Tryptophan Synthase: Structure and Function of the Monovalent Cation Site

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• 作者：Adam T. Dierkers ; Dimitri Niks ; Ilme Schlichting ; Michael F. Dunn
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：November 24, 2009
• 年：2009
• 卷：48
• 期：46
• 页码：10997-11010
• 全文大小：1278K
• 年卷期：v.48,no.46(November 24, 2009)
• ISSN：1520-4995

文摘

The monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of substrate channeling. In vitro, MVCs affect the equilibrium distribution of intermediates formed in the reaction of l-Ser with the α₂β₂ complex; the MVC-free, Cs⁺-bound, and NH₄⁺-bound enzymes stabilize the α-aminoacrylate species, E(A-A), while Na⁺ binding stabilizes the l-Ser external aldimine species, E(Aex₁). Two probes of β-site reactivity and conformation were used herein, the reactive indole analogue, indoline, and the l-Trp analogue, l-His. MVC-bound E(A-A) reacts rapidly with indoline to give the indoline quinonoid species, E(Q)_indoline, which slowly converts to dihydroiso-l-tryptophan. MVC-free E(A-A) gives very little E(Q)_indoline, and turnover is strongly impaired; the fraction of E(Q)_indoline formed is <3.5% of that given by the Na⁺-bound form. The reaction of l-Ser with the MVC-free internal aldimine species, E(Ain), initially gives small amounts of an active E(A-A) which converts to an inactive species on a slower, conformational, time scale. This inactivation is abolished by the binding of MVCs. The inactive E(A-A) appears to have a closed β-subunit conformation with an altered substrate binding site that is different from the known conformations of tryptophan synthase. Reaction of l-His with E(Ain) gives an equilibrating mixture of external aldimine and quinonoid species, E(Aex)_his and E(Q)_his. The MVC-free and Na⁺ forms of the enzyme gave trace amounts of E(Q)_his (1% of the β-sites). The Cs⁺ and NH₄⁺ forms gave 17 and 14%, respectively. The reactivity of MVC-free E(Ain) was restored by the binding of an α-site ligand. These studies show MVCs and α-site ligands act synergistically to modulate the switching of the β-subunit from the open to the closed conformation, and this switching is crucial to the regulation of β-site catalytic activity. Comparison of the structures of Na⁺ and Cs⁺ forms of the enzyme shows Cs⁺ favors complexes with open indole binding sites poised for the conformational transition to the closed state, whereas the Na⁺ form does not. The β-subunits of Cs⁺ complexes exhibit preformed indole subsites; the indole subsites of the open Na⁺ complexes are collapsed, distorted, and too small to accommodate indole.