We are using the tryptophan synthase multienzyme alpha2beta2 complex as a model system for investigating how protein-protein interaction and protein-ligand interaction affect enzyme structure and function. Our previous X-ray crystallographic studies of the wild type alpha2beta2 complex from Salmonella typhimurium reveal that the active sites of the alpha and beta subunits are 25 A apart and are connected by a tunnel. Thus reciprocal communication between the active sites of the heterologous subunits occurs over 25 A and must involve protein conformational changes. We are using the crystal structure as a framework for selecting enzyme residues for site-directed mutagenesis. During the past year we have used several experimental techniques to investigate intersubunit communication and putative conformational changes in the wild type and mutant enzymes. (1) X-ray crystallographic studies have refined the structures of the wild type alpha2beta2 complex and of two mutant forms and have detected conformational changes upon ligand binding. (2) Steady state and rapid kinetic studies have shown the four mutations in the alpha subunit prevent a conformational changes that affects channeling and intersubunit communication. Mutations in the interaction site between the alpha and beta subunits alter subunit association and intersubunit communication. (3) Differential scanning calorimetry is clarifying the effects of ligands and of subunits association on thermal unfolding. (4) Fluorescence spectroscopy has shown that alpha subunit ligands and limited proteolysis of loop 6 in the alpha subunit alter intersubunit communication and the interaction of Nile Re with a hydrophobic site within the beta subunit in the alpha2beta2 complex. (5) Spectroscopic studies have elucidated the mechanistic roles of beta subunit Lys-87 in pyridoxal phosphate-dependent reactions. Lys-87 serves critical roles in ransimination, catalysis and product release. Addition of ammonia to an enzyme-serine intermediate results in """"""""chemical rescue"""""""" of the mutant enzyme and results in the formation of a stable aminocrylate intermediate at the active site key intermediate which triggers the activation of the alpha subunit.
