Affinities of phosphorylated substrates for the E. coli tryptophan synthase α-subunit: Roles of Ser-235 and helix-8′ dipole

The roles of Ser-235 and helix-8′ (residues 235–242) in the functional binding and turnover of phosphorylated substrates by the α-subunit of the E. coli tryptophan synthase (TSase) α₂β₂-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 K_ms of mutant α-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 nonpeptidyl amido-containing residue (proline, mutant SP235) exhibit 40- to 180-fold K_m increases for IGP and D -glyceraldehyde-3-phosphate; no K_m defects for indole were observed. k_cat 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 α-subunits; however, the SP235 results are consistent with the suggested phosphate binding role for the Ser-235 peptidyl amide 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 K_ms 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. K_m increases for phosphorylated substrates are substantial (up to 40-fold) and are dependent upon the presence of L -serine at the β-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 inter-subunit-induced conformational changes due to ligand (L -serine) binding to the β-subunit. © 1995 Wiley-Liss, Inc.     

Growth and morphogenesis of embryonic mouse organs on biopore membrane

Research supported by the American Heart Association, the Wesley Foundation, and NASA (NAGW 1197 and 2328).