans have another enzyme, called methionine synthase reductase that repairs vitamin B12 and restores methionine synthase activity. Second, methionine synthase has an important role in human health, as it reduces the amount of a toxic compound in the body that can damage cells and cause cardiovascular disease. The enzyme also produces compounds that are essential for making DNA and protein. Impairment of the enzyme's activity prevents the ability of cells to divide and grow. Third, methionine synthase is very complex structurally, as it is composed of four individual rigid units that are linked together by flexible connectors. Two of the units bind to substrate molecules, another houses vitamin B12 and the fourth interacts with the repair enzyme, methionine synthase reductase. For the enzyme to function, all four units must move in specific order at a specific time over relatively large distances. Moreover, methionine synthase reductase is also composed of mobile units that must engage and disengage with each other and with specific units of methionine synthase at key times. The complexity associated with coordinating movement of these units is rare amongst enzymes. We want to measure the distance between individual units at specific times, and determine how far and how fast they travel. We also want to understand what controls movement of these individual units and how their movement is synchronised. To address these questions, we will determine the molecular architecture of both enzymes, (i.e. where each atom of the enzyme is located in a three dimensional space), as individual entities and when they are bound to each other. We will also try to solve the molecular architecture of the individual units. From this information, we will be able to examine unique structural features of the enzymes that are associated with movement. To measure distance between units at key times, we will use different techniques for each enzyme. Methionine synthase reductase contains intrinsic probes one in each of the mobile units that can be used to measure distance between the units. Under a certain physical state, these two probes contain an electron that acts like a miniature magnet. Surrounded by a larger magnet and a microwave frequency, these probes can report on their immediate environment, (i.e. neighbouring atoms) as well as the distance to each other. Methionine synthase does not contain intrinsic probes; therefore, we will attach them artificially to the surface of the enzyme. By shining laser light at a particular frequency, energy is transferred from one probe to the next. However, the amount of energy transferred is dependent on the distance between the probes. Since we are able to measure the energy transferred over time, we can observe the time-dependent distance between the probes. Moreover, we will have the capability to observe single enzyme molecules, and we will be able to determine if they behave similarly or not. This relatively new technique can reveal a wealth of information of the structural and functional properties of enzymes. This will be the first time it is applied to such a complex enzyme system.