Affinities of phosphorylated substrates for the E. coli tryptophan synthase alpha-subunit: roles of Ser-235 and helix-8' dipole.Proteins. 1995 Feb; 21(2):130-9.P
The roles of Ser-235 and helix-8' (residues 235-242) in the functional binding and turnover of phosphorylated substrates by the alpha-subunit of the E. coli tryptophan synthase (TSase) alpha 2 beta 2-holoenzyme complex are examined. Previous crystallographic analyses indicated that this region was one of several near the phosphate moiety of the physiological substrate, indole-3-glycerol phosphate (IGP). The peptidyl amido group of Ser-235 was suggested to H-bond to the phosphate group; a helix macrodipole binding role was suggested for helix-8'. The activities and substrate Kms of mutant alpha-subunits altered in this region by site-specific mutagenesis are reported here. Substitutions at Ser-235 by an acidic (glutamic acid, mutant SE235), basic (lysine, mutant SK235), or a non-peptidyl amido-containing residue (proline, mutant SP235) exhibit 40- to 180-fold Km increases for IGP and D-glyceraldehyde-3-phosphate; no Km defects for indole were observed. kcat values for SP235, SE235, and SK235 are 100, 70, and 40%, respectively, of the wild-type value. Steric considerations may explain the results with the SE235 and SK235 mutant alpha-subunits; however, the SP235 results are consistent with the suggested phosphate binding role for the Ser-235 peptidyl amido group during catalysis, A helix-8' dipole role was explored following proline substitutions separately at the first six (of eight) residues. Proline substitutions at positions-1 through -4 in helix-8' have normal indole Kms and catalytic activities in all four TSase reactions, suggesting no major global structural changes in these proteins. By these criteria, substitutions at positions-5 and -6 lead to significant structural alterations. Km increases for phosphorylated substrates are substantial (up to 40-fold) and are dependent upon the presence of L-serine at the beta-subunit active site. In the absence of L-serine, substitution only at the first position results in binding defects; in the presence of L-serine, substitutions at the first, second and third positions, show binding defects of decreasing magnitude, sequentially. Substitutions at the fourth and fifth position have no effect on substrate binding. It is suggested that during catalysis a helix dipole effect on binding may be exerted but only via intersubunit-induced conformational changes due to ligand (L-serine) binding to the beta-subunit.