Indole protects tryptophan indole-lyase, but not tryptophan synthase, from inactivation by trifluoroalanine.
Trifluoroalanine is a mechanism-based inactivator of Escherichia coli tryptophan indole-lyase (tryptophanase) and E. coli tryptophan synthase (R. B. Silverman and R. H. Abeles, 1976, Biochemistry 15, 4718-4723). We have found that indole is able to prevent inactivation of tryptophan indole-lyase by trifluoroalanine. The protection of tryptophan indole-lyase by indole exhibits saturation kinetics, with a KD of 0.03 mM, which is comparable to the KI for inhibition of pyruvate ion formation (0.01 mM) and the Km for L-tryptophan synthesis. Fluoride electrode measurements indicate the formation of 28 mol of fluoride ion per mole of enzyme during inactivation of tryptophan indole-lyase, and 121 mol of fluoride ion are formed per mole of enzyme in the presence of 2 mM indole during the same incubation period. 19F NMR spectra of reaction mixtures of tryptophan indole-lyase and trifluoroalanine showed evidence only for fluoride ion formation, in either the absence or the presence of indole, and difluoropyruvic acid was not detected. The partition ratio, kcat/kinact, is estimated to be 9. Tryptophan indole-lyase in the presence of trifluoroalanine exhibits visible absorption peaks at 446 and 478 nm, which decay at the same rate as inactivation. However, in the presence of 1 mM indole and trifluoralanine, tryptophan indole-lyase exhibits a peak only at 420 nm, and the spectra show a gradual increase at 300-310 nm with incubation. In contrast, tryptophan synthase is not protected by indole from inactivation by trifluoroalanine, and the absorption peak at 408 nm for the tryptophan synthase-trifluoroalanine complex is unaffected by indole. These results demonstrate that inactivation of tryptophan indole-lyase occurs via a catalytically competent species, probably the beta,beta-difluoro-alpha-aminoacrylate intermediate, which can be partitioned from inactivation to products by a reactive aromatic nucleophile, indole.
Phillips
R S
RS
Department of Chemistry, College of Pharmacy, University of Georgia, Athens 30602.
Dua
R K
RK
D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005459:Fluorides / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013053:Spectrophotometry; D014367:Tryptophan Synthase / Q000037:antagonists & inhibitors*; D014368:Tryptophanase / Q000037:antagonists & inhibitors*
D007211:Indoles; D011773:Pyruvates; C016164:3,3,3-trifluoroalanine; D019289:Pyruvic Acid; C030374:indole; D014368:Tryptophanase; D014367:Tryptophan Synthase; D000409:Alanine; D005459:Fluorides