Loop closure and intersubunit communication in tryptophan synthase
Loop closure and intersubunit communication in tryptophan synthase
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DOI：
10.1021/bi9728957
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发表时间：
1998-04-21
期刊：
影响因子：
2.9
通讯作者：
Schlichting, I
中科院分区：
文献类型：
--
作者：
Schneider, TR;Gerhardt, E;Schlichting, I
测定了鼠伤寒沙门氏菌野生型色氨酸合酶α（2）β（2）复合物的晶体结构，以研究在β活性位点形成的氨基丙烯酸酯中间体对α反应的变构激活机制。使用流动池，在丝氨酸和α位点抑制剂5-氟吲哚丙醇磷酸盐（F-IPP）存在下，在稳态条件下在晶体中产生β反应的氨基丙烯酸酯（A-A）中间体（TRPSA-AF-IPP）。氨基丙烯酸酯和β-亚基辅因子磷酸吡哆醛（PLP）之间的席夫碱的构象模型。将TRPSA-AF-IPP结构与在F-IPP不存在（TRPS）和存在（TRPSF-IPP）下测定的酶结构进行比较。F-IPP的α-亚基结合的详细模型。与Hyde等[（1988）J.Biol.Chem.263，17857-17871]和Rhee等[（1997）Biochemisty 36，7664-7680]的发现相反，我们发现α-位点单独配体的存在足以使α-活性位点顶上的环α L 6闭合。该环的一部分，α Thr 183，不仅对定位催化性α Asp 60很重要，而且对协调F-IPP结合后环α L2的伴随排序也很重要。在这三种结构的基础上，建立了α-和β-活性位点之间的通信途径。该途径的中心元件是一个新定义的刚性但可移动的结构域，其一侧通过环α L2与α亚基相互作用，另一侧与β活性位点相互作用。这些发现为理解色氨酸合酶的变构特性提供了结构基础。
Crystal structures of wild-type tryptophan synthase alpha(2) beta(2) complexes from Salmonella typhimurium were determined to investigate the mechanism of allosteric activation of the alpha-reaction by the aminoacrylate intermediate formed at the beta-active site. Using a flow cell, the aminoacrylate (A-A) intermediate of the beta-reaction (TRPSA-AF-IPP) was generated in the crystal under steady state conditions in the presence of serine and the alpha-site inhibitor 5-fluoroindole propanol phosphate (F-IPP). A model for the conformation of the Schiff base between the aminoacrylate and the beta-subunit cofactor pyridoxal phosphate (PLP) is presented. The TRPSA-AF-IPP structure is compared with structures of the enzyme determined in the absence (TRPS) and presence (TRPSF-IPP) of F-IPP. A detailed model for binding of F-IPP to the alpha-subunit is presented. In contrast to findings by Hyde et al. [(1988) J. Biol. Chem. 263, 17857-17871] and Rhee et al. [(1997) Biochemisty 36, 7664-7680], we find that the presence of an alpha-site alone ligand is sufficient for loop alpha L6 closure atop the alpha-active site. Part of this loop, alpha Thr183, is important not only for positioning the catalytic alpha Asp60 but also for coordinating the concomitant ordering of loop alpha L2 upon F-IPP binding. On the basis of the three structures, a pathway for communication between the alpha- and beta-active sites has been established. The central element of this pathway is a newly defined rigid, but movable, domain that on one side interacts with the alpha-subunit via loop alpha L2 and on the other side with the beta-active site. These findings provide a structural basis for understanding the allosteric properties of tryptophan synthase.