THE BETA-SUBUNIT OF TRYPTOPHAN SYNTHASE - CLARIFICATION OF THE ROLES OF HISTIDINE-86, LYSINE-87, ARGININE-148, CYSTEINE-170, AND CYSTEINE-230

  • 15 April 1989
    • journal article
    • research article
    • Vol. 264  (11) , 6280-6287
Abstract
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 imporved 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 mehtod is also used in the accompanying paper to purify nine .alpha.2.beta.2 complexes containing mutant forms of the .alpha. subunit.