Catalytic and Anticatalytic Snapshots of a Short-Form ATP Phosphoribosyltransferase
Catalytic and Anticatalytic Snapshots of a Short-Form ATP Phosphoribosyltransferase
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DOI：
10.1021/acscatal.8b00867
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发表时间：
2018-06-01
期刊：
影响因子：
12.9
通讯作者：
da Silva, Rafael G.
中科院分区：
文献类型：
--
作者：
Alphey, Magnus S.;Fisher, Gemma;da Silva, Rafael G.
催化的别构调节是控制通量的生物合成酶的常见调节策略。酶ATP磷酸核糖基转移酶（ATPPRT）催化组氨酸生物合成中的第一个反应，ATP和5-磷酸-α-D-核糖基-1-焦磷酸（PRPP）的镁依赖性缩合生成N-1-（5-磷酸-β-D-核糖基）-ATP（PRATP）和焦磷酸（PP）。ATPPRT被该途径的最终产物组氨酸变构抑制。异源八聚体ATPPRT由四个催化亚基（HisG（s））和四个调节亚基（HisZ）组成，参与复杂的催化调节。HisZ在不存在组氨酸的情况下增强HisG催化，而在其存在下介导变构抑制。在这里，我们报告了HisGs结构的脱辅基酶和复合物与底物（PRPP，PRPP-ATP，PRPP-ADP），产品（PRATP），和抑制剂（AMP），沿着与ATPPRT全酶结构的复合物与底物（PRPP，PRPP-ATP，PRPP-ADP）和产品（PRATP）。这10个晶体结构提供了北极嗜冷杆菌ATPPRT的催化循环和变构活化的原子视图。在这两个三元复合物与PRPP-ATP，腺嘌呤环被发现在一个anticatalytic方向，旋转180度的催化旋转异构体。Arg 32与ATP和PRPP的磷酸基团相互作用，使底物接近以进行催化。负电荷排斥被夹在两种基质的α-和β-磷酸基团之间的镁离子进一步衰减。HisZ结合形成异源八聚体使HisG亚基在Michaelis复合物中以更紧密的二聚体更靠近在一起，这使来自相邻HisG分子的ArgS 6平衡，用于PPi离去基团在过渡态的交叉亚基稳定。全酶的静电预组织活性位点可能使适应过渡态所需的重组能量最小化。这为变构活化提供了结构基础，其中化学通过促进离去基团离开而加速。
Allosteric modulation of catalysis is a common regulatory strategy of flux-controlling biosynthetic enzymes. The enzyme ATP phosphoribosyltransferase (ATPPRT) catalyzes the first reaction in histidine biosynthesis, the magnesium-dependent condensation of ATP and 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) to generate N-1-(5-phospho-beta-D-ribosyl)-ATP (PRATP) and pyrophosphate (PP,). ATPPRT is allosterically inhibited by the final product of the pathway, histidine. Hetero-octameric ATPPRT consists of four catalytic subunits (HisG(s)) and four regulatory subunits (HisZ) engaged in intricate catalytic regulation. HisZ enhances HisG(s) catalysis in the absence of histidine while mediating allosteric inhibition in its presence. Here we report HisGs structures for the apoenzyme and complexes with substrates (PRPP, PRPP-ATP, PRPP-ADP), product (PRATP), and inhibitor (AMP), along with ATPPRT holoenzyme structures in complexes with substrates (PRPP, PRPP-ATP, PRPP-ADP) and product (PRATP). These 10 crystal structures provide an atomic view of the catalytic cycle and allosteric activation of Psychrobacter arcticus ATPPRT. In both ternary complexes with PRPP-ATP, the adenine ring is found in an anticatalytic orientation, rotated 180 degrees from the catalytic rotamer. Arg32 interacts with phosphate groups of ATP and PRPP, bringing the substrates in proximity for catalysis. The negative charge repulsion is further attenuated by a magnesium ion sandwiched between the alpha- and beta-phosphate groups of both substrates. HisZ binding to form the hetero-octamer brings HisG(s) subunits closer together in a tighter dimer in the Michaelis complex, which poises ArgS6 from the adjacent HisG(s) molecule for cross-subunit stabilization of the PPi leaving group at the transition state. The more electrostatically preorganized active site of the holoenzyme likely minimizes the reorganization energy required to accommodate the transition state. This provides a structural basis for allosteric activation in which chemistry is accelerated by facilitating leaving group departure.