Site-specific mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. Changing arginine 179 to leucine alters the reciprocal transmission of substrate-induced conformational changes between the alpha and beta 2 subunits.
Arginine 179 of the alpha subunit of tryptophan synthase of Salmonella typhimurium was changed to leucine by site-directed mutagenesis. The mutant alpha subunit was expressed in S. typhimurium, purified and crystallized as the alpha 2 beta 2 complex, and characterized by kinetic studies under steady-state reaction conditions. The rate of cleavage of indole 3-glycerol phosphate (alpha reaction) is reduced by 60% in the mutant alpha 2 beta 2 complex, whereas the rate of L-tryptophan synthesis from indole and L-serine (beta reaction) is unchanged. Thus, arginine 179 is not obligatory for catalysis, for binding of indole 3-glycerol phosphate, or for interaction of the alpha and beta 2 subunits. However, changing arginine 179 to leucine does have striking effects on ligand-dependent properties of this multienzyme complex. Ligands of the alpha subunit (DL-alpha-glycerophosphate and indole 3-propanol phosphate) which strongly inhibit the beta reaction of the native alpha 2 beta 2 complex have a slight stimulatory effect on the beta reaction of the mutant alpha 2 beta 2 complex. Likewise, L-serine, a ligand of the beta subunit which produces a 5-fold reduction in the Km for the alpha ligand indole 3-glycerol phosphate in the native alpha 2 beta 2 complex, has no effect on the mutant alpha 2 beta 2 complex. These results suggest that arginine 179 of the alpha subunit plays a role in the reciprocal transmission of substrate-induced conformational changes which occur between native alpha and beta 2 subunits in the alpha 2 beta 2 complex.
D000595:Amino Acid Sequence; D001120:Arginine; D004274:DNA, Recombinant; D005994:Glycerophosphates / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine; D046911:Macromolecular Substances; D009154:Mutation; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014367:Tryptophan Synthase / Q000235:genetics / Q000378:metabolism*
D004274:DNA, Recombinant; D005994:Glycerophosphates; D007211:Indoles; D046911:Macromolecular Substances; C041272:indolepropanol phosphate; D012694:Serine; D001120:Arginine; C029620:alpha-glycerophosphoric acid; D014367:Tryptophan Synthase; D007930:Leucine