Grytsyk Natalia, Sugihara Junichi, Kaback H Ronald, Hellwig Petra
Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg CNRS, 67081 Strasbourg, France.
Department of Physiology, University of California, Los Angeles, CA 90095-7327.
Proc Natl Acad Sci U S A. 2017 Feb 14;114(7):1530-1535. doi: 10.1073/pnas.1621431114. Epub 2017 Feb 1.
Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and a H across the cytoplasmic membrane of (galactoside/H symport). One of the most important aspects of the mechanism is the relationship between protonation and binding of the cargo galactopyranoside. In this regard, it has been shown that protonation is required for binding. Furthermore when galactoside affinity is measured as a function of pH, an apparent pK (pK) of ∼10.5 is obtained. Strikingly, when Glu325, a residue long known to be involved in coupling between H and sugar translocation, is replaced with a neutral side chain, the pH effect is abolished, and high-affinity binding is observed until LacY is destabilized at alkaline pH. In this paper, infrared spectroscopy is used to identify Glu325 in situ. Moreover, it is demonstrated that this residue exhibits a pK of 10.5 ± 0.1 that is insensitive to the presence of galactopyranoside. Thus, it is apparent that protonation of Glu325 specifically is required for effective sugar binding to LacY.
乳糖通透酶（LacY）是最大的膜转运蛋白家族的一个范例，它催化半乳糖苷和一个H+跨细胞质膜的偶联转运（半乳糖苷/H+同向转运）。该机制最重要的方面之一是货物半乳糖吡喃糖苷的质子化与结合之间的关系。在这方面，已经表明结合需要质子化。此外，当测量半乳糖苷亲和力作为pH的函数时，可得到约10.5的表观pK（pK app）。引人注目的是，当长期以来已知参与H+和糖转运偶联的残基Glu325被中性侧链取代时，pH效应消失，并且观察到高亲和力结合，直到LacY在碱性pH下不稳定。在本文中，红外光谱用于原位鉴定Glu325。此外，证明该残基表现出10.5±0.1的pK，对半乳糖吡喃糖苷的存在不敏感。因此，很明显，Glu325的质子化是半乳糖有效结合到LacY所必需的。
