CRYOCRYSTALLOGRAPHY AND MICROSPECTROPHOTOMETRY OF A MUTANT (ALPHA-D60N) TRYPTOPHAN SYNTHASE ALPHA(2)BETA(2) COMPLEX REVEALS ALLOSTERIC ROLES OF ALPHA-ASP6O

We have investigated the role of Asp60 of the alpha-subunit in alloste ric communication between the tryptophan synthase alpha- and beta-subu nits. Crystallographic and microspectrophotometric studies have been c arried out on a mutant (alpha D60N) tryptophan synthase alpha(2)beta(2 ) complex which has no observable alpha-activity, but has substantial beta-activity. Single-crystal polarized absorption spectra indicate th at the external aldimine is the predominant L-serine intermediate and that the amount of the intermediate formed is independent of pH, monov alent cations, and allosteric effecters. The three-dimensional structu re is reported for this mutant enzyme complexed with indole 3-propanol phosphate bound to the alpha-site and L-serine bound to the beta-site (alpha D60N-IPP-Ser), and this structure is compared with that of the unliganded mutant enzyme (alpha D60N). In the complex, L-serine forms a stable external aldimine with the pyridoxal phosphate coenzyme at t he active site of the beta-subunit. The conformation of the unliganded mutant is almost identical to that of the wild type enzyme. However, the structure of the mutant complexed with IPP and serine exhibits lig and-induced conformational changes much smaller than those observed pr eviously for another mutant enzyme in the presence of the same ligands (beta K87T-IPP-Ser) [Rhee, S., Parris, K. D., Hyde, C. C., Ahmed, S. A., Miles, E. W., and Davies, D. R. (1997) Biochemistry 36, 7664-7680] . The alpha D60N-IPP-Ser alpha(2)beta(2) complex does not undergo the following ligand-induced conformational changes: (1) the closure of th e alpha-subunit loop 6 (residues 178-191), (2) the movement of the mob ile subdomain (residues 93-189) of the beta-subunit, and (3) the rotat ion of the alpha-subunit relative to the beta-subunit. These observati ons show that alpha Asp60 plays important roles in the closure of loop 6 and in allosteric communication between the alpha- and beta-subunit s.