Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism

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Abstract
The interaction of tryptophan synthase and tryptophanase with the tryptophan analogs oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan was examined. Since these analogs have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan. Oxindolyl-L-analine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 .mu.M) 10- to 100-fold lower than the corresponding Km of KI values for L-tryptophan. Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the .alpha.2.beta.2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or .alpha.-carbanion intermediate. Spectrophotometric titration data give half-saturation values of 5 and 25 .mu.M, which are comparable to the KI values obtained in kinetic experiments. The finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form .alpha.-carbanion intermediates with oxindolyl-L-alanine. The indolenine tautomer of L-tryptophan is probably an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct.