Allosteric regulation of substrate channeling in tryptophan synthase:: Modulation of the L-Serine reaction in stage I of the ß-reaction by α-site ligands
Allosteric regulation of substrate channeling in tryptophan synthase:: Modulation of the L-Serine reaction in stage I of the ß-reaction by α-site ligands
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DOI：
10.1021/bi7003872
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发表时间：
2007-07-03
期刊：
影响因子：
2.9
通讯作者：
Dunn, Michael F.
中科院分区：
文献类型：
--
作者：
Ngo, Huu;Kimmich, Novelle;Dunn, Michael F.
在色氨酸合酶双酶复合物中，α 位点底物裂解产生的吲哚通过 25 埃长的通道引导至 β 位点。在 β 位点内，吲哚和 L-Ser 与 5'-磷酸吡哆醛发生两阶段反应，生成 L-Trp。在阶段 I，L-Ser 形成外部醛亚胺 E(Aex(1))，其转化为 α-氨基丙烯酸酯醛亚胺 E(A-A)。在 β 位点形成 E(A-A) 可将 α 位点激活 > 30 倍。在第二阶段，吲哚与 E(A-A) 反应生成 L-Trp。 α位配体（ASL）的结合对β位点的底物反应产生强烈的变构效应：第一阶段形成的中间体的分布向E（A-A）方向移动，并且ASL的结合触发β位点的构象变化，形成对L-Ser亲和力增加的状态。在这里，我们将新 ASL 作为 I 期变构效应器的行为与天然产物 D-甘油醛 3-磷酸的行为进行了比较。快速动力学和动力学同位素效应表明，这些 ASL 的结合亲和力范围从微摩尔到毫摩尔，并且 E(Aex(1)) 转化为 E(A-A) 的速率决定步骤增加了 8-10 倍。为了导出第一阶段基于结构的机制，确定了与不同 ASL 复合的 E(Aex(1)) 和 E(A-A) 态的 X 射线结构，并与带有内部醛亚胺的 ASL 复合物的结构进行了比较。
In the tryptophan synthase bienzyme complex, indole produced by substrate cleavage at the alpha-site is channeled to the beta-site via a 25 angstrom long tunnel. Within the beta-site, indole and L-Ser react with pyridoxal 5'-phosphate in a two-stage reaction to give L-Trp. In stage I, L-Ser forms an external aldimine, E(Aex(1)), which converts to the alpha-aminoacrylate aldimine, E(A-A). Formation of E(A-A) at the beta-site activates the alpha-site > 30-fold. In stage II, indole reacts with E(A-A) to give L-Trp. The binding of alpha-site ligands (ASLs) exerts strong allosteric effects on the reaction of substrates at the beta-site: the distribution of intermediates formed in stage I is shifted in favor of E(A-A), and the binding of ASLs triggers a conformational change in the beta-site to a state with an increased affinity for L-Ser. Here, we compare the behavior of new ASLs as allosteric effectors of stage I with the behavior of the natural product, D-glyceraldehyde 3-phosphate. Rapid kinetics and kinetic isotope effects show these ASLs bind with affinities ranging from micro- to millimolar, and the rate-determining step for conversion of E(Aex(1)) to E(A-A) is increased by 8-10-fold. To derive a structure-based mechanism for stage I, X-ray structures of both the E(Aex(1)) and E(A-A) states complexed with the different ASLs were determined and compared with structures of the ASL complexes with the internal aldimine.