The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230.
Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit.
Miles
E W
EW
Section on Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Kawasaki
H
H
Ahmed
S A
SA
Morita
H
H
Morita
H
H
Nagata
S
S
D001120:Arginine; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine; D004252:DNA Mutational Analysis; D006639:Histidine; D008239:Lysine; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D013057:Spectrum Analysis; D014367:Tryptophan Synthase / Q000235:genetics / Q000502:physiology*
D011994:Recombinant Proteins; D006639:Histidine; D011732:Pyridoxal Phosphate; D001120:Arginine; D014367:Tryptophan Synthase; D008239:Lysine; D003545:Cysteine