Division of Medicinal Chemistry, College of Pharmacy, Department of Chemistry and Biochemistry and Institute of Cellular and Molecular Biology, University of Texas, Austin, Texas 78712-0128, USA.
J Biol Chem. 2012 Feb 10;287(7):4602-8. doi: 10.1074/jbc.M111.312538. Epub 2011 Dec 20.
UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(β) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).
尿苷二磷酸半乳糖差向异构酶 (UGM) 需要还原型黄素腺嘌呤二核苷酸 (FAD(red)) 来催化尿苷二磷酸半乳糖 (UDP-Galp) 和尿苷二磷酸半乳糖呋喃糖 (UDP-Galf) 的可逆互变。最近对 UGM 的结构和机制研究为催化循环中存在 FAD-Galf/p 加合物作为中间体提供了证据。这些发现与涉及 FAD(red)的 N5 对 UDP-Galf/p 的 C1 的亲核攻击的路易斯酸碱化学一致。在这项研究中，我们使用了各种 FAD 类似物，通过位置同位素交换 (PIX) 和线性自由能关系研究来表征 FAD(red)在 UGM 催化循环中的作用。PIX 研究表明，用 5-脱氮黄素腺嘌呤二核苷酸 (5-deaza-FAD(red)) 重建的 UGM 无法催化 UDP-Galp 的桥接 C1-OP(β)氧的 PIX，表明 FAD(red)的 N5 原子在此过程中直接发挥作用。此外，对 k(cat)与 FAD(red)的 N5 的亲核性的动力学线性自由能关系的分析给出了斜率 ρ = -2.4 ± 0.4。这些发现最符合 UGM 的化学机制，该机制涉及 FAD(red)的 N5 通过 S(N)2 型取代从 UDP-Galf/p 中取代 UDP。
