CHARACTERIZATION OF THE FUNCTIONAL-ROLE OF A FLEXIBLE LOOP IN THE ALPHA-SUBUNIT OF TRYPTOPHAN SYNTHASE FROM SALMONELLA-TYPHIMURIUM BY RAPID-SCANNING, STOPPED-FLOW SPECTROSCOPY AND SITE-DIRECTED MUTAGENESIS

The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha2beta2 bienzyme complex has been investigate d utilizing rapid-scanning (RSSF) and single-wavelength (SWSF) stopped -flow spectroscopies. Loop 6 is an extended sequence of residues which connects beta-strand 6 with, alpha-helix 6 in the beta/alpha-barrel f old of the alpha-subunit. Substitution of Leu for Arg179 near the base of loop 6 does not significantly affect either the association of the alpha- and beta-subunits to form the bienzyme complex or the kinetics of the reaction of indole with L-serine (L-Ser) to form L-tryptophan (L-Trp), the process catalyzed by the wild-type beta-subunit [Kawasaki , H., Bauerle, R., Zon, G., Ahmed, S., & Miles, E. W. (1987) J. Biol. Chem. 262, 10678-10683]. However, the alpha-subunit-specific ligand gl ycerol phosphate (GP), which is an inhibitor of the wild-type beta-rea ction, is a much less effective inhibitor of the alphaR179L-catalyzed beta-reaction. Equilibrium titration studies show that the affinity of GP for the alpha-site when either L-Ser or glycine is bound at the be ta-site has been reduced by nearly 100- and 200-fold, respectively. SW SF analysis of the reaction of IGP and L-Ser to form L-Trp catalyzed b y the bienzyme complex revealed a 15-fold reduction in the binding aff inity of the alpha-site substrate 3-indole-D-glycerol 3'-phosphate (IG P) in the reaction catalyzed by the alphaR179L mutant as compared to t he wild-type enzyme. These studies show that loop 6 is important both for ligand binding to the alpha-site and for the ligand-induced confor mational transition of the alpha-subunit from an ''open'' to a ''close d'' structure. Modeling studies, based on extensive structural homolog y of the alpha-subunit with the glycolytic enzyme triosephosphate isom erase (TIM), predict that closure of loop 6 induced by ligand binding at the alpha-active site would effectively sequester the bound substra te from the solvent and trap indole, produced from the cleavage of IGP , within the confines of the bienzyme complex. This conformational tra nsition would promote the diffusion of indole to the beta-active site via the interconnecting tunnel and would help ensure the close coordin ation of alpha- and beta-subunit catalytic activities.