AFFINITIES OF PHOSPHORYLATED SUBSTRATES FOR THE ESCHERICHIA-COLI TRYPTOPHAN SYNTHASE ALPHA-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 a-subunit of the E. coli tryptophan synthase (TSase) alpha(2) beta(2)-holoenzyme c omplex are examined. Previous crystallographic analyses indicated that this region was one of several near the phosphate moiety of the physi ological substrate, indole-3-glycerol phosphate (IGP). The peptidyl am ido group of Ser-235 was suggested to H-bond to the phosphate group; a helix macrodipole binding role was suggested for helix-8'. The activi ties and substrate K(m)s of mutant alpha-subunits altered in this regi on by site-specific mutagenesis are reported here. Substitutions at Se r-235 by an acidic (glutamic acid, mutant SE235), basic (lysine, mutan t SK235), or a nonpeptidyl amido-containing residue (proline, mutant S P235) exhibit 40- to 180-fold K-m increases for IGP and D-glyceraldehy de-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 th e 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-2 35 peptidyl amido group during catalysis. A helix-8' dipole role was e xplored following proline substitutions separately at the first six (o f eight) residues. Proline substitutions at positions-1 through -4 in helix-8' have normal indole K(m)s and catalytic activities in all four TSase reactions, suggesting no major global structural changes in the se proteins. By these criteria, substitutions at positions-5 and -6 le ad to significant structural alterations. K-m increases for phosphoryl ated substrates are substantial (up to 40-fold) and are dependent upon the presence of L-serine at the beta-subunit active site. In the abse nce of L-serine, substitution only at the first position results in bi nding defects; in the presence of L-serine, substitutions at the first , second and third positions show binding defects of decreasing magnit ude, 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 intersub unit-induced conformational changes due to ligand (L-serine) binding t o the beta-subunit. (C) 1995 Wiley-Liss, Inc.