Loop closure and intersubunit communication in tryptophan synthase.
Crystal structures of wild-type tryptophan synthase alpha2beta2 complexes from Salmonella typhimurium were determined to investigate the mechanism of allosteric activation of the alpha-reaction by the aminoacrylate intermediate formed at the beta-active site. Using a flow cell, the aminoacrylate (A-A) intermediate of the beta-reaction () was generated in the crystal under steady state conditions in the presence of serine and the alpha-site inhibitor 5-fluoroindole propanol phosphate (F-IPP). A model for the conformation of the Schiff base between the aminoacrylate and the beta-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 alpha-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 alpha-site alone ligand is sufficient for loop alphaL6 closure atop the alpha-active site. Part of this loop, alphaThr183, is important not only for positioning the catalytic alphaAsp60 but also for coordinating the concomitant ordering of loop alphaL2 upon F-IPP binding. On the basis of the three structures, a pathway for communication between the alpha- and beta-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 alpha-subunit via loop alphaL2 and on the other side with the beta-active site. These findings provide a structural basis for understanding the allosteric properties of tryptophan synthase.
Schneider
T R
TR
Department for Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany.
Gerhardt
E
E
Lee
M
M
Liang
P H
PH
Anderson
K S
KS
Schlichting
I
I
D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000495:Allosteric Site; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D008024:Ligands; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000201:enzymology; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D014367:Tryptophan Synthase / Q000737:chemistry*
D008024:Ligands; D012694:Serine; C015102:dehydroalanine; D014367:Tryptophan Synthase; D000409:Alanine