LOOP CLOSURE AND INTERSUBUNIT COMMUNICATION IN TRYPTOPHAN SYNTHASE

Crystal structures of wild-type tryptophan synthase alpha(2) beta(2) c omplexes from Salmonella typhimurium were determined to investigate th e mechanism of allosteric activation of the alpha-reaction by the amin oacrylate intermediate formed at the beta-active site. Using a flow ce ll, the aminoacrylate (A-A) intermediate of the beta-reaction (TRPSA-A F-IPP) was generated in the crystal under steady state conditions in t he presence of serine and the alpha-site inhibitor 5-fluoroindole prop anol phosphate (F-IPP). A model for the conformation of the Schiff bas e between the aminoacrylate and the beta-subunit cofactor pyridoxal ph osphate (PLP) is presented. The TRPSA-AF-IPP structure is compared wit h structures of the enzyme determined in the absence (TRPS) and presen ce (TRPSF-IPP) of F-IPP. A detailed model for binding of F-IPP to the alpha-subunit is presented. In contrast to findings by Hyde et al. [(1 988) J. Biol. Chem. 263, 17857-17871] and Rhee et al. [(1997) Biochemi sty 36, 7664-7680], we find that the presence of an alpha-site alone l igand is sufficient for loop alpha L6 closure atop the alpha-active si te. Part of this loop, alpha Thr183, is important not only for positio ning the catalytic alpha Asp60 but also for coordinating the concomita nt ordering of loop alpha L2 upon F-IPP binding. On the basis of the t hree structures, a pathway for communication between the alpha- and be ta-active sites has been established. The central element of this path way is a newly defined rigid, but movable, domain that on one side int eracts with the alpha-subunit via loop alpha L2 and on the other side with the beta-active site. These findings provide a structural basis f or understanding the allosteric properties of tryptophan synthase.