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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Intramolecular domain-domain association/dissociation and phosphoryl transfer in the mannitol transporter of Escherichia coli are not coupled
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Intramolecular domain-domain association/dissociation and phosphoryl transfer in the mannitol transporter of Escherichia coli are not coupled

机译：大肠杆菌的甘露醇转运蛋白中的分子内域-域缔合/解离和磷酰基转移不偶联

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摘要

The Escherichia coli mannitol transporter (II~(Mtl)) comprises three domains connected by flexible linkers: a transmembrane domain (C) and two cytoplasmic domains (A and B). II~(Mtl) catalyzes three successive phosphoryl-transfer reactions: one intermolecular (from histidine phosphocarrier protein to the A domain) and two intramolecular (from the A to the B domain and from the B domain to the incoming sugar bound to the C domain). A key functional requirement of II~(Mtl) is that the A and B cytoplasmic domains be able to rapidly associate and dissociate while maintaining reasonably high occupancy of an active stereospecific AB complex to ensure effective phosphoryl transfer along the pathway. We have investigated the rate of intramolecular domain-domain association and dissociation in IIBA~(Mtl) by using ~1H relaxation dispersion spectros-copy in the rotating frame. The open, dissociated state (comprising an ensemble of states) and the closed, associated state (comprising the stereospecific complex) are approximately equally populated. The first-order rate constants for intramolecular association and dissociation are 1.7 (±0.3) x 10~4 and 1.8 (±0.4) x 10~4 s~(-1), respectively. These values compare to rate constants of ≈ 500 s~(-1) for A → B and B → A phosphoryl transfer, derived from qualitative line-shape analysis of ~1H-~(15)N correlation spectra taken during the course of active catalysis. Thus, on average, ≈ 80 association/ dissociation events are required to effect a single phosphoryi-transfer reaction. We conclude that intramolecular phosphoryl transfer between the A and B domains of II~(Mtl) is rate-limited by chemistry and not by the rate of formation or dissociation of a stereospecific complex in which the active sites are optimally apposed.
机译：大肠杆菌甘露醇转运蛋白（II-（Mt1））包括通过柔性接头连接的三个结构域：跨膜结构域（C）和两个胞质结构域（A和B）。 II〜（Mtl）催化三个连续的磷酸基转移反应：一个分子间（从组氨酸磷酸载体蛋白到A结构域）和两个分子内（从A到B结构域，从B结构域到结合到C结构域的传入糖） ）。 II_（Mt1）的关键功能要求是A和B胞质结构域能够快速缔合和解离，同时保持合理的高活性立体特异性AB复合物占有率，以确保沿该途径有效的磷酸基转移。我们已通过在旋转框架中使用〜1H弛豫分散光谱法研究了IIBA〜（Mtl）中分子内域-域缔合和解离的速率。打开的，解离的状态（包含状态集合）和关闭的，关联的状态（包含立体特定复合体）近似相等地填充。分子内缔合和解离的一级速率常数分别为1.7（±0.3）x 10〜4和1.8（±0.4）x 10〜4 s〜（-1）。这些值与A→B和B→A磷酰基转移的≈500 s〜（-1）的速率常数进行比较，该速率常数是通过对活性过程中〜1H-〜（15）N相关光谱的定性线形分析得出的催化。因此，平均而言，需要约80个缔合/解离事件来进行单个磷酸转移反应。我们得出结论，II-（Mt1）的A和B结构域之间的分子内磷酸基转移是受化学速率限制的，而不是受立体构象复合物的形成或解离的速率限制的，其中活性位点被最佳地并置。

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