Suh Jeong-Yong, Iwahara Junji, Clore G Marius
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3153-8. doi: 10.1073/pnas.0609103104. Epub 2007 Feb 21.
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 (1)H relaxation dispersion spectroscopy 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 approximately 500 s(-1) for A --> B and B --> A phosphoryl transfer, derived from qualitative line-shape analysis of (1)H-(15)N correlation spectra taken during the course of active catalysis. Thus, on average, approximately 80 association/dissociation events are required to effect a single phosphoryl-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.
大肠杆菌甘露醇转运蛋白（II(Mtl)）由通过柔性连接子相连的三个结构域组成：一个跨膜结构域（C）和两个细胞质结构域（A和B）。II(Mtl)催化三个连续的磷酸转移反应：一个分子间反应（从组氨酸磷酸载体蛋白到A结构域）和两个分子内反应（从A结构域到B结构域以及从B结构域到与C结构域结合的进入糖）。II(Mtl)的一个关键功能要求是A和B细胞质结构域能够快速缔合和解离，同时保持活性立体特异性AB复合物的合理高占有率，以确保沿该途径进行有效的磷酸转移。我们通过在旋转坐标系中使用（1）H弛豫分散光谱研究了IIBA(Mtl)中分子内结构域-结构域缔合和解离的速率。开放的、解离状态（包括一系列状态）和封闭的、缔合状态（包括立体特异性复合物）的丰度大致相等。分子内缔合和解离的一级速率常数分别为1.7（±0.3）×10(4)和1.8（±0.4）×10(4) s(-1)。这些值与从活性催化过程中获取的（1）H-（15）N相关光谱的定性线形分析得出的A→B和B→A磷酸转移的速率常数约500 s(-1)相比。因此，平均而言，单次磷酸转移反应需要大约80次缔合/解离事件。我们得出结论，II(Mtl)的A和B结构域之间的分子内磷酸转移受化学作用限制，而非受活性位点最佳并列的立体特异性复合物形成或解离速率的限制。
