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.
We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Since these analogues 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-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or 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 microM, which are comparable to the KI values obtained in kinetic experiments. Our 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. These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct.
D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D013058:Mass Spectrometry; D000078183:Oxindoles; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013053:Spectrophotometry; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014368:Tryptophanase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000078183:Oxindoles; C044832:oxindolylalanine; D011732:Pyridoxal Phosphate; C044338:2,3-dihydrotryptophan; D014364:Tryptophan; D008190:Lyases; D014368:Tryptophanase; D014367:Tryptophan Synthase; D000409:Alanine