Evidence for two conformers of the beta subunit of tryptophan synthase in solution.
To explain our finding that the dimeric beta subunit of tryptophan synthase is only 50% inactivated by beta-chloro-L-alanine (Ahmed, S. A., Ruvinov, S. B., Kayastha, A. M., and Miles, E. W. (1991) J. Biol. Chem. 266, 21548-21557), we have extended our investigation using spectroscopic, steady-state kinetic, and electrophoretic methods. The spectroscopic properties of the half-active beta 2 dimer and the reactivation after alkali treatment show that the inactivation proceeds by an "enamine" mechanism. Although the fully active beta 2 dimer associates with the tryptophan synthase alpha subunit to form alpha 2 beta 2 complex, the inactive beta subunits in the half-active enzyme associate weakly or not at all with the alpha subunit. Our results provide evidence for two conformers of the beta subunit in solution: one is rapidly inactivated by beta-chloro-L-alanine and the other is not inactivated. Thermal inactivation studies and non-denaturing polyacrylamide gel electrophoresis of the half-active enzyme show that the beta 2 dimer exists in both homologous and heterologous combinations of these two forms. After removal of the reaction products and unreacted beta-chloro-L-alanine from the half-active beta 2 dimer by gel filtration, further incubation with beta-chloro-L-alanine results in the loss of 50% of the remaining activity. This result suggests that the subunits undergo rearrangement via an intermediate monomer form to regenerate the two conformers of the active beta subunit. This mechanism of rearrangement is supported by our finding that the extent of inactivation increases at lower concentrations of the beta 2 dimer.
Ahmed
S A
SA
Laboratory of Biochemical Pharmacology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Miles
E W
EW
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D013053:Spectrophotometry; D013379:Substrate Specificity; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D015091:beta-Alanine; C006977:3-chloroalanine; D014367:Tryptophan Synthase