Effects of Native and Permethylated Cyclodextrins on the Catalytic Activity of L-Tryptophan indole lyase

Journal of Inclusion Phenomena (Impact Factor: 1.4). 01/2006; 54(3):283-288. DOI:10.1007/s10847-005-9003-z

ABSTRACT The impact of native and permethylated α-, β-, and γ-cyclodextrins on the L-tryptophan indole lyase-catalyzed decomposition of L-tryptophan was investigated by means of spectrophotometric measurements. The inhibitory effects of cyclodextrins on the catalytic
activity of the enzyme are shown. The observed inhibition is of mixed type, i.e. competitive and non-competitive. This phenomenon
is supposed to be the result of host–guest complex formation involving cyclodextrins and L-tryptophan, and probably between aromatic amino acid residues on the surface of the investigated enzyme. Therefore cyclodextrins
were found to have an impact on the maximal velocity and on the Michaelis constant of the described catalysis. The competitive
inhibition does not only depend on the stabilities of inclusion complexes, but mainly on their structures.

0 0
  • [show abstract] [hide abstract]
    ABSTRACT: An X-ray diffraction study was carried out on anhydrous permethyl-alpha-cyclodextrin crystallized from aq solution at 40 degrees C, space group P2(1)2(1)2(1) with a = 15.424(9), b = 18.167(7), c = 23.128(8) A, vol = 6481(5) A3. The molecule is boat-shaped with the narrow end of the cyclodextrin cavity closed by two diametrically opposing glucose residues. These are strongly tilted towards the molecular axis, so that their primary methoxy groups form van der Waals contacts across the molecular opening. The remaining cavity volume is occupied by the methoxy group of a neighboring molecule (self-inclusion). From solutions containing 1 M NaCl, anhydrous permethyl-alpha-cyclodextrin crystallizes isomorphously at room temperature.
    Carbohydrate Research 03/1996; 282(1):53-63. · 2.04 Impact Factor
  • [show abstract] [hide abstract]
    ABSTRACT: 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.
    Biochemistry 01/1985; 23(25):6228-34. · 3.38 Impact Factor
  • [show abstract] [hide abstract]
    ABSTRACT: Tryptophan indole-lyase from Escherichia coli catalyzes the reversible cleavage of l-tryptophan to indole and ammonium pyruvate. This reaction is mechanistically interesting since it involves the elimination of an aromatic carbon leaving group. We have been studying the mechanism of tryptophan indole-lyase using rapid-scanning stopped-flow spectrophotometry. Recently, we demonstrated that the rate constant for α-aminoacrylate intermediate formation from α-2H-l-tryptophan exhibits an isotope effect of 3.0 (Sloan, M. J.; Phillips, R. S. Biochemistry 1996, 35, 16165−16173). We have confirmed this previous result (Dk = 2.99 ± 0.30) and we have now found that β,β-di-2H-l-tryptophan also exhibits a secondary isotope effect (Dk = 1.17 ± 0.03) on the elimination reaction. Furthermore, α,β,β-tri-2H-l-tryptophan exhibits a multiple isotope effect (Dk = 4.42 ± 0.67) on the elimination of indole. In addition, there is a significant solvent isotope effect (Dk = 1.79 ± 0.11) on indole elimination in D2O. This solvent isotope effect combines with the effect of α-deuterium, since elimination of α-2H-l-tryptophan in D2O exhibits Dk = 4.30 ± 0.16. In addition, the rate constant for indole elimination shows a linear Eyring plot between 5 and 35 °C. In the direction of tryptophan synthesis, the reaction of the α-aminoacrylate intermediate with indole to form a quinonoid intermediate also exhibits a kinetic isotope effect for 3-2H-indole, with Dk = 1.88 ± 0.19. In contrast to our expectations, the results suggest that the proton transfer and carbon−carbon bond cleavage in the elimination reaction are very nearly simultaneous and that the indolenine structure is a transient intermediate which occupies a very shallow well on the reaction coordinate, or a transition state, in the reaction of Trpase.
    ChemInform 01/2010; 31(20).