Department of Chemistry, Wesleyan University , Middletown, Connecticut 06459, United States.
Biochemistry. 2013 Aug 6;52(31):5158-60. doi: 10.1021/bi400807r. Epub 2013 Jul 23.
Heptosyltransferase I (HepI), the enzyme responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide, is a member of the GT-B structural class of enzymes. Crystal structures have revealed open and closed conformations of apo and ligand-bound GT-B enzymes, implying that large-scale protein conformational dynamics play a role in their reaction mechanism. Here we report transient kinetic analysis of conformational changes in HepI reported by intrinsic tryptophan fluorescence and present the first real-time evidence of a GT-B enzyme undergoing a substrate binding-induced transition from an open to closed state prior to catalysis.
七磷酸葡萄糖基转移酶 I（HepI）是一种酶，负责将 l-甘油基-d-甘露庚糖转移到脂多糖生长核心区域的 3-脱氧-α-d-甘露-oct-2-烯吡喃糖酸（Kdo）上，它是 GT-B 结构类酶的成员。晶体结构揭示了 apo 和配体结合的 GT-B 酶的开放和闭合构象，这意味着大规模的蛋白质构象动力学在它们的反应机制中发挥作用。在这里，我们通过本征色氨酸荧光报告了 HepI 构象变化的瞬态动力学分析，并首次实时证明 GT-B 酶在催化前经历了一个从开放状态到闭合状态的底物结合诱导转变。
