Novel allosteric effecters of the tryptophan synthase alpha(2)beta(2) complex identified by computer-assisted molecular modeling

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) c omplex catalyzing the formation of L-tryptophan. The functional properties of one subunit are allosterically regulated by ligands of the other subunit . Molecules tailored for binding to the alpha-active site were designed usi ng as a starting model the three-dimensional structure of the complex betwe en the enzyme from Salmonella typhimurium and the substrate analog indole-3 -propanol phosphate. On the basis of molecular dynamics simulations, indole -3-acetyl-X, where X is glycine, alanine, valine and aspartate, and a few o ther structurally related compounds were found to be good candidates for li gands of the alpha-subunit. The binding of the designed compounds to the al pha-active site was evaluated by measuring the inhibition of the alpha-reac tion of the enzyme from Salmonella typhimurium. The inhibition constants we re found to vary between 0.3 and 1.7 mM. These alpha-subunit ligands do not bind to the beta-subunit, as indicated by the absence of effects on the ra te of the beta-reaction in the isolated beta(2) dimer. A small inhibitory e ffect on the activity of the alpha(2)beta(2) complex was caused by indole-3 -acetyl-glycine and indole-3-acetyl-aspartate whereas a small stimulatory e ffect was caused by indole-3-acetamide. Furthermore, indole-3-acetyl-glycin e, indole-3-acetyl-aspartate and indole-3-acetamide perturb the equilibrium of the catalytic intermediates formed at the beta-active site, stabilizing the alpha-aminoacrylate Schiff base. These results indicate that (i) indol e-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide bind t o the alpha-subunit and act as allosteric effecters whereas indole-3-acetyl -valine and indole-3-acetyl-alanine only bind to the alpha-subunit, and (ii ) the terminal phosphate present in the already known allosteric effecters of tryptophan synthase is not strictly required for the transmission of reg ulatory signals. (C) 2000 Elsevier Science B.V. All rights reserved.