Allostery and Substrate Channeling in the Tryptophan Synthase Bienzyme Complex: Evidence for Two Subunit Conformations and Four Quaternary States

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• 作者：Dimitri Niks ; Eduardo Hilario ; Adam Dierkers ; Huu Ngo ; Dan Borchardt ; Thomas J. Neubauer ; Li Fan ; Leonard J. Mueller ; Michael F. Dunn
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：September 17, 2013
• 年：2013
• 卷：52
• 期：37
• 页码：6396-6411
• 全文大小：984K
• 年卷期：v.52,no.37(September 17, 2013)
• ISSN：1520-4995

文摘

The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the 伪- and 尾-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the 伪- and 尾-catalytic cycles. ¹⁹F NMR studies of bound 伪-site substrate analogues, N-(4鈥?trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4鈥?trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of 尾-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while 伪-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of 鈭?.12 ppm. X-ray crystal structures show the F6 and F9 CF₃ groups located at the 伪鈥撐?subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in ¹⁹F chemical shift results primarily from changes in the 伪-site ligand conformation. Structures of 伪-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the 伪- and 尾-subunits have closed conformations wherein access of ligands into the 伪- and 尾-sites from solution is blocked. Internal and external aldimine structures show the 伪- and 尾-subunits with closed and open global conformations, respectively. These results establish that 尾-subunits exist in two global conformational states, designated open, where the 尾-sites are freely accessible to substrates, and closed, where the 尾-site portal into solution is blocked. Switching between these conformations is critically important for the 伪尾-catalytic cycle.