Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT
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- 作者：Christopher J. Law ; Qiang Yang ; Celine Soudant ; Peter C. Maloney ; Da-Neng Wang
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：October 30, 2007
- 年：2007
- 卷：46
- 期：43
- 页码：12190 - 12197
- 全文大小：142K
- 年卷期：v.46,no.43(October 30, 2007)
- ISSN：1520-4995
文摘
Secondary active transport of substrate across the cell membrane is crucial to many cellularand physiological processes. The crystal structure of one member of the secondary active transporterfamily, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli,suggests a mechanism for substrate translocation across the membrane that involves a rocker-switch-typemovement of the protein. This rocker-switch mechanism makes two specific predictions with respect tokinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of thetransporter to a substrate is temperature-independent. In this work, we directly tested these two predictionsby transport kinetics and substrate-binding experiments, integrating the data on this single system into acoherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiportreaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstitutedinto proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophanfluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increaseddramatically with temperature. In contrast, neither the apparent Michaelis constant (Km) nor the apparentsubstrate-binding dissociation constant (Kd) showed temperature dependence. Moreover, GlpT-catalyzedG3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJmol-1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporteris delicately poised between the inward- and outward-facing conformations. When these results are takentogether, they are in agreement with a rocker-switch mechanism for GlpT.