Brzović P S, Hyde C C, Miles E W, Dunn M F
Department of Biochemistry, University of California at Riverside 92521-0129.
Biochemistry. 1993 Oct 5;32(39):10404-13. doi: 10.1021/bi00090a016.
The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha 2 beta 2 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 alpha R179L-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 alpha R179L 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.
利用快速扫描（RSSF）和单波长（SWSF）停流光谱技术，对色氨酸合酶α2β2双酶复合物α亚基中柔性环（环6）的功能进行了研究。环6是一段延伸的残基序列，它在α亚基的β/α桶状折叠结构中连接β链6和α螺旋6。在环6基部附近用亮氨酸取代精氨酸179，对α亚基和β亚基形成双酶复合物的缔合过程，以及吲哚与L-丝氨酸（L-Ser）反应生成L-色氨酸（L-Trp）（该过程由野生型β亚基催化）的动力学均无显著影响[川崎，H.，鲍勒尔，R.，宗，G.，艾哈迈德，S.，&迈尔斯，E.W.（1987年）《生物化学杂志》262，10678 - 10683]。然而，α亚基特异性配体甘油磷酸（GP），它是野生型β反应的抑制剂，对αR179L催化的β反应的抑制效果要差得多。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。对双酶复合物催化IGP与L-Ser反应生成L-Trp的过程进行SWSF分析发现，与野生型酶相比，在αR179L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对于配体与α位点的结合以及配体诱导的α亚基从“开放型”到“封闭型”结构的构象转变都很重要。基于α亚基与糖酵解酶磷酸丙糖异构酶（TIM）广泛的结构同源性进行的建模研究预测，在α活性位点结合配体诱导环6闭合，将有效地使结合的底物与溶剂隔离，并将IGP裂解产生的吲哚捕获在双酶复合物的范围内。这种构象转变将促进吲哚通过连接通道扩散到β活性位点，并有助于确保α亚基和β亚基催化活性的紧密协调。
