Crystal Structure, SAXS and Kinetic Mechanism of Hyperthermophilic ADP-Dependent Glucokinase from Thermococcus litoralis Reveal a Conserved Mechanism for Catalysis

ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Termococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.ADP依赖型葡萄糖激酶（ADP-dependent glucokinases）是一类隶属于核糖激酶超家族（ribokinase superfamily）的独特激酶，主要存在于嗜热古菌（hyperthermophilic archaea）中。针对这类酶，学界尚未就其配体结合相关的结构转变幅度，以及此类转变是否对酶功能具有实际意义达成共识。我们以利托尔热球菌（Termococcus litoralis）来源的ADP依赖型葡萄糖激酶为模型，研究底物结合时X射线晶体结构（X-ray crystallographic structures）中观察到的构象变化，并将其与溶液中测定的构象变化进行对比，以阐明这类构象变化与葡萄糖激酶功能之间的相互作用。初始速率研究（initial velocity studies）结果显示，该酶的催化过程遵循有序序贯催化机制（sequential ordered mechanism），这一机制与酶在溶液及晶体状态下所经历的结构转变高度吻合。综合所有实验数据，我们解析了覆盖完整反应循环的开-闭构象转变，并鉴定出负责该转变的相应氨基酸残基（aminoacids residues）簇。本研究结果为ADP依赖型激酶家族中保守的通用作用机制提供了分子层面的理论依据。