ALLOSTERIC INTERACTIONS COORDINATE CATALYTIC ACTIVITY BETWEEN SUCCESSIVE METABOLIC ENZYMES IN THE TRYPTOPHAN SYNTHASE BIENZYME COMPLEX
ALLOSTERIC INTERACTIONS COORDINATE CATALYTIC ACTIVITY BETWEEN SUCCESSIVE METABOLIC ENZYMES IN THE TRYPTOPHAN SYNTHASE BIENZYME COMPLEX
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DOI：
10.1021/bi00130a014
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发表时间：
1992-04-21
期刊：
影响因子：
2.9
通讯作者：
DUNN, MF
中科院分区：
文献类型：
--
作者：
BRZOVIC, PS;NGO, K;DUNN, MF
来自肠细菌的色氨酸合酶是α-2-β-2双酶复合物，其催化从3-吲哚-D-甘油3 '-磷酸（IGP）和L-丝氨酸（L-Ser）生物合成L-色氨酸（L-Trp）的最后两个反应。双酶复合物在异源亚基之间表现出相互配体介导的变构相互作用[Houben，K.，& Dunn，M. F.（1990）Biochemistry 29，2421-2429]，但变构和催化之间的关系尚未完全确定。我们已经利用快速扫描停流（RSSF）紫外可见光谱研究变构和催化的α-β-反应中的双酶复合物从鼠伤寒沙门氏菌催化的关系。当L-Ser和IGP与α-2-β-2复合物同时混合时发生的稳态前光谱变化表明，IGP与α位点的结合加速了β位点L-Ser形成α-氨基丙烯酸酯[E（A-A）]。通过使用L-Ser类似物，我们在本文中表明，E（A-A）中间体的形成是触发激活α亚基的构象转变的化学信号。β-亚基配体，如L-Trp，在β-位点反应形成共价中间体，但不能形成E（A-A），不刺激α-亚基的活性。滴定实验表明，G3 P和GP在α位点的亲和力取决于β活性位点上存在的化学中间体的性质。这些结果表明，双酶复合物中异源亚基之间的配体依赖性变构相互作用用于协调α和β活性位点处的催化事件，以确保L-Trp的有效合成。我们提出，这些配体依赖性变构现象是伴随着在“开放”和“封闭”的构象，控制配体的亲和力和催化活性之间的α-和β-亚基的构象转变。
Tryptophan synthase from enteric bacteria is an alpha-2-beta-2 bienzyme complex that catalyzes the final two reactions in the biosynthesis of L-tryptophan (L-Trp) from 3-indole-D-glycerol 3'-phosphate (IGP) and L-serine (L-Ser). The bienzyme complex exhibits reciprocal ligand-mediated allosteric interactions between the heterologous subunits [Houben, K., & Dunn, M. F. (1990) Biochemistry 29, 2421-2429], but the relationship between allostery and catalysis had not been completely defined. We have utilized rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy to study the relationship between allostery and catalysis in the alpha-beta-reaction catalyzed by the bienzyme complex from Salmonella typhimurium. The pre-steady-state spectral changes that occur when L-Ser and IGP are mixed simultaneously with the alpha-2-beta-2 complex show that IGP binding to the alpha-site accelerates the formation of alpha-aminoacrylate [E(A-A)] from L-Ser at the beta-site. Through the use of L-Ser analogues, we show herein that the formation of the E(A-A) intermediate is the chemical signal which triggers the conformational transition that activates the alpha-subunit. Beta-subunit ligands, such as L-Trp, that react to form covalent intermediates at the beta-site, but are incapable of E(A-A) formation, do not stimulate the activity of the alpha-subunit. Titration experiments show that the affinity of G3P and GP at the alpha-site is dependent upon the nature of the chemical intermediate present at the beta-active site. These results show that ligand-dependent allosteric interactions between heterologous subunits in the bienzyme complex serve to coordinate catalytic events at the alpha- and beta-active sites to ensure the efficient synthesis of L-Trp. We propose that these ligand-dependent allosteric phenomena are accompanied by conformational transitions in both the alpha- and beta-subunits between "open" and "closed" conformations that control ligand affinity and catalytic activity.