The tryptophan synthase alpha-2,beta-2 multienzyme complex is an excellent model system for investigating enzyme mechanism, protein-protein interaction, metabolite channeling, and ligand-dependent site-site interactions. Our recent determination by x-ray crystallography of the three-dimensional structure of the tryptophan synthase complex alpha-2,beta-2 from salmonella typhimurium provides a useful framework for further exploration of the relationships between protein structure and protein function. We are using the three-dimensional structure to select enzyme residues for site-directed mutagenesis. Our studies with wild type and mutant forms have led to the following findings during the last year: (1) Single turnover quench-flow kinetic studies demonstrate that indole is a channeled intermediate and L-serine modulates the rate of indole formation. (2) Certain mutations alter the kinetics of indole formation and the rate of passage of indole through the channel. (3) Alteration of a flexible loop in the alpha subunit by mutagenesis or by proteolytic cleavage modifies allosteric effects and the reciprocal communication between subunits. (4) Certain mutations in the active site and tunnel region of the beta subunit alter the reaction and substrate specificity and lead to substrate-induced inactivation. (5) Scanning microcalorimetric and circular dichroism studies of the thermal unfolding of wild type and mutant forms of tryptophan synthase complex and of its subunits reveal effects of subunit interaction and of single amino acid residues on protein stability. (6) Spectroscopic studies of chromophoric intermediates formed between pyridoxal phosphate and substrates at the active site of the beta subunit demonstrate that the equilibrium distribution of intermediates is affected by pH, by ligands of the alpha subunit, and by mutation.
