- 學位論文
高解析度之阿拉伯芥六碳糖激酶晶體結構與催化機制之探討
High-resolution Crystal Structures of Hexokinases from Arabidopsis thaliana Reveal the Mechanism of Enzyme Catalysis
摘要
六碳糖激酶是參與生物體內醣類代謝的重要酵素，其主要功能為執行糖解作用的第一個反應—以三磷酸腺苷將葡萄糖磷酸化。除了酵素催化與醣類代謝的功能之外，阿拉伯芥的六碳糖激酶 (AtHXK) 甚至能感知葡萄糖濃度的變化，並且傳遞葡萄糖引起的訊息進而影響整個植物體的生理現象。在本篇研究中共解出六個AtHXK1與AtHXK2的晶體結構，每個結構各為不同的型態：(一) AtHXK1與葡萄糖-6-磷酸的複合體; (二) AtHXK1與葡萄糖、鎂離子、二磷酸腺苷的複合體; (三) AtHXK1與2-脫氧葡萄糖、鎂離子、二磷酸腺苷的複合體; (四) AtHXK2的未結合態; (五) AtHXK2與葡萄糖的複合體; (六) AtHXK2與甘露糖、鎂離子、二磷酸腺苷的複合體。型態一的結構為AtHXK1的酵素-產物複合體。在型態二與型態三的結構中，AtHXK1的活性位包含鎂離子、與鎂離子進行配位鍵結的六個水分子以及二磷酸腺苷。同時，藉由這兩種型態結構的解析，首次阿拉伯芥的六碳糖激酶與金屬離子以及核苷酸分子之間的交互作用模式得以被直接觀察。型態四的結構為AtHXK2的未結合態，其整體構型為展開型。型態五的結構為AtHXK2的酵素-受質複合體，其整體構型為閉合型。型態六的結構呈現AtHXK2與金屬離子以及核苷酸分子之間的交互作用模式，其整體構型為閉合型。藉由這三種型態結構的比較可得知，葡萄糖的結合會引發AtHXK2的結構產生構型變化。同時，藉由這三種型態結構的解析，AtHXK2在催化酵素反應的過程中所經歷的蛋白質結構的變化因而能被直接觀察到。此外，相較於葡萄糖，二磷酸腺苷與AtHXK2的結合則並未使其蛋白質結構產生明顯的構型變化。更進一步的，本篇研究以AtHXK1與AtHXK2的二磷酸腺苷複合體結構探討阿拉伯芥的六碳糖激酶在催化酵素反應時的作用機制，以及其餘四個阿拉伯芥的六碳糖激酶家族成員所呈現的功能性分歧的起源。
並列摘要
Hexokinase catalyzes the reaction of glucose phosphorylation by consuming ATP, which mainly serves as the gateway enzyme in glycolysis. Beyond the roles in enzyme catalysis and glucose metabolism, the hexokinase from Arabidopsis thaliana (AtHXK) is an identified sugar sensor that detects fluctuation of glucose concentration. Furthermore, it transmits glucose signal and participates in glucose signaling to alter physiological phenomena of the plant. In this study, the crystal structures of AtHXK1 and AtHXK2 in different forms are presented: (I) AtHXK1 binary complex with glucose 6-phosphate; (II) AtHXK1 quaternary complex with glucose, Mg2+, and ADP; (III) AtHXK1 quaternary complex with 2-deoxyglucose, Mg2+, and ADP; (IV) AtHXK2 apo-form; (V) AtHXK2 binary complex with glucose; (VI) AtHXK2 quaternary complex with mannose, Mg2+, and ADP. Structure form I represents the state of enzyme-product complex of AtHXK1. Structures form II and III include well-recognized Mg2+ ion, coordinating water molecules, and ADP within active site of AtHXK1, which provide the first direct view for interacting patterns of metal ion and nucleotide to Arabidopsis hexokinase. Structure form IV is the apo-form of AtHXK2 in open state. Structure from V is the enzyme-substrate complex of AtHXK2 in closed state. Structure form VI obviously presents the interacting patterns of metal ion and nucleotide to AtHXK2, which is also in closed state. The three structures show that AtHXK2 has an obvious domain reorientation induced by glucose binding, which provides direct view for conformational change of protein structure during the catalytic process. However, ADP binding does not induce such change as glucose does. The obtained ADP quaternary complex structures of AtHXK1 and AtHXK2 shall allow the catalytic mechanism taken by Arabidopsis hexokinase being proposed, and further applied for better understanding about the functional diversities exhibited by the other four Arabidopsis hexokinase family proteins.