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首页> 外文期刊>Biochemistry >BETA-SITE COVALENT REACTIONS TRIGGER TRANSITIONS BETWEEN OPEN AND CLOSED CONFORMATIONS OF THE TRYPTOPHAN SYNTHASE BIENZYME COMPLEX
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BETA-SITE COVALENT REACTIONS TRIGGER TRANSITIONS BETWEEN OPEN AND CLOSED CONFORMATIONS OF THE TRYPTOPHAN SYNTHASE BIENZYME COMPLEX

机译：TRYPTOPHAN合酶双酶复合物的开式和闭式构象的贝塔共价反应触发跃迁

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The tryptophan synthase bienzyme complex (alpha(2) beta(2)) from Salmonella typhimurium catalyzes the final two steps in the biosynthesis of L-Trp. To investigate the roles played by conformational change in tryptophan synthase catalysis, the fluorophore 8-anilino-1-naphthalenesulfonate (ANS) is used to identify conformational states. The binding of ANS to the alpha(2) beta(2) bienzyme complex is accompanied by a dramatic enhancement of ANS fluorescence and a shift of the emission maximum from 520 to 482 nm. The ANS binding isotherm is biphasic and consists of a class of moderately high-affinity, noninteracting sites with a stoichiometry of 1 site/alpha beta dimeric unit (K-d' = 62 +/- 15 mu M) and a much weaker set of non-specific interactions with K-d' > 1 mM. Our findings show that the affinity of the enzyme for ANS is strongly decreased (> 10-fold) by interactions at two loci 30 Angstrom apart: (i) the binding of the alpha-site ligands, 3-indole-D-glycerol 3'-phosphate or alpha-glycerol phosphate (GP) or (ii) reaction at the beta-subunit to form either the alpha-aminoacrylate Schiff base, E(A-A), or quinonoid species, E(Q). In contrast, formation of the L-Ser and L-Trp external aldimines E(Aex(1)) and E(Aex(2)) at the beta-site causes a 2-3-fold decrease in the affinity of the enzyme for ANS. The combination of E(A-A) or E(Q) with GP brings about almost complete displacement of ANS, indicating that these interactions drive a conformation change in alpha beta subunit pairs which prevents the binding of ANS. These results are consistent with a model which postulates that alpha beta subunit pairs undergo ligand-mediated transitions between open and closed conformations during the catalytic cycle. Consistent with the kinetic data showing that binding of alpha-site ligands increases the affinity of the beta site for L-Ser and that formation of E(A-A) activates the or reaction [Brzovic, P. S., Ngo, K., & Dunn, M. F. (1992) Biochemistry 31, 3831-3839], while mutations in a subunit loops 2 and 6 prevent the ligand-mediated transition to a closed structure [Brzovic, P. S., Hyde, C. C., Miles, E. W., & Dunn, M. F. (1993) Biochemistry 32, 10404-10413], we conclude that reciprocal ligand-mediated allosteric interactions between the heterologous subunits promote conformational transitions between open and closed structures in alpha beta subunit pairs which function to coordinate catalytic activities and facilitate the channeling of indole between the two catalytic sites.
机译：来自鼠伤寒沙门氏菌的色氨酸合酶双酶复合物（alpha（2）beta（2））催化L-Trp的生物合成中的最后两个步骤。为了研究色氨酸合酶催化中构象变化所起的作用，使用了荧光团8-苯胺基-1-萘磺酸盐（ANS）来鉴定构象状态。 ANS与alpha（2）beta（2）双酶复合物的结合伴随着ANS荧光的显着增强和发射最大值从520 nm移至482 nm。 ANS结合等温线是两相的，由一类中等高度亲和力的非相互作用位点组成，其化学计量比为1个位点/αβ二聚体单位（Kd'= 62 +/- 15μM）和一组弱得多的非- Kd'> 1 mM的特定相互作用。我们的发现表明，通过两个相距30埃的相互作用，酶对ANS的亲和力大大降低（> 10倍）：（i）α位配体，3-吲哚-D-甘油3'的结合β-亚磷酸酯或α-甘油磷酸酯（GP）或（ii）在β亚基上反应形成α-氨基丙烯酸Schiff碱E（AA）或醌类物质E（Q）。相反，在β位形成L-Ser和L-Trp外部醛亚胺E（Aex（1））和E（Aex（2））会导致酶的亲和力降低2-3倍ANS。 E（A-A）或E（Q）与GP的结合可导致ANS几乎完全置换，表明这些相互作用驱动了α-β亚基对的构象变化，从而阻止了ANS的结合。这些结果与一个模型相符，该模型假定在催化周期中，αβ亚基对在开放和封闭构象之间经历了配体介导的过渡。与动力学数据一致，该数据表明，α位配体的结合增加了β位对L-Ser的亲和力，并且E（AA）的形成激活或反应[Brzovic，PS，Ngo，K.，＆Dunn，MF （1992）Biochemistry 31，3831-3839]，而亚基环2和6中的突变则阻止了配体介导的向封闭结构的过渡[Brzovic，PS，Hyde，CC，Miles，EW，＆Dunn，MF（1993）。 [Biochemistry 32，10404-10413]，我们得出结论，异源亚基之间的相互配体介导的变构相互作用促进了αβ亚基对中开放结构和封闭结构之间的构象转变，其功能是协调催化活性并促进两个催化之间吲哚的通道化网站。

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