Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits
Lim, W.K.; Shin, H.J.; Milton, D.L.; Hardman, J.K.
Journal of Bacteriology 173(6): 1886-1893
ISSN/ISBN: 0021-9193 PMID: 2001993 Accession: 007741395
In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase .alpha. subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant .alpha. subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability properties in crude extracts. With the exception of mutant .alpha. subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 and 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant .alpha. subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant .alpha. subunits alered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specific mutagenesis of the Salmonella typhimurium tryptophan synthetase .alpha. subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant .alpha. subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the .beta.2 subunit. Also among the mutant .alpha. subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the .alpha. polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth requirement of the trpA mutant host. Heat stability and potential folding-rate alterations are found in both enzymatically active and defective mutant .alpha. subunits. Tyr-4, Pro-28, Ser-33, Gly-44, Asp-46, Arg-89, Pro-96, and Cys-118 may be important for these properties, especially for folding. Two regions, one near Thr-24 and another near Met-101, have been also tentatively identified as important for increasing stability.