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Zusammenfassung:
Crystal structures of wild−type tryptophan synthase α2β2 complexes from Salmonella typhimurium were determined to investigate the mechanism of allosteric activation of the α−reaction by the aminoacrylate intermediate formed at the β−active site. Using a flow cell, the aminoacrylate (A−A) intermediate of the β−reaction () was generated in the crystal under steady state conditions in the presence of serine and the α−site inhibitor 5−fluoroindole propanol phosphate (F−IPP). A model for the conformation of the Schiff base between the aminoacrylate and the β−subunit cofactor pyridoxal phosphate (PLP) is presented. The structure is compared with structures of the enzyme determined in the absence (TRPS) and presence (TRPSF−IPP) of F−IPP. A detailed model for binding of F−IPP to the α−subunit is presented. In contrast to findings by Hyde et al. [(1988) J. Biol. Chem. 263,17857−17871] and Rhee et al. [(1997) Biochemistry 36, 7664−7680], we find that the presence of an α−site alone ligand is sufficient for loop αL6 closure atop the α−active site. Part of this loop, αThr183, is important not only for positioning the catalytic αAsp60 but also for coordinating the concomitant ordering of loop αL2 upon F−IPP binding. On the basis of the three structures, a pathway for communication between the α− and β−active sites has been established. The central element of this pathway is a newly defined rigid, but movable, domain that on one side interacts with the α−subunit via loop αL2 and on the other side with the β−active site. These findings provide a structural basis for understanding the allosteric properties of tryptophan synthase
