ABSTRACT
Conformational changes in the ß2α2 and ß6α6 loops in the alpha subunit of tryptophan synthase (αTS) are important for enzyme catalysis and coordinating substrate channeling with the beta subunit (ßTS). It was previously shown that disrupting the hydrogen bond interactions between these loops through the T183V substitution on the ß6α6 loop decreases catalytic efficiency and impairs substrate channeling. Results presented here also indicate that the T183V substitution decreases catalytic efficiency in Escherchia coli αTS in the absence of the ßTS subunit. Nuclear magnetic resonance (NMR) experiments indicate that the T183V substitution leads to local changes in the structural dynamics of the ß2α2 and ß6α6 loops. We have also used NMR chemical shift covariance analyses (CHESCA) to map amino acid networks in the presence and absence of the T183V substitution. Under conditions of active catalytic turnover, the T183V substitution disrupts long-range networks connecting the catalytic residue Glu49 to the αTS-ßTS binding interface, which might be important in the coordination of catalytic activities in the tryptophan synthase complex. The approach that we have developed here will likely find general utility in understanding long-range impacts on protein structure and dynamics of amino acid substitutions generated through protein engineering and directed evolution approaches, and provide insight into disease and drug-resistance mutations.
