CHARACTERIZATION OF THE FUNCTIONAL-ROLE OF A FLEXIBLE LOOP IN THE ALPHA-SUBUNIT OF TRYPTOPHAN SYNTHASE FROM SALMONELLA-TYPHIMURIUM BY RAPID-SCANNING, STOPPED-FLOW SPECTROSCOPY AND SITE-DIRECTED MUTAGENESIS
CHARACTERIZATION OF THE FUNCTIONAL-ROLE OF A FLEXIBLE LOOP IN THE ALPHA-SUBUNIT OF TRYPTOPHAN SYNTHASE FROM SALMONELLA-TYPHIMURIUM BY RAPID-SCANNING, STOPPED-FLOW SPECTROSCOPY AND SITE-DIRECTED MUTAGENESIS
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DOI：
10.1021/bi00090a016
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发表时间：
1993-10-05
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;HYDE, CC;DUNN, MF
利用快速扫描（RSSF）和单波长（SWSF）停流光谱研究了色氨酸合酶α 2 β 2双酶复合物α亚基中柔性环（环6）的功能。环6是残基的延伸序列，其在α-亚基的β/α-桶折叠中连接β-链6与α-螺旋6。在环6的碱基附近用Leu取代Arg 179不会显著影响α-和β-亚基形成双酶复合物的结合或吲哚与L-丝氨酸（L-Ser）形成L-色氨酸（L-Trp）的反应动力学，该反应由野生型β-亚基催化[川崎，H.，鲍尔河，Zon，G.，艾哈迈德，S.，& Miles，E. W.（1987）J.Biol.Chem.262，10678-10683]。然而，作为野生型β-反应抑制剂的α-亚基特异性配体甘油磷酸（GP）是α R179 L催化的β-反应的有效性低得多的抑制剂。平衡滴定研究表明，当L-Ser或甘氨酸结合在β位点时，GP对α位点的亲和力分别降低了近100倍和200倍。由双酶复合物催化的IGP和L-Ser形成L-Trp的反应的SWSF分析显示，与野生型酶相比，在由alphaR 179 L突变体催化的反应中，α位点底物3-吲哚-D-甘油3 ′-磷酸（IGP）的结合亲和力降低了15倍。这些研究表明，环6对于配体结合到α位点和对于配体诱导的α亚基从“开放”结构到“封闭"结构的构象转变都是重要的。建模研究，广泛的结构同源性的基础上的α-亚基与糖酵解酶磷酸丙糖异构酶（TIM），预测闭环6诱导的配体结合在α-活性位点将有效地螯合结合底物的溶剂和陷阱吲哚，产生的分裂的IGP，在双酶复合物的范围内。这种构象转变将促进吲哚通过相互连接的通道扩散到β-活性位点，并有助于确保α-和β-亚基催化活性的密切协调。
The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha2beta2 bienzyme complex has been investigated utilizing rapid-scanning (RSSF) and single-wavelength (SWSF) stopped-flow spectroscopies. Loop 6 is an extended sequence of residues which connects beta-strand 6 with, alpha-helix 6 in the beta/alpha-barrel fold of the alpha-subunit. Substitution of Leu for Arg179 near the base of loop 6 does not significantly affect either the association of the alpha- and beta-subunits to form the bienzyme complex or the kinetics of the reaction of indole with L-serine (L-Ser) to form L-tryptophan (L-Trp), the process catalyzed by the wild-type beta-subunit [Kawasaki, H., Bauerle, R., Zon, G., Ahmed, S., & Miles, E. W. (1987) J. Biol. Chem. 262, 10678-10683]. However, the alpha-subunit-specific ligand glycerol phosphate (GP), which is an inhibitor of the wild-type beta-reaction, is a much less effective inhibitor of the alphaR179L-catalyzed beta-reaction. Equilibrium titration studies show that the affinity of GP for the alpha-site when either L-Ser or glycine is bound at the beta-site has been reduced by nearly 100- and 200-fold, respectively. SWSF analysis of the reaction of IGP and L-Ser to form L-Trp catalyzed by the bienzyme complex revealed a 15-fold reduction in the binding affinity of the alpha-site substrate 3-indole-D-glycerol 3'-phosphate (IGP) in the reaction catalyzed by the alphaR179L mutant as compared to the wild-type enzyme. These studies show that loop 6 is important both for ligand binding to the alpha-site and for the ligand-induced conformational transition of the alpha-subunit from an ''open'' to a ''closed'' structure. Modeling studies, based on extensive structural homology of the alpha-subunit with the glycolytic enzyme triosephosphate isomerase (TIM), predict that closure of loop 6 induced by ligand binding at the alpha-active site would effectively sequester the bound substrate from the solvent and trap indole, produced from the cleavage of IGP, within the confines of the bienzyme complex. This conformational transition would promote the diffusion of indole to the beta-active site via the interconnecting tunnel and would help ensure the close coordination of alpha- and beta-subunit catalytic activities.